Protein binding of tirilazad (U-74006) in human, Sprague-Dawley rat, beagle dog and cynomolgus monkey serum.

Determination of the serum protein binding of tirilazad across species was required to predict the pharmacokinetic behavior of this new drug. Equilibrium dialysis and ultrafiltration techniques, commonly used to study serum protein binding, were shown to be unsuitable for tirilazad due to high nonspecific binding and low aqueous solubility, resulting in unbound drug levels that were nondetectable with current analytical methodology. Ultracentrifugation appeared to offer a technique with which unbound tirilazad could be measured; however, after extensive studies, the apparent lipid partitioning behavior of tirilazad into low density and very low density lipoproteins showed that ultracentrifugation was also unsuitable for determination of the true unbound fraction of tirilazad. Fractions of tirilazad measured in the supernatant were highly correlated with total triglycerides and very low density lipoproteins in the sera of all species analyzed. Studies with delipidized human serum yielded a nonsaturable binding isotherm with free fractions of less than 0.6 +/- 0.02% (mean +/- S.D.) over a concentration range of 4.6 to 55.6 micrograms/ml (normal human in vivo range, 0.01-20 micrograms/ml). These data indicated that, as the triglyceride content of the sera increases, portions of tirilazad bound to serum proteins shift into the lipid phase of lipoproteins. What effect this has on the true unbound fraction is unknown and does not seem to be ascertainable with current technology.