RESUMEN
Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Descubrimiento de Drogas , Preparaciones Farmacéuticas/sangre , Cromatografía Liquida , Diálisis/instrumentación , Diálisis/métodos , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Interacciones Farmacológicas , Estabilidad de Medicamentos , Humanos , Técnicas In Vitro , Límite de Detección , Preparaciones Farmacéuticas/química , Reproducibilidad de los Resultados , Solubilidad , Espectrometría de Masas en TándemRESUMEN
It was found that the presence of 5 mM Cu(2+) caused precipitation of protein present in hen's egg white to a large extent. About 85% of lysozyme activity remained in the supernatant and the enzyme was purified by approx. 13-fold. A further gel-filtration step on Sephadex G-75 resulted in an overall yield of 80% for the enzyme with 655-fold purification, and showed a single band on SDS/PAGE.
