An in vitro toolbox to accelerate anti-malarial drug discovery and development.

BACKGROUND: Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. METHODS: Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS. RESULTS: Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. CONCLUSIONS: This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

Clostridium neurotoxin fragments as potential targeting moieties for liposomal gene delivery to the CNS.

Targeted transfection of the CNS with synthetic, nonviral vectors represents a huge technical challenge. The approach explored here attempts to combine self-assembly ABCD nanoparticles (Kostarelos and Miller, Chem. Soc. Rev. 2005, 34, 970), with the potential of Clostridium neurotoxin fragments to effect receptor-mediated transfection of neuronal cells. Cationic liposome-plasmid DNA complexes were first modified with a PEG stealth layer, before the addition of C-terminal fragments of tetanus toxin (TH(C)), botulinum toxin (BH(C)) or the truncated C-terminal domain of TH(C) as biological "targeting" ligands. First-generation nanoparticles were identified for the transfection of two neuronal cell lines (human SH-5YSY and rat/mouse hybrid N18-RE105); control studies were also performed with HeLa cells. ABCD nanoparticle transfections of the neuronal cell lines were up to 30-fold higher than corresponding control transfections with nanoparticles that lacked the protein ligand. We also demonstrate apparent receptor-mediated uptake by means of competition-binding and real-time confocal experiments. By contrast, nanoparticle transfection of HeLa cells appeared to involve alternative nonspecific enhanced cellular uptake mechanism(s). Receptor-mediated and nonspecific mechanisms appear to be in competition, potentially harming the capacity of receptor-mediated delivery to effect proper targeted delivery of nucleic acids to cells ex vivo and in vivo.