PHARMACOKINETICS OF GENETICALLY MODIFIED T-PA IN RAT

To better understand the non-saturable mechanism responsible for the rapid systemic clearance of t-pa, previously demonstrated in all animal species tested so far, we have synthesized a genetically altered form of t-pa and analyzed the pharmacokinetic profile and organ distribution of this mutant. The mutant t-pa has been deleted in the fibronectin finger and epidermal growth factor domain, plus genetically modified to prevent N-linked glycosylation from occurring. Mammalian cells secreting either wild-type or mutant t-pa were radiolabeled with 35S-metnionine. The metabolically labeled t-pa was purified to homogeneity from conditioned medium and injected into the tail vein of rats. Multiple plasma samples were taken, post infusion, and analyzed for levels of acid-precipitable radioactivity. All rats were sacrificed fifteen minutes post infusion to determine the distribution of radioactivity in the liver, kidney or lung. The clearance profile of wild-type t-pa (n=8) was biphasi.e. with an initial alpha-phase half-life of 0.8 minutes (r=0.99) for approximately 80% of the protein and a beta-phase half-life of 12.2 minutes (r=0.97). Essentially all of the radioactivity recovered at fifteen minutes post-infusion was found in the liver. The mutant was analyzed (n=3) in identicle fashion and found to have only a single clearance phase with a 15.4 minute half-life (r=-97). Radioactivity determinations performed on organs showed proportionally less protein to be resident in the liver with a substantial increase in radioactivity observed in the kidneys. In conclusion, we have synthesized a genetic variant of t-pa which has a significantly increased half-life in the rat.