Purification of metal-dependent lysine deacetylases with consistently high activity.

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.

Formulation and evaluation of biodegradable nanoparticles for the oral delivery of fenretinide.

Fenretinide is an anticancer drug with low water solubility and poor bioavailability. The goal of this study was to develop biodegradable polymeric nanoparticles of fenretinide with the intent of increasing its apparent aqueous solubility and intestinal permeability. Three biodegradable polymers were investigated for this purpose: two different poly lactide-co-glycolide (PLGA) polymers, one acid terminated and one ester terminated, and one poly lactide-co-glycolide/polyethylene glycol (PLGA/PEG) diblock copolymer. Nanoparticles were obtained by using an emulsification solvent evaporation technique. The formulations were characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and particle size analysis. Dissolution studies and Caco-2 cell permeation studies were also carried out for all formulations. Ultra high performance liquid chromatography coupled with mass spectrometry (UPLC/MS) and ultraviolet detection was used for the quantitative determination of fenretinide. Drug loading and the type of polymer affected the nanoparticles' physical properties, drug release rate, and cell permeability. While the acid terminated PLGA nanoparticles performed the best in drug release, the ester terminated PLGA nanoparticles performed the best in the Caco-2 cell permeability assays. The PLGA/PEG copolymer nanoparticles performed better than the formulations with ester terminated PLGA in terms of drug release but had the poorest performance in terms of cell permeation. All three categories of formulations performed better than the drug alone in both drug release and cell permeation studies.

Preparation and in vitro evaluation of hydrophilic fenretinide nanoparticles.

Fenretinide is an effective anti-cancer drug with high in vitro cytotoxicity and low in vivo systemic toxicity. In clinical trials, fenretinide has shown poor therapeutic efficacy following oral administration - attributed to its low bioavailability and solubility. The long term goal of this project is to develop a formulation for the oral delivery of fenretinide. The purpose of this part of the study was to prepare and characterize hydrophilic nanoparticle formulations of fenretinide. Three different ratios of polyvinyl pyrrolidone (PVP) to fenretinide were used, namely, 3:1, 4:1, and 5:1. Both drug and polymer were dissolved in a mixture of methanol and dichloromethane (2:23 v/v). Rotary evaporation was used to remove the solvents, and, following reconstitution with water, a high pressure homogenizer was used to form nanoparticles. The particle size and polydispersity index were measured before and after lyophilization. The formulations were studied by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray powder diffraction (XRPD). The effectiveness of the formulations was assessed by release studies and Caco-2 cell permeability assays. As the PVP content increased, the recovered particle size following lyophilization became more consistent with the pre-lyophilization particle size, especially for those formulations with less lactose. The DSC scans of the formulations did not show any fenretinide melting endotherms, indicating that the drug was either present in an amorphous form in the formulation or that a solid solution of the drug in PVP had formed. For the release studies, the highest drug release among the formulations was 249.2±35.5ng/mL for the formulation with 4:1 polymer-to-drug. When the permeability of the formulations was evaluated in a Caco-2 cell model, the mean normalized flux for each treatment group was significantly higher (p<0.05) from the fenretinide control. The formulation containing 4:1 polymer-to-drug ratio and 6:5 lactose-to-formulation ratio emerged as the optimal choice for further evaluation as a potential oral delivery formulation for fenretinide.