Treating gout with pegloticase, a PEGylated urate oxidase, provides insight into the importance of uric acid as an antioxidant in vivo

Abstract

A high plasma urate concentration (PUA), related to loss of urate oxidase in evolution, is postulated to protect humans from oxidative injury. This hypothesis has broad clinical relevance, but support rests largely on in vitro data and epidemiologic associations. Pegloticase therapy generates H(2)O(2) while depleting urate, offering an in vivo test of the antioxidant hypothesis. We show that erythrocytes can efficiently eliminate H(2)O(2) derived from urate oxidation to prevent cell injury in vitro; during therapy, disulfide-linked peroxiredoxin 2 dimer did not accumulate in red blood cells, indicating that their peroxidase capacity was not exceeded. To assess oxidative stress, we monitored F2-Isoprostanes (F2-IsoPs) and protein carbonyls (PC), products of arachidonic acid and protein oxidation, in plasma of 26 refractory gout patients receiving up to five infusions of pegloticase at 3-wk intervals. At baseline, PUA was markedly elevated in all patients, and plasma F2-IsoP concentration was elevated in most. Pegloticase infusion rapidly lowered mean PUA to < or = 1 mg/dL in all patients, and PUA remained low in 16 of 21 patients who completed treatment. F2-IsoP levels did not correlate with PUA and did not increase during 15 wk of sustained urate depletion. There also was no significant change in the levels of plasma PC. Because refractory gout is associated with high oxidative stress in spite of high PUA, and profoundly depleting uric acid did not increase lipid or protein oxidation, we conclude that urate is not a major factor controlling oxidative stress in vivo.

Conflict of interest statement

Conflict of interest statement: M.S.H., J.S.S., and L.J.R. have acted as paid consultants to Savient Pharmaceuticals. Duke University, M.S.H. and S.J.K., and Mountain View Pharmaceuticals hold patent rights in pegloticase and its use, which have been licensed to Savient Pharmaceuticals. Duke University, M.S.H., and S.J.K. will receive royalties from sales if pegloticase receives Food and Drug Administration approval.

Figures

Fig. 1.

Catalase and erythrocytes protect CEM T-lymphoblastoid cells from H2O2 generated by urate plus pegloticase. CEM cell viability determined by trypan blue exclusion (A) and fold increase in CEM cell number (B) was followed during culture for 72 h in medium containing the following additions: Control, no additions; (A) uric acid (U) 0.3 mM, pegloticase (P) 40 mU/mL, catalase (C) 4,000 mU/mL; (B) uric acid (U), 0.5 mM; pegloticase (P) 40 mU/mL; RBC, 4 × 107/mL. (A) and (B) each show the mean results of two independent experiments.

Fig. 2.

Effects of H2O2 and urate oxidation on Prx2 in erythrocytes. (A) Reversible effect of H2O2 on Prx2 in a dilute suspension of erythrocytes. RBC (5 × 106/mL in PBS, 5 mM glucose) were incubated at 37 °C with 5 μM H2O2 for 5 min; then, at the indicated times, aliquots were removed for immunoblot analysis of Prx2 monomer (mon) and dimer (dim) distribution. (B) Effect of urate plus pegloticase on Prx2 in reconstituted blood. Washed RBC from a healthy individual were resuspended in two volumes of plasma containing 13.7 mg/dL urate (0.82 mM); after the addition of 30 mU/mL pegloticase, the suspension was incubated at 37 °C. At the indicated times, aliquots were removed for measuring urate concentration in plasma and for analysis of Prx2 in RBC. (C) Analysis of Prx2 in erythrocytes during treatment with pegloticase (8 mg every 3 wk). Blood samples drawn immediately before (“pre”) and 2 h after (“+2h”) infusions of pegloticase on the indicated days of treatment were processed to determine PUA. The remaining RBC were washed with 100 mM NEM, and then frozen at −80 °C for en batch immunoblot analysis of Prx2. As a positive control, 10 μL of RBC from the final sample (day 70) was removed before the NEM wash and incubated for 10 min in 100 μL of PBS, 5 mM glucose, 75 μM H2O2 to generate Prx2 dimer.

Fig. 3.

PUA (A) and F2-IsoP (B). In each panel, the data in the first four columns from the left were obtained from all 26 patients before (“pre”) and at the indicated times after the first infusion of pegloticase. Data in the last two columns on the right were obtained 7 d after the last infusion of pegloticase (“L + 7d”), and at a follow-up visit (“F-up”) 7 wk after the last infusion (i.e., the fifth dose for 21 patients, and the second or third dose for four patients who discontinued treatment earlier, as explained in Materials and Methods). The horizontal lines represent the mean, and vertical bars represent SEM. *P < 0.0001 vs. pretreatment. In A, red symbols in the last two columns indicate patients with “transient” responses to pegloticase; blue symbols indicate patients with “persistent” responses, as defined in Results.

Fig. 4.

Resolution of prominent digital tophi (s.c. MSU deposits) in a persistently hypouricemic patient. (A) Before treatment with pegloticase. (B) After five infusions of pegloticase, 8 mg every 3 wk.

Fig. 5.

Relative change in plasma F2-IsoP concentration from baseline during pegloticase therapy. For each patient, the F2-IsoP concentration at each sampling point was expressed as a percent of baseline (“pre”) value; the mean percent of baseline is plotted (red symbols). For comparison, the mean PUA also is plotted (blue symbols). Error bars = SEM. (Left) Data for all 26 patients for the first week of treatment, when all patients became hypouricemic. (Right) Data obtained 7 and 49 d after the fifth infusion of pegloticase for the 21 patients who completed the trial; these data are plotted separately for the 16 patients who were persistently hypouricemic (solid symbols, dashed lines) and for the five patients who were transiently hypouricemic but became hyperuricemic again before the fifth infusion (open symbols, no connecting lines).