BIOLOGICAL-ACTIVITY OF UREASE FORMULATED IN POLOXAMER 407 AFTER INTRAPERITONEAL INJECTION IN THE RAT

The advent of genetic engineering has resulted in a proliferation of protein pharmaceuticals available for a variety of therapeutic needs. However, the formulation and delivery of these proteins remain an intriguing challenge. Polymer-based protein drug delivery systems continue to be investigated, although many of the fabrication techniques used to incorporate proteins into the polymer matrix or device result in irreversible inactivation (denaturation) of the proteins. A well-characterized model enzyme, urease, was formulated in 33% (w/w) poloxamer 407 (Pluronic F-127) vehicle and injected intraperitoneally (ip) into rats in an attempt to achieve both preservation of biological activity and sustained release of the protein. The resulting ammonia concentration in plasma-time profiles were compared with those for rats injected with an identical dose (27.6 units of activity per 200 g of body weight) of urease dissolved in pH 7 phosphate buffer. Neither a pH 7 phosphate buffer solution nor poloxamer 407 (33%, w/w) dissolved in pH 7 phosphate buffer, when injected ip into rats, resulted in elevated ammonia levels in plasma. The time to reach a maximum ammonia level in plasma was increased approximately threefold following the injection of the urease-poloxamer 407 formulation, compared with that in control rats administered an identical dose of urease in solution. In addition, hyperammonemia was extended almost threefold in treated rats compared with control rats, without untoward effects. However, prolonged hyperammonemia in animals receiving an ip injection of the urease-poloxamer 407 formulation may have potentially resulted from the reduced clearance of ammonia and ammonium ion in the proximal tubules of the rats. Thus, it was not possible to definitively assign the threefold increase in the time to the maximum ammonia level in plasma to either the sustained release of urease from the semisolid urease-poloxamer 407 matrix in the peritoneal cavity or a poloxamer 407-induced decrease in the elimination of ammonia by the rat kidney. The half-life of elimination for poloxamer 407 in the urine of rats following an ip injection of poloxamer 407 vehicle alone (1.7 g/kg) was estimated to be 20.9 +/- 0.9 h. On the basis of a 1-g dose of a potential protein-poloxamer 407 formulation containing 33% (w/w) poloxamer 407 being injected intramuscularly or subcutaneously into a 70-kg patient (dose of poloxamer 407, 4.7 x 10(-3) g/kg), it is anticipated that poloxamer 407 would not interfere with the renal elimination of a protein cleared predominantly by the kidney. Thus, our preliminary results suggest that poloxamer 407 may potentially be a useful vehicle for preserving the biological activity of select recombinant-derived protein pharmaceuticals that are administered extravascularly. In addition, the potential exists for such a protein-poloxamer 407 formulation to sustain the rate of input of the therapeutic protein into the systemic circulation following subcutaneous or intramuscular injection.