FACTORS INFLUENCING THE PROTEIN-BINDING OF YH-439 USING AN EQUILIBRIUM DIALYSIS TECHNIQUE - A NEW HEPATOPROTECTIVE AGENT

Various factors influencing the plasma protein binding of YH-439 to 4% human serum albumin (HSA) were evaluated using the equilibrium dialys is method at the initial YH-439 concentration of 2 mu g mL(-1). It too k approximately 12 h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% of dextran ( 'the buffer') using a Spectra/Por 2 membrane (molecular weight cut-off , 12000-14000) in a water bath shaker kept at 37 degrees C and at a ra te of 50 oscillations min(-1). YH-439 was fairly stable both in 4% HSA and in the 'buffer' for up to 24 h incubation. The binding of YH-439 to 4% HSA was constant (974 +/- 0.55%) at YH-439 concentrations rangin g from 0.5 to 10 mu g mL(-1). However, the extent of binding was depen dent on HSA concentrations: the values were 90.7, 94.7, 96.7, 97.0, 97 .0, 97.1, and 97.5% at HSA concentrations of 0.5, 1, 2, 3, 4, 5, and 6 %, respectively. The plasma protein binding decreased with increasing incubation temperature: the binding values were 98.2, 97.6, 97.2, and 96.8% when incubated at 10, 21, 26, and 37 degrees C, respectively. Th e binding of YH-439 was also influenced by the chloride concentration in the buffer: the binding values were 94.5, 97.0, and 96.8% for the c hloride concentrations of 0, 0.249, and 0.546%, respectively. The bind ing of YH-439 was also dependent on the buffer pH: the percentages of free fraction were 6.0, 4.1, 3.8, 2.8, 2.7 and 2.8% for the buffer pHs of 5.0, 6.0, 6.5, 7.0, 7.4, and 8.0, respectively. The free fraction of YH-439 was slightly increased by the addition of heparin (up to 40 U mL(-1)), sodium azide (NaN3, up to 0.5%), and its metabolites. The p rotein binding of YH-439 was influenced neither by AAG, acetylsalicyli c acid, or sulphisoxazole, nor by the addition of citrate or EDTA. The free fractions of YH-439 in rabbit (4.2%) and dog (4.7%) plasma seeme d to be higher than in rats (2.9%) and humans (3.1%).