ABSTRACT
Permeability data from MDR1-MDCKII and PAMPA-BBB assays were compared to data from in situ brain perfusion to evaluate the accuracy of in vitro assays in predicting in vivo blood-brain barrier (BBB) permeability. PAMPA-BBB significantly correlated to in situ brain perfusion, however, MDR1-MDCKII had no correlation with in situ brain perfusion. PAMPA-BBB also significantly correlated with MDR1-MDCKII. The differential correlation of PAMPA-BBB and MDR1-MDCKII to in situ brain perfusion appears to be mainly due to the difference in membrane characteristics rather than binding to brain tissue. The MDR1-MDCKII cell membrane has lower ratios of: phospholipid to cholesterol, unsaturated to saturated acyl chains, and phosphatidyl-choline (PC) to sphingomyelin (SM) than brain endothelial cells, making it a poor passive permeability model for BBB. The BBB is more hydrophobic, rigid, and less fluidic than MDR1-MDCKII cell membrane. PAMPA-BBB more closely matches the BBB membrane in these characteristics and is a more accurate passive diffusion permeability model for BBB than MDR1-MDCKII. PAMPA-BBB is high throughput, low cost and has good prediction of in vivo BBB permeability, and therefore, it is a valuable tool in drug discovery to screen compounds for the rate of brain penetration.
Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Cell Culture Techniques , Membranes, Artificial , Perfusion , Biological Transport , Brain/cytology , Cell Line , Drug Discovery/economics , Drug Discovery/methods , Humans , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Sensitivity and SpecificityABSTRACT
High throughput metabolic stability assays are widely implemented in drug discovery to guide structural modification, predict in vivo performance, develop structure-metabolic stability relationships, and triage compounds for in vivo animal studies. However, these methods are often developed and validated using commercial drugs. Many drug discovery compounds differ from commercial drugs, with many having high lipophilicity, high molecular weight and low solubility. The impact of very low solubility on metabolic stability assay results was explored. Two metabolic stability assays, the 'aqueous dilution method' and the 'cosolvent method, were compared. For commercial drugs and most discovery compounds having reasonable drug-like properties, the two methods gave comparable results. For highly lipophilic, insoluble drug discovery compounds, the 'aqueous dilution method' gave artificially higher stability results. The cosolvent method performs compound dilutions in solutions with higher organic solvent content and adds solutions directly to microsomes to assist with solubilization, minimize precipitation and reduce non-specific binding to plastics. This method is more applicable in drug discovery where compounds of a wide range of solubility are studied.
Subject(s)
Microsomes, Liver/metabolism , Pharmaceutical Preparations/chemistry , Acetonitriles/chemistry , Animals , Dimethyl Sulfoxide/chemistry , Male , Mice , Mice, Inbred C57BL , Pharmaceutical Preparations/metabolism , Rats , Rats, Sprague-Dawley , SolubilityABSTRACT
Screening of solution stability provides an early alert on potential liabilities of drug candidates so that strategies can be developed to overcome the challenges. A fully automated solution stability assay has been developed to accelerate traditional manual operation. The assay uses the advanced capabilities of a high-performance liquid chromatography instrument that is present in many pharmaceutical research laboratories. The samples are prepared automatically by a temperature-controlled autosampler. The samples are delivered to the stability matrices, mixed, incubated, and injected at selected time points during the reaction time course. This automated process occurs without operator intervention, thus allowing 96 experiments to be run with 0.5 h of a scientist's time compared to 8 h for the same study when performed manually. Automation not only eliminates the manual operation but also improves accuracy and throughput. The assay protocol has been optimized to achieve homogenous mixing and eliminate carryover. The assay is robust, flexible, and high throughput. It can be used to study stability for a large number of samples under multiple incubation conditions and has a wide range of applications in drug discovery and development, such as screening compound stability in biological assay media, obtaining a stability-pH profile, surveying compound stability in physiological fluids, and performing development forced degradation and excipient compatibility.
