Preparation of polyethylene glycol-tissue plasminogen activator adducts that retain functional activity: characteristics and behavior in three animal species

Abstract

Conditions were defined for the derivatization of recombinant tissue plasminogen activator (rt-PA) with polyethylene glycol (PEG) so as to retain functional activity as a possible means of producing a t-PA species with a prolonged circulating lifetime. Derivatives with a wide range of retention of activities were prepared by varying the concentration and species of activated PEG. The specific activities of the PEG-rt-PA derivatives were dependent on the method of assay. Assays using preformed fibrin gave higher estimates of retention of activity than assays using soluble components. Plasma elimination studies in mice and rats indicated prolonged circulating lifetimes for the radiolabeled PEG-rt-PA derivatives after a rapid clearance and distribution phase; however, the disappearance of functional activity was much more rapid than the disappearance of radiolabeled material. The PEG-rt-PA derivatives appeared to accumulate in tissues above their interstitial fluid concentrations and were rapidly inactivated, apparently by reaction with the plasma protease inhibitors. These results were consistent with the inactivation of the PEG-rt-PA derivatives in rat plasma in vitro. A somewhat longer half-life (t1/2) of the one derivative studied was observed in dogs (t1/2, 16 minutes) as compared with the rat (t1/2, five minutes). This was sufficient to confer thrombolytic activity upon the derivative (administered by bolus injection) in contrast to native rt-PA. The potential of PEG-modified rt-PA as a long-lived thrombolytic agent in humans will depend, however, on whether there will be a further extension of the t1/2 because of a reduction in clearance and/or a reduction in the rate of inactivation.

Serum albumin–protamine conjugate for biocompatible platform for targeted delivery of therapeutic macromolecules

“…However, covalent attachment of PEG on the protein surface, ironically, often results in diminished drug activity because of steric hindrance created by PEG molecules that prevent the target molecule to interact and produce pharmacological action, as was the case with tPA. 22 Our proposed HSA-protamine conjugate is designed to address the above mentioned limitations in which the bulky albumin moiety will serve as a "camouflage" for the drug in the systemic circulation, whereas the positively charged protamine will facilitate formation of an electrostatic complex with the drug. Depending on the therapeutic application, a triggering agent can be chosen to disrupt the construct and liberate free drug at the site of action.…”

“…The initial rate of hydrolysis of S-2251 by plasmin generated from plasminogen by tPA was determined by measuring the absorbance at 405 nm at different time intervals using a micro-plate reader (SynergyMx, Biotek). The slope of absorbance against square of time (DA min 22 ) was expressed as the initial rate of S-2251 hydrolysis. To assess heparin-mediated triggered release, the assay was also performed in the presence of 0.4 m/mL of heparin.…”

Engineered microparticles and nanoparticles for fibrinolysis

“…40 Berger and Pizzo showed that PEGylated tPA circulates 10 times longer in mice, rats, and beagles than tPA, though the active half-life is only increased threefold because of inhibition by PAI-1 and PAI-2. 41 In all of these reports, the fibrinolytic rate of the PEGylated PA is slightly accelerated compared to the non-PEGylated control, likely due to reduced inhibition.…”

Vascular Nanomedicine: Current Status, Opportunities, and Challenges

“…glycol)-based modification (PEGylation) of fibrinolytic agents such as tissue plasminogen activator (tPA), streptokinase (SK), urokinase (UK), and staphylokinase (SAK) has been reported. [62][63][64][65][66] In some studies, it was found that the "protective" property of PEG-SK conjugates could be improved by increasing the molecular weight of PEG. 63 PEGylated systems of truncated SK variants with promising in vitro fibrinolytic activity and enhanced in vivo circulation residence time have also been reported recently, although the in vivo fibrinolytic capacity of these systems were not reported.…”

“…64 Recombinant tPA has also been conjugated to PEG and the PEG-tPA product was reported to show partial enhancement of circulation lifetime and bioactivity compared with free tPA in canine models. 62 The PEGylation strategy has also been reported for anticoagulant molecules. For example, PEG has been conjugated to the thrombin inhibitor hirudin and these PEG-hirudin systems have been evaluated in vitro, in vivo, and even in clinical studies in human patients.…”