PEG-uricase in the management of treatment-resistant gout and hyperuricemia☆
Introduction
Gout is characterized by persistent hyperuricemia, which results in the deposition of monosodium urate monohydrate (MSU) crystals in the joints and periarticular structures and of uric acid calculi in the urinary tract (“kidney stones”) [1]. While gout has been recognized for several millennia [2], its increasing incidence during the past few decades [3], [4] and the growing recognition of the renal and cardiovascular consequences of persistent hyperuricemia [5], [6], [7], [8], [9] have enhanced the urgency of developing more effective treatments. For a significant number of patients with gout and other conditions in which hyperuricemia is poorly controlled with available drugs, polymer-coupled uricase and other drugs in development offer new hope of improved clinical outcomes.
Unlike nearly all other mammals, humans and higher apes lack the enzyme uricase (urate oxidase, E.C. 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, a more soluble product that is readily excreted in the urine [10]. The lack of uricase in humans results in plasma uric acid concentrations that are much higher than in most mammals. When these concentrations exceed the solubility limit of about 7 mg/dL at physiological pH, uric acid may nucleate to form crystals in tissues and joints [11]. Shedding of MSU crystals into the synovial cavity induces acute inflammatory responses known clinically as gout flares. The persistence of crystals in the synovial fluid and synovial membrane induces chronic inflammation. In addition to gout, another condition associated with hyperuricemia is tumor lysis syndrome, in which the induction of effective chemotherapy for hematologic cancers such as leukemia and lymphoma may result in dramatic elevations of plasma uric acid [12], [13], [14], [15]. Hyperuricemia also occurs with high frequency during the months and years following organ transplantation, when renal function and hence the excretion of uric acid are compromised by immunosuppressive agents, such as cyclosporine A, used to prevent rejection of the transplanted organ [16], [17], [18], [19] and by diuretic drugs administered after kidney transplantation.
A major contribution to the management of gout and other consequences of hyperuricemia was the introduction in the 1960s of allopurinol, a precursor of oxypurinol that is a potent inhibitor of xanthine oxidase, which catalyzes the last steps in the metabolism of purines to uric acid [20], [21]. Intractable gout can result from allergic reactions to allopurinol, some of which are life-threatening or fatal [22], [23], [24], [25] or from an inadequate response to the highest tolerated doses of the available drugs. Patients suffering from treatment-resistant gout often have painful gout flares and/or tophi (inflamed deposits of MSU crystals), as do patients whose serum uric acid levels are lowered to just below 6 mg/dL [26]. Resolution of tophi occurs when sub-saturating serum urate levels are maintained for long periods, but this rarely happens in response to the inhibition of uric acid synthesis by allopurinol [27]. More rapid resolution of tophi is observed when serum uric acid levels are lowered further by the administration of more effective urate-lowering therapy, e.g., uricosuric agents with or without allopurinol [28].
During the past seven decades, there have been numerous attempts to manage treatment-resistant gout by the administration of uricase extracted from various sources or expressed in recombinant organisms. In the early 1940s, Oppenheimer and colleagues injected uricase into hens, cocks and one patient [29], [30]. London and Hudson [31] were also among the pioneers who administered purified uricase to two human patients. Recognition of the immunogenicity of uricase in humans led to efforts to prepare poly(ethylene glycol) conjugates of uricase (PEG-uricase) [32], [33], [34], [35], [36], [37], [38] and to the first clinical study of PEG-uricase in five patients with advanced solid tumors [39]. The first U.S. patent claiming “water-soluble non-immunogenic polypeptide compositions,” which issued in 1979, described the synthesis of PEG-uricase as Example I [32]. Nevertheless, it has taken another quarter century to overcome the challenges presented by the development of a form of PEG-uricase that fulfills the requirements for a useful drug:
- 1.
sufficient reduction of the immunogenicity of this non-human enzyme to permit repeated dosing;
- 2.
retention of sufficient enzymatic activity to be effective at a reasonable dose, e.g., retention of at least 75% of the intrinsic activity in the conjugate;
- 3.
sufficient solubility under physiological conditions to enable reasonable bioavailability;
- 4.
reproducible and cost-effective synthesis of a conjugate with adequate stability during storage and shipping and in vivo; and
- 5.
a sufficiently long half-life in patients to permit a convenient schedule of administration, e.g., once or twice a month.
All of the above objectives appear to have been fulfilled by a PEGylated form of recombinant mammalian uricase that was developed jointly by scientists from Mountain View Pharmaceuticals, Inc. and Duke University [40], [41], [42], [43]. Savient Pharmaceuticals, Inc. (East Brunswick, NJ) holds a worldwide exclusive license to this form of PEG-uricase, for which the trademark Puricase® was registered by Mountain View Pharmaceuticals, Inc. Savient expects to complete its Phase 3 clinical trials of Puricase® in North America during 2007.
Section snippets
Preclinical development of PEGylated recombinant mammalian uricase
The collaborative development of PEGylated recombinant mammalian uricase by scientists at Duke University and Mountain View Pharmaceuticals, Inc. (MVP) began during 1995, with support during 1996–1998 from the U.S. National Institutes of Health. Drs. Michael S. Hershfield and Susan J. Kelly of Duke constructed various recombinant mammalian uricases, among which the selected amino acid sequences resembled that of porcine uricase most closely, but contained several residues from the baboon
Phase 1
In the first reported open-label Phase 1 trial of PEG-uricase (Puricase®) sponsored by Savient, 13 treatment-failure gout patients, including 11 with tophaceous gout, received single subcutaneous (s.c.) injections of PEG-uricase containing 4, 8, 12 or 24 mg of protein [62]. By day 7 after injection, the plasma uric acid concentration had decreased from a mean of about 11 mg/dL to a mean of about 3 mg/dL. Adverse reactions included injection-site reactions in three patients and gout flares in
Anti-PEG antibodies
In a family of patent applications first published in 2004, Martinez et al. [56] showed that rabbits immunized with recombinant mammalian uricase coupled to an average of about two strands of 10-kDa methoxyPEG (mPEG) per subunit produced antibodies against both the enzyme and the polymer component. It should be noted that the conjugate used for these immunizations was known to contain an insufficient number of strands of PEG per subunit to inhibit the binding of the conjugate to anti-uricase
Induction vs. maintenance therapy with PEG-uricase
As in many chemotherapy protocols, e.g. as reported by Hainsworth et al. [74], it is possible that a relatively high dose of PEG-uricase will be needed to induce rapid resolution of tophi or other signs and symptoms of severe gout, while either lower and/or less frequent doses of PEG-uricase will be sufficient for maintenance therapy [11]. Once the accumulation of uric acid deposits in the joints and tissues of patients who have been treated suboptimally for years has been depleted by an
Alternative drugs for treatment-resistant gout
While the focus of this review is the development and early clinical trials of PEGylated recombinant mammalian uricase, several classes of alternative urate-lowering drugs should be mentioned. Drugs that are currently marketed or in clinical trials for the treatment of chronic hyperuricemia include: 1) uricosuric drugs, such as probenecid, sulfinpyrazone and benzbromarone (marketed in Japan); 2) uricostatic drugs, which are xanthine oxidase inhibitors such as allopurinol, oxypurinol and
Conclusions
Converting an enzyme that is totally foreign to the human body into a clinically useful drug presents formidable obstacles. While the covalent attachment of poly(ethylene glycol) (PEGylation) may appear to be an obvious approach, most of the PEGylated proteins that are currently in clinical use have amino acid sequences that are identical or nearly identical to those of a natural human protein. Nevertheless, it has been possible to mask the immunogenicity of porcine-like uricase and to achieve
Acknowledgments
We are grateful to Dr. L. David Williams, John A. French and Alexa L. Martinez of Mountain View Pharmaceuticals, Inc. for their essential contributions to many aspects of the preclinical research described in this review; to Dr. Marc Whitlow of Berlex Biosciences for providing the program Add_PEG, and to Shyam S. Bhaskaran and Monika A. Sobczyk of Mountain View Pharmaceuticals, Inc., for their skill in adapting the Add_PEG program to produce the structure of PEG-uricase shown in Fig. 1D.
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This review is part of the Advanced Drug Delivery Reviews theme issue on “Peptide and Protein PEGylation III: Advances in Chemistry and Clinical Applications”.