Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase

Abstract

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 +/- 2.1 mg/dl (mean +/- SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 +/- 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.

Figure 1

Plasma uricase activity and plasma uric acid concentration after subcutaneous injection of PEG-uricase. A single 8 mg injection of PEG-modified recombinant mammalian urate oxidase (PEG-uricase) was administered. The horizontal axis indicates days after dosing.

Figure 2

Relationship of dose of PEG-uricase and serum or plasma uric acid concentration. (a) Data for each dose cohort. (b) Data for two groups of subjects with distinct pharmacokinetic patterns: 'long-circulating' (plasma uricase activity (pUox) present three weeks after injection (n = 8)) and 'early elimination' (pUox undetectable beyond 10 days after injection (n = 5)). The horizontal axis indicates days after injection of PEG-modified recombinant mammalian urate oxidase (PEG-uricase); 'pre*', serum obtained at screening, just before the two-week allopurinol washout period; 'd 0', plasma obtained immediately before PEG-uricase injection.

Figure 3

Two pharmacokinetic patterns after single subcutaneous injections of PEG-uricase. (a) 'Long-circulating' group: eight subjects with uricase activity present in plasma at 21 days after injection. (b) 'Early elimination' group: five subjects with undetectable plasma uricase activity beyond 10 days after injection. The keys indicate the dose of PEG-modified recombinant mammalian urate oxidase (PEG-uricase).

Figure 4

Time course of appearance of IgM and IgG antibodies against PEG-uricase, and of plasma uricase activity, in 'early elimination' subjects 002, 011, and 013. Subject 002 received 4 mg, and subjects 011 and 013 each received 12 mg, of PEG-modified recombinant mammalian urate oxidase (PEG-uricase).

Figure 5

Titer of ELISA for IgG antibody against PEG-uricase. The data shown are for the five subjects who showed early elimination of plasma uricase activity. PEG-uricase, PEG-modified recombinant mammalian urate oxidase.

Figure 6

Competition ELISA to determine the specificity of IgG antibody against PEG-uricase. Increasing amounts of the indicated materials were added to aliquots of plasma obtained from subject 013 on day 14 after subcutaneous injection of 12 mg of PEG-modified recombinant mammalian urate oxidase (PEG-uricase). After incubation overnight, these mixtures were tested at a 1:60 dilution in the ELISA for IgG antibody against PEG-uricase. (a) 'Uricase' is the unmodified recombinant protein used in PEG-uricase, and 'PEG 10000' is 10 K monomethoxyPEG (mPEG) conjugated with glycine instead of uricase protein. 'PEG-PNP' is bacterial purine nucleoside phosphorylase conjugated with mPEG of molecular mass 5 kDa, prepared as described [18]. (b) 'PEG 350' and 'PEG 2000' are unconjugated, nonactivated mPEGs of molecular masses 350 and 2,000 Da. 'PEG 5000' and 'PEG 10000' are p-nitrophenyl carbonate-activated mPEGs of the indicated molecular masses conjugated with glycine.

Figure 7

Comparison of ELISAs performed with mPEGs and PEG-uricase. ELISA plates were coated with 2.5 μg of PEG-modified recombinant mammalian urate oxidase (PEG-uricase) or with 25 μg of p-nitrophenyl carbonate-activated monomethoxyPEGs (mPEGs) of molecular mass 5 kDa (mPEG-5K) or 10 kDa (mPEG-10K). After washing, the plates were blocked with 1% BSA, 1% glycine in PBS. The indicated plasma samples obtained on day 0 or day 21 after subcutaneous injection of PEG-uricase were then assayed with each substrate at a dilution of 1:50 as described in the Materials and methods section for the PEG-uricase ELISA.