Preclinical Pharmacokinetics Of Recombinant Urate Oxidase And Immunogenicity And Bioactivity Of Recombinant Urate Oxidase Modified With PEG

AIM:To develop an immunoradiometric assay for the pharmacokinetic study of recombinant urate oxidase after intravenous administration in rhesus monkeys.To study the pharmacokinetics property following single dosing and multiple dosing of rUOX via the route of intravenous administration in rhesus monkeys.To study the immunogenicity and bioactivity of PEG-rUOX.METHODS:The plasma drug concentration was determined by immunoradiometric assay (IRMA). The pharmacokinetic parameters were calculated by noncompartment model.An indirect ELISA method was utilized to determine the level of rUOX and PEG-rUOX antibody.An UV to study the in vitro bioactivity of PEG-rUOX.RESULTS:The limit of quantification was found to be 0.5ng.ml^-1 and the detection range was from 0.5 to 400 ng·ml^-1. The intra- and inter-day precision (CV) of analysis were <15%, and accuracy were within±0.53%. The recovery of urate oxidase in different animal plasma were all >80%.After intravenous administration of urate oxidase at dose of 0.15, 0.6, and 2.4mg·kg^-1 and intravenous administration of Fasturtec at dose of 0.15 mg·kg^-1, the plasma level of urate oxidase decreased with time and was down to the base at 24h after intravenous administration. The mean AUC_0-t values were 5741±2297, 15528±5076, 61099±22635 ng·h·mL^-1, respectively. T_1/2 values for urate oxidase at dose of 0.15, 0.6, 2.4 mg·kg^-1 were 2.1±0.4h, 1.9±0.2h, 2.2±0.2h . The total body clearance (CL) were 30.0±14.7, 41.5±13.3, 42.5±13.1mL·kg^-1·h^-1, and the volume of distribution (Vd) were 41.9±16.2, 81.0±34.3, 66.7±12.6mL·kg^-1 at three doses respectively. The AUC increased with the increasing doses for intravenous administration, and had good linearity(r>0.99972, P<0.05). Whatever the dose, the CL, T_1/2 and V were roughly invariant. There was no significantly different between low dose of urate oxidase and low dose of Fasturtec on the trend of kinetics and on the plasma concentrations by T test. Moreover, there was no accumulation of urate oxidase in rhesus monkeys.The levels of PEG-rUOX antibody were significantly lower than that of rUOX in SD rats.The bioactivity of PEG-rUOX and rUOX were all lower than that of Rasburicase.CONCLUSION:The IRMA assay was sensitive, and specific for the pharmacokinetics study of urate oxidase and successfully to determine urate oxidase pharmacokinetic parameters after intravenous administration in rhesus monkeys. The kinetic characteristic of urate oxidase in rhesus monkeys was linearity kinetics.The PEG- rUOX was more excellent than rUOX in clinical application.We have to carry further study of the bioactivity of PEG- rUOX.DISCUSSION:To evaluate urate oxidase pharmacokinetic parameters, a precise, sensitive, and specific method allowing the measurement of plasma concentrations of the enzyme was required. Here we describe the selection of monoclonal antibodies (m- Abs) directed against the recombinant urate oxidase to develop a sensitive and specific immunometric assay in plasma. The general advantages of such a IRMA performed in microtiter plates compared to other classical methods are the easy handling of large amount of samples at the same time, the possibility of automation, the need of less material, simple and convenient to operate.For rhesus monkey, endogenous urate oxidase had no effect on pharmacokinetic experiment for absence of urate oxidase because of making some measures, such as diluting the plasma sample by blank plasma.As a demonstration of the potential utility of the radiommunometric assay, pharmacokinetic studies of urate oxidase in rhesus monkeys were performed after intravenous administration. Whatever the dose, the CL, T^1/2 and V were roughly invariant, demonstrating the linearity of the pharmacokinetics within the dose in rhesus monkeys. It can be seen that the small values of CL and V after urate oxidase was intravenously administered to thesus monkeys at the dose of 0.15, 0.6, and 2.4 mg·kg^-1. These results suggested urate oxidase had a low clearance and a low volume of distribution, similar to Dussossoy D et al reported that the small values of CL and V after Fasturtec was intravenously administered to baboons at the dose of 0.15,0.45,1.5mg·kg^-1. V was approximately equal to plasma volume by assessing. This suggests that the tissue distribution of urate oxidase is very restricted. It was important that the pharmacokinetic parameter of testing rUOX is similar to that of Fasturtec. These studies have shown the linear pharmacokinetics characteristic of urate oxidase and can provide reference for clinical administraton. The PEG- rUOX was more excellent than rUOX in clinical application by assessing the immunogenicity verus that of non-PEG-rUOX. Although the results showed the bioactivity of PEG- rUOX and rUOX, we could not deny the clinical application feasibility. We have to carry further study of the bioactivity of PEG- rUOX.