Introduction

Gout is a disease of purine metabolism as well as an autoinflammatory disease, affecting the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and the interleukin-1 (IL-1) pathway. It results from long-standing hyperuricemia [defined as serum urate (SU) levels > 6.8 mg/dL], which leads to monosodium urate (MSU) crystal deposition [1]. Oral urate-lowering therapy (ULT) is key to the treatment of gout. However, many patients receiving oral ULT do not achieve the desired target SU reduction, partly because some patients cannot tolerate or have contraindications to their use, mainly due to comorbidities, such as chronic kidney disease (CKD). This may lead to refractory or uncontrolled gout. These are severe gout patients. These are also patients with a high disease burden, including frequent gout flares (defined as two or more flares annually), widespread tophaceous deposits, and severe pain.

Exogenous uricases, considered enzyme replacement therapy, are a therapeutic option for patients with refractory or uncontrolled gout. Uricase treatment reduces hyperuricemia, leading to rapid resolution of tophi and improved quality of life [2–4].

This review discusses approved and investigational uricases and the clinical challenges associated with their use; loss of efficacy of anti-drug antibodies (ADAs), infusion reactions (IRs), anaphylaxis, and flares.

Uricase in evolution/uricase structure

Urate (also called monosodium urate) is the salt of a uric acid sodium salt. In most mammals, SU levels are highly regulated by the uricase (urate oxidase) enzyme [5]. In species other than humans and some non-human primates, uricase converts urate to allantoin, which is more water-soluble, thus leading to enhanced renal excretion. The uricase gene activity became silenced in a common ancestor of the ape/human lineage, which led to uric acid being the end-product of human purine metabolism [6–8]. Uricases are synthesized primarily in the liver in mammalian species but are also found in the gastrointestinal tract, vascular, and other tissues. Uricase is a tetramer of identical subunits. Each monomer comprises two domains. Integrating the two identical domains produces a folded monomer consisting of an antiparallel β-sheet of eight sequential strands, with helices on the sheet’s concave side [9, 10]. The kidney and the gut must eliminate uric acid to maintain urate homeostasis since human tissues have a very limited ability to metabolize uric acid, making humans susceptible to hyperuricemia and gout [11].

Conjugation of polyethylene glycols

Polyethylene glycol (PEG) consists of repeating ethylene glycol units (-CH₂-CH₂-O-) and is synthesized to be highly hydrophilic, possessing a large exclusion volume in aqueous solutions. Enhancing the pharmacological and pharmaceutical properties of proteins through PEG conjugation, known as PEGylation, significantly improves their clinical utility by extending their circulation time, thus increasing their half-life [12]. This process can involve attaching PEG to specific amino-acid residues (e.g., lysine side chains in pegloticase) or targeting unique predetermined sites on the protein. PEGylation also decreases immunogenicity and enhances the solubility and stability of proteins. Consequently, PEGylation enhances both in vitro and in vivo stability, leading to longer circulation times and fewer adverse effects [13]. Due to their foreign nature to the human immune system, exogenous uricases require PEGylation to reduce their immunogenicity, which can also extend their half-life.

However, while free PEG exhibits minimal to no immunogenicity, PEGylation of proteins can induce an immune response, primarily via a T cell-dependent pathway, leading to the production of anti-PEG antibodies (Abs). Anti-PEG Abs are prevalent even among people not exposed to PEGylated drugs, with Ab prevalence ranging from 23% to 72% [14]. This is likely due to daily exposure to PEG-containing products such as cosmetics, toothpaste, shampoos, sunscreens, and processed foods, accounting for the considerable variation in anti-PEG Ab titers [15]. Pegloticase, a PEG-conjugated protein, exhibits this phenomenon, as mentioned below [16, 17]. Factors contributing to Ab formation against PEGylated drugs include the route of drug administration, dosage, pre-existing Abs, the patient’s immune status, and genetic factors [18].

Anti-PEG Abs, especially after repeated administration, can diminish the therapeutic efficacy and safety of PEGylated drugs by decreasing subsequent doses’ circulation time [19]. These ADAs can neutralize the therapeutic effect of PEGylated drugs and accelerate their clearance from the blood, further reducing their clinical effectiveness [20].

These contrasting effects highlight the complexities and challenges associated with PEGylation, emphasizing the need for continued research to optimize the clinical benefits of PEG-conjugated treatments.

Uricases

It is important to note that all uricases are contraindicated in those with glucose‐6‐phosphate dehydrogenase (G6PD) deficiency since uricases can trigger life-threatening hemolysis and methemoglobinemia [21]. G6PD deficiency is an X‐linked disorder that affects over 400 million people worldwide of primarily Mediterranean, African, and Asian ancestry [22]. Males of African or Mediterranean ancestry are at higher risk of G6PD deficiency. This is true for all uricases. Hence, the regulatory agencies [The US Food and Drug Administration (FDA) and The European Medicine Agency (EMA)] require (label) screening for G6PD deficiency in all patients before the first uricase infusion.

Uricases approved and in development are discussed below (Table 1).

Approved uricases

Polyethylene glycol immunogenicity—anti drug antibodies when using pegloticase

The immunogenicity associated with pegloticase is directed against the PEG moiety [16]. In contrast, Abs to uricase was infrequent. The ADAs lead to reduced efficacy, increased risk for IRs, and decreased treatment duration of PEGylated uricase [4, 37].

During the pivotal trials of pegloticase, anti-pegloticase, and anti-PEG Abs were observed following prolonged exposure to pegloticase. In its Phase III randomized controlled trials (RCTs), ADAs were detected in 89% of patients at least once during the 6-month study period. Among the patients receiving pegloticase, 41% (69 out of 169) developed high titer anti-pegloticase Abs (> 1:2,430), and 40% (67 out of 169) developed anti-PEG Abs [38]. Anti-pegloticase Abs typically emerged within 2–3 months of treatment, resulting in a failure to achieve sustained SU lowering, reduced efficacy, and an increased risk of IRs [38]. Up to 50% of patients had anti-PEG Abs even without prior exposure to pegloticase [17].

Age may influence Ab formation. 61% of patients who were 70 or older were pegloticase responders; 50% of patients ≥ 60 years old were responders, and 30% of patients < 60 years old were responders (P = 0.015) [37].

For patients undergoing uricase therapy, it is crucial to discontinue oral urate-lowering therapies and monitor SU levels as a biomarker for developing anti-drug Abs.

Infusion reactions

IRs are defined as occurring during or within 2 h after the end of an infusion. In the pivotal Phase III trials, IRs were observed in 26% of patients receiving pegloticase every two weeks, compared to 5% in the placebo group [4], with 3% of IRs happening during the first infusion. In 88% of these cases, a loss of urate-lowering efficacy was noted prior to the patient’s first IR [37]. This led to the recommendation to discontinue pegloticase if SU levels rose above 6 mg/dL before the next infusion. IRs were the second most common AE following gout flares, with symptoms including chest discomfort, flushing, and dyspnea. Some IRs met the FDA’s criteria for anaphylaxis. Managing IR symptoms involved strategies such as slowing the infusion rate, pausing and resuming the infusion at a slower pace, and administering antihistamines, fluids, steroids, and analgesics.

The pivotal pegloticase trials did not include stopping rules. Reviewing the pivotal trial data, it became clear that SU is a biomarker for predicting IRs. Once the drug is started, pre-infusion SU can be used as a surrogate for the presence of anti-pegloticase Abs [39]. Hence, one must monitor the SU levels before each infusion and stop pegloticase treatment when the SU levels are > 6 mg/dL before two consecutive infusions.

Immunomodulation

The major limitation of pegloticase is immunogenicity [38, 39]. Although pegloticase effectively lowered SU, in the pivotal trials, only 42% of patients maintained an SU lower than 6.0 mg/dL at six months of therapy, likely due to the development of ADAs. Concomitant immunomodulation administered with pegloticase significantly improves the durability and efficacy of the response, as well as safety. Administering immunomodulation with disease-modifying antirheumatic drugs (DMARDs), including methotrexate, azathioprine, leflunomide, and mycophenolate mofetil (MMF), leads to reduced ADA production, decreased IRs and increased treatment response [39–44].

The MIRROR (Methotrexate to Increase Response Rates in Patients with Uncontrolled Gout Receiving Pegloticase) study was a randomized, double-blind, placebo-controlled trial focused on patients with uncontrolled gout. The primary measure of success was the percentage of patients maintaining SU levels under 6 mg/dL for at least 80% of the time during the sixth month. The study’s findings indicated a 71% response rate in the group treated with pegloticase and methotrexate, compared to 38.5% in the pegloticase and placebo group [48]. Moreover, patients who received pegloticase with methotrexate experienced a significant reduction in IRs, with only 4% (4 of 96) having IRs vs 31% (15 of 49) in the placebo group [48].

The RECIPE (Reducing Immunogenicity to Pegloticase) trial assigned patients with uncontrolled gout to either MMF (1 gram twice daily, n = 22) or placebo (n = 10) with a 2-week run-in period before starting biweekly pegloticase [41]. After 12 weeks, MMF was discontinued. The primary endpoint of having SU at or below 6 mg/dL at 12 weeks was achieved by 86% (19 of 22) of patients in the MMF group, compared to 40% (4 of 10) in the placebo group. At 24 weeks, 68% of those in the MMF group-maintained SU-lowering, whereas only 30% of those in the placebo group did. Notably, no IRs were reported in the MMF group, while 30% occurred in the placebo group [41].

In the TRIPLE (Tolerization Reduces Intolerance to Pegloticase and Prolongs the Urate Lowering Effect) study, participants were treated with azathioprine (starting at 1.25 mg/kg daily for one week, increasing to 2.5 mg/kg daily) and maintenance of daily 2.5 mg/kg with pegloticase infusion (n = 12). Interim analysis showed that six patients completed the treatment, while two continued this treatment after the study, all maintaining SU levels below 6 mg/dL. Of the four patients not included in this analysis, two lost urate-lowering efficacy, one experienced an IR, and one was intolerant to azathioprine [44].

Following the MIRROR trial results, the US FDA expanded the pegloticase label to endorse co-treatment with methotrexate for patients with uncontrolled gout. Combining pegloticase with immunomodulators like methotrexate is gaining popularity due to its increased efficacy and reduced ADA formation [42, 45]. Most patients respond positively to this co-treatment, showing improved outcomes while on immunomodulation therapy [17].

Uricases in late-stage development

Uricases in early-stage development

Minimizing flares with anti-inflammatory prophylaxis

The exact mechanism by which ULT can trigger gout flares has yet to be well understood. Flares may occur due to preexisting MSU crystals’ chemical or physical changes when ULT induces rapid SU lowering [50]. In patients who experience fast and dramatic SU lowering, such as that seen with uricase treatment, a high incidence of flares has been observed. Thus, it is unsurprising that flares were the most commonly reported AEs due to pegloticase infusions. Up to 80% of patients on pegloticase treatment had flares during the first three months of therapy, despite flare prophylaxis initiated one week before the first infusion and continued throughout the study [4, 50]. The pilot study for rasburicase showed a similarly high incidence of flares [25].

While gout flares observed during pegloticase therapy are documented as AEs, they actually signify evidence of the drug’s effectiveness. Individuals undergoing pegloticase treatment are more prone to experiencing gout flares because of the rapid reduction in SU levels [4, 51]. As a result, the frequent occurrence of gout flares during pegloticase treatment highlights the importance of implementing prophylactic measures to prevent flares in patients receiving pegloticase.

The choice of anti-inflammatory drugs for prophylaxis is limited to low-dose colchicine (0.5, 0.6 or 1.0 mg daily) and non-steroidal anti-inflammatory drugs (NSAIDs). Colchicine is the only FDA-approved drug for anti-inflammatory prophylaxis. However, it is often contraindicated, non-efficacious, or intolerable in patients with gout [52]. NSAIDs, too, are not recommended due to possible AEs, especially in patients with comorbidities. In the pivotal pegloticase trials during months 1–3, gout flares were higher for pegloticase-treated patients than in the placebo group [4]. However, with continued treatment during months 4–6, a significant reduction in flares was observed, as was the case in the SEL-212 studies.

The drug choice for anti-inflammatory prophylaxis depends on the patient’s comorbidities. In patients with CKD 3–5 and patients with heart disease, NSAID use is not advised. In CKD 3–5, the dosage of colchicine may be limited. Pegloticase infusions are preceded by methylprednisolone to prevent IRs and it has been suggested that methylprednisolone may have some prophylactic effect on flare prevention.

IL-1 is a key cytokine in gouty inflammation [53]. Although IL-1 inhibition is a logical target for preventing gout flares IL-1 inhibitors have not been approved by the EMA or FDA for flare prophylaxis. Instead, canakinumab, a neutralizing monoclonal Ab, is approved to treat flares [49]. While one should note that a single subcutaneous injection of canakinumab 150 mg used to treat severe flares also demonstrates a long-lasting effect over three months when compared to an intramuscular injection of 40 mg triamcinolone acetonide; this apparent flare prophylactic effect is off-label [54]. It has been shown that a single dose of canakinumab 150 mg subcutaneously resolves flares within 48 h and prevents new gout flares in patients initiating pegloticase + methotrexate [55].

Conclusions

Humans lack a functional uricase. Exogenous uricases, or “uricase enzyme replacement therapy”, mimic the uricases found in other species. Uricases are increasingly used to treat uncontrolled gout. However, they are immunogenic, leading to ADA formation. Formation of ADAs against the uricase enzyme and/or the PEG moiety leads to IRs and decreased efficacy. Innovative approaches are needed to develop therapeutic pegylated uricases with improved properties such as soluble expression, neutral pH solubility, and high expression in E. coli as well as low potential for immunogenicity [11]. Coadministration of methotrexate with pegloticase and rapamycin-containing nanoparticles with pegadricase suppresses ADA production leading to improved long-term uricase efficacy.

Current uricases on the market include pegloticase and rasburicase. Pegloticase is indicated for uncontrolled gout, whereas rasburicase is approved to prevent tumor lysis syndrome. The EMA has recommended uricase-based treatments in its gout treatment guidelines, but despite widespread interest in new developments in this therapeutic area, there is no option in Europe for such treatment; it is currently available only in the US.

Which is better pegloticase or SEL-212? This is unclear. Pegadricase did not exhibit a statistical superiority in reducing SU < 6 mg/dL for at least 80% of the time during months 3 and 6 when compared with pegloticase (53% pegadricase vs 46% pegloticase, P = 0.181) [28, 29]. The main limitations of pegloticase are gout flares and IRs following ADA formation. Using pegloticase and pegadricase in combination with immunomodulation decreases ADA formation and improves SU-lowering and treatment outcomes. Uricases in development will likely be studied with immunomodulation, leading to enhanced urate-lowering efficacy and increased use in refractory and uncontrolled gout patients.