Gene therapy approaches in the non-human primate model of Parkinson's disease.

The field of gene therapy has recently witnessed a number of major conceptual changes. Besides the traditional thinking that comprises the use of viral vectors for the delivery of a given therapeutic gene, a number of original approaches have been recently envisaged, focused on using vectors carrying genes to further modify basal ganglia circuits of interest. It is expected that these approaches will ultimately induce a therapeutic potential being sustained by gene-induced changes in brain circuits. Among others, at present, it is technically feasible to use viral vectors to (1) achieve a controlled release of neurotrophic factors, (2) conduct either a transient or permanent silencing of any given basal ganglia circuit of interest, (3) perform an in vivo cellular reprogramming by promoting the conversion of resident cells into dopaminergic-like neurons, and (4) improving levodopa efficacy over time by targeting aromatic L-amino acid decarboxylase. Furthermore, extensive research efforts based on viral vectors are currently ongoing in an attempt to better replicate the dopaminergic neurodegeneration phenomena inherent to the progressive intraneuronal aggregation of alpha-synuclein. Finally, a number of incoming strategies will soon emerge over the horizon, these being sustained by the underlying goal of promoting alpha-synuclein clearance, such as, for instance, gene therapy initiatives based on increasing the activity of glucocerebrosidase. To provide adequate proof-of-concept on safety and efficacy and to push forward true translational initiatives based on these different types of gene therapies before entering into clinical trials, the use of non-human primate models undoubtedly plays an instrumental role.

Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum.

Identification of G protein-coupled receptors and their specific function in a given neuron becomes essential to better understand the variety of signal transduction mechanisms associated with neurotransmission. We hypothesized that angiotensin II type 1 (AT1) and dopamine D2 receptors form heteromers in the central nervous system, specifically in striatum. Using bioluminescence resonance energy transfer, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors. Heteromerization did not affect cAMP signaling via D2 receptors but attenuated the coupling of AT1 receptors to Gq. A common feature of heteromers, namely cross-antagonism, i.e. the blockade of the signaling of one receptor by the blockade of the partner receptor, was tested in co-transfected cells. Candesartan, the selective AT1 receptor antagonist, was able to block D2-receptor mediated effects on cAMP levels, MAP kinase activation and ß-arrestin recruitment. This effect of candesartan, which constitutes a property for the dopamine-angiotensin receptor heteromer, was similarly occurring in primary cultures of neurons and rat striatal slices. The expression of heteromers in striatum was confirmed by robust labeling using in situ proximity ligation assays. The results indicate that AT1 receptors are expressed in striatum and form heteromers with dopamine D2 receptors that enable drugs selective for the AT1 receptor to alter the functional response of D2 receptors.

Drug solubilization and delivery from cyclodextrin-Pluronic aggregates.

Colloidal systems based on Pluronic F127 (PF127) and hydroxypropyl-beta-cyclodextrin (HPbetaCD) have been characterized with a view to their potential use as delivery systems of hydrophobic drugs. Complexation of PF127 and HPbetaCD was evaluated by surface tension measurements, 1H-NMR spectroscopy and transmission electron microscopy. The critical micellar concentration, CMC, at 25 degrees C of PF127 (0.39 mM in pH 5.8 and 7.4 phosphate buffers, and 0.59 mM in pH 4.5 acetic/acetate and lactic/lactate buffers) was shifted to higher values by the addition of 38.17 mM HPbetaCD (CMC(app) = 1.18 mM). This is related to the threading of HPbetaCD onto the PF127 chains, as confirmed by 1H NMR experiments. HPbetaCD at this concentration notably raised the sol-gel transition temperature; the minimum PF127 concentration required for providing gelling systems in physiological environments being 13.4 mM. Both HPbetaCD and PF127 by themselves are able to notably increase the solubility of sertaconazole (SN). At HPbetaCD concentrations below 80 mM, an additive effect of both components on SN solubility was observed. At greater HPbetaCD concentrations, a non-additive increase occurred, which is related to the complexation of some PF127 unimers with HPbetaCD molecules, decreasing the total number of micelles and HPbetaCD cavities available for interacting with SN. The 13.4 mM PF127/38.17 mM HPbetaCD system, able to increase up to 100 times the SN solubility in pH5.8 phosphate buffer, showed temperature-dependent drug diffusion coefficients, able to control the release for one week at 37 degrees C.

Effect of cyclodextrins on the solubility and antimycotic activity of sertaconazole: experimental and computational studies.

This study investigated the effects of the complexation of sertaconazole nitrate with different cyclodextrin (CD) derivatives (alpha-CD, beta-CD, gamma-CD, hydroxypropyl-beta-CD, and hydroxypropyl-gamma-CD) on the aqueous solubility and antimycotic activity of the drug. Phase solubility studies indicated that the solubility of sertaconazole in enzyme-free simulated gastric- and enzyme-free simulated enteric fluids was significantly increased in the presence of cyclodextrins. The observed order of solubility increasing effect was: gamma-CD > HPgamma-CD > HPbeta-CD > beta-CD > alpha-CD. Solid-state sertaconazole-cyclodextrin complexes were prepared by freeze drying, and characterized by X-ray powder difractometry, differential scanning calorimetry (DSC), and infrared spectroscopy (FTIR). Freeze-dried complexes showed markedly higher solubility than both physical mixtures and sertaconazole alone. The antimycotic activities of sertaconazole-cyclodextrin complexes in solution were evaluated by inhibition zone assays with Candida albicans. The activity ranking agrees with the solubility ranking observed for these complexes, with the gamma-CD-sertaconazole complex showing the strongest antimycotic activity. Finally, molecular modeling studies were carried out using the MM2 force field method, for complexes in vacuum and in water. This enable indentification of the preferred orientation of sertaconazole in the gamma-CD cavity and of the main structural features responsible for the enhancement of its solubility and antimycotic activity.