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Improving the activity of uricase and lowering its immunogenicity remain significant challenges in the enzyme replacement management of hyperuricemia and related inflammatory diseases. Herein, an immunogenicity-masking strategy based on engineered red blood cells (RBCs) was developed for effective uricase delivery against both hyperuricemia and gout. The dynamic membrane of RBCs enabled high resistance to protease inactivation and hydrogen peroxide accumulation. Benefiting from these advantages, a single infusion of RBC-loaded uricase (Uri@RBC) performed prolonged blood circulation and sustained hyperuricemia management. Importantly, RBCs masked the immunogenicity of uricase, leading to the maintenance of UA-lowering performance after repeated infusion through reduced antibody-mediated macrophage clearance. In an acute gout model, Uri@RBC profoundly alleviated joint edema and inflammation with minimal systemic toxicity. This study supports the employment of immunogenicity-masking tools for efficient and safe enzyme delivery, and this strategy may be leveraged to improve the usefulness of enzyme replacement therapies for managing a wide range of inflammatory diseases., Competing Interests: Declaration of competing interest These authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Serum urate (SU) lowering with PEGylated uricases in gout can reduce flares and tophi. However, treatment-emergent anti-drug antibodies adversely affect safety and efficacy and the currently approved PEGylated uricase pegloticase requires twice-monthly infusions. Investigational SEL-212 therapy aims to promote uricase-specific tolerance via monthly sequential infusions of a proprietary rapamycin-containing nanoparticle (ImmTOR) and pegadricase., Methods: COMPARE was a randomized, phase 2, open-label trial of SEL-212 vs pegloticase in adults with refractory gout. SEL-212 [ImmTOR (0.15 mg/kg) and pegadricase (0.2 mg/kg)] was infused monthly or pegloticase (8 mg) twice monthly for 6 months. The primary endpoint was the proportion of participants with SU <6 mg/dl for ≥80% of the time during 3 and 6 months. Secondary outcomes were mean SU, gout flares, number of tender and/or swollen joints and safety., Results: During months 3 and 6 combined, numerically more participants achieved and maintained a SU <6 mg/dl for ≥80% of the time with SEL-212 vs pegloticase (53.0% vs 46.0%, P = 0.181). The percentage reductions in SU levels were statistically greater during months 3 and 6 with SEL-212 vs pegloticase (-73.79% and -47.96%, P = 0.0161). Reductions in gout flare incidence and number of tender and/or swollen joints were comparable between treatments. There were numerical differences between the most common treatment-related adverse events of interest with SEL-212 and pegloticase: gout flares (60.2% vs 50.6%), infections (25.3% vs 18.4%) and infusion-related reactions (15.7% vs 11.5%), respectively. Stomatitis (and related terms) was experienced by eight participants (9.6%) with SEL-212 and none with pegloticase. Stomatitis, a known event for rapamycin, was associated with ImmTOR only., Conclusions: SEL-212 efficacy and tolerability were comparable to pegloticase in refractory gout. This was associated with a substantial reduction in treatment burden with SEL-212 due to decreased infusion frequency vs pegloticase., Clinical Trial Registration: NCT03905512., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

The present study aims to immobilize the uricase enzyme on magnetic nanowires and to examine its potential for use in the treatment of gout. For this, Au/Ni/Au nanowires were synthesized using a polycarbonate membrane template by the sequential electrodeposition of Au, Ni, and Au, respectively. The uricase enzyme was covalently attached to these nanowires and was also coated with PEG. Optimum enzymatic conditions, kinetic parameters, thermal, storage, and operational stability were determined by performing enzymatic activity tests of free and immobilized uricase. Additionally, the efficacy of both enzyme preparations in artificial human serum and the presence of protease was also investigated. Experimental results showed that immobilized uricase showed higher stability than free uricase in all studied conditions. The potential of immobilized uricase to oxidize uric acid in artificial serum was also investigated and it was found that immobilized preparation demonstrated approximately 6 times higher activity than that of the free enzyme. The results of this study showed that uricase-attached nanowires oxidized uric acid effectively and are promising in the treatment of gout., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Approximately 15% of US adults have circulating levels of uric acid above its solubility limit, which is causally linked to the disease gout. In most mammals, uric acid elimination is facilitated by the enzyme uricase. However, human uricase is a pseudogene, having been inactivated early in hominid evolution. Though it has long been known that uric acid is eliminated in the gut, the role of the gut microbiota in hyperuricemia has not been studied. Here, we identify a widely distributed bacterial gene cluster that encodes a pathway for uric acid degradation. Stable isotope tracing demonstrates that gut bacteria metabolize uric acid to xanthine or short chain fatty acids. Ablation of the microbiota in uricase-deficient mice causes severe hyperuricemia, and anaerobe-targeted antibiotics increase the risk of gout in humans. These data reveal a role for the gut microbiota in uric acid excretion and highlight the potential for microbiome-targeted therapeutics in hyperuricemia., Competing Interests: Declaration of interests D.D. is a co-founder of Federation Bio. M.H.M. is a member of the Scientific Advisory Board of Hexagon Bio and co-founder of Design Pharmaceuticals., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Gout is a form of inflammatory arthritis that results from elevated serum uric acid levels and the deposition of urate crystals in multiple joints. The inflammatory response during an acute gout attack is mediated by the activation of the NLRP3 inflammasome, leading to the release of IL-1β and inducing a localized tissue inflammatory response. Urate lowering therapies such as Pegloticase effectively reduce serum uric acid levels but are generally associated with an increase in acute gout flares. In this study, we developed a long-acting anti-inflammatory recombinant uricase by sequential fusing interleukin-1 receptor antagonist (IL-1Ra) and albumin-binding domain (ABD) with the N-terminal end of Arthrobacter globiformis uricase (AgUox). The recombinant uricase has longer in vivo half-life, and significantly alleviates monosodium urate (MSU) crystals induced inflammation in mouse model compared with the wild-type AgUox. This long-acting anti-inflammatory recombinant uricase has the potential to be developed as an effective urate lowering therapy with better safety profiles., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures ('tophi') composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient 'hyperuricemic' larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis.-/- larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis.

Protein therapeutics, except for antibodies, have a short plasma half-life and poor stability in circulation. Covalent coupling of polyethylene glycol (PEG) to protein drugs addresses this limitation. However, unlike previously thought, PEG is immunogenic. In addition to induced PEG antibodies, ≈70% of the US population has pre-existing anti-PEG antibodies. Both induced and preexisting anti-PEG antibodies result in accelerated drug clearance, reduced clinical efficacy, and severe hypersensitivity reactions that have limited the clinical utility of uricase, an enzyme drug for treatment for refractory gout that is decorated with a PEG corona. Here, the authors synthesize a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) conjugate of uricase that decorates the protein with multiple polymer chains to create a corona to solve these problems. The resulting uricase-POEGMA is well-defined, has high bioactivity, and outperforms its PEG counterparts in its pharmacokinetics (PK). Furthermore, the conjugate does not induce anti-POEGMA antibodies and is not recognized by anti-PEG antibodies. These findings suggest that POEGMA conjugation may provide a solution to the immunogenicity and antigenicity limitations of PEG while improving upon its PK benefits. These results transcend uricase and can be applied to other PEGylated therapeutics and the broader class of biologics with suboptimal PK., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Microbial urate oxidase has emerged as a potential source of therapeutic properties for hyperuricemia in arthritic gout and renal disease. The thermostability and long-term thermal tolerance of the enzyme need to be established to prolong its therapeutic effects. Here, we present the biochemical and structural aspects of a hyperthermostable urate oxidase (TbUox) from the thermophilic microorganism Thermobispora bispora. Enzymatic characterization of TbUox revealed that it was active over a wide range of temperatures, from 30 to 70 °C, with optimal activity at 65 °C and pH 8.0, which suggests its applicability under physiological conditions. Moreover, TbUox exhibits high thermostability from 10 to 65 °C, with Tm of 70.3 °C and near-neutral pH stability from pH 7.0 to 8.0 and high thermal tolerance. The crystal structures of TbUox revealed a distinct feature of the C-terminal loop extensions that may help with protein stability via inter-subunit interactions. In addition, the high thermal tolerance of TbUox may be contributed by the extensive inter-subunit contacts via salt bridges, hydrogen bonds, and hydrophobic interactions. The findings in this study provide a molecular basis for the thermophilic TbUox urate oxidase for application in hyperuricemia and gout therapy., (Copyright © 2021. Published by Elsevier B.V.)

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg 2+ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.

Objective: To examine the efficacy and safety of pegloticase in combination with methotrexate (MTX) in patients with uncontrolled gout in an exploratory, open-label clinical trial (ClinicalTrials.gov: NCT03635957) prior to a randomized, controlled trial., Methods: A multicenter, open-label efficacy and safety study of pegloticase with MTX co-treatment was conducted in patients with uncontrolled gout. Patients were administered oral MTX (15 mg/week) and folic acid (1 mg/day) 4 weeks prior to and throughout pegloticase treatment. The primary study outcome was the proportion of responders, defined as serum uric acid (sUA) < 6 mg/dL for ≥ 80% of the time during Month 6 (Weeks 20, 22, and 24). All analyses were performed on a modified intent-to-treat population, defined as patients who received ≥ 1 pegloticase infusion., Results: Seventeen patients were screened and 14 patients (all men, average age 49.3 ± 8.7 years) were enrolled. On Day 1, mean sUA was 9.2 ± 2.5 mg/dL, and 12 of the 14 patients had visible tophi. At the 6-month timepoint, 11/14 (78.6%, 95% CI 49.2-95.3%) met the responder definition, with 3 patients discontinuing after meeting protocol-defined treatment discontinuation rules (preinfusion sUA values > 6 mg/dL at 2 consecutive scheduled visits). All patients tolerated MTX. No new safety concerns were identified., Conclusion: In this study, an increased proportion of patients maintained therapeutic response at 6 months when treated concomitantly with MTX and pegloticase as compared to the previously reported 42% using pegloticase alone. These results support the need for a randomized study of MTX or placebo with pegloticase to validate these open-label findings.

Arthrobacter globiformis Uricase (AgUricase) is a homotetrameric uricase with the potential for therapeutic use in treating hyperuricemia-related diseases. To achieve sufficient therapeutic effects, it is essential for this enzyme to have high thermostability and long half-life in physiological condition. To improve the thermostability of this enzyme, we introduced a series of cysteine pair mutations into the AgUricase subunits based on its structural model and studied the thermostability of the mutant enzymes with introduced disulfide bridges. Two intersubunit cysteine pair mutations, K12C-E286C and S296C-S296C, were found to markedly increase the melting temperatures of the corresponding mutant enzymes compared with WT AgUricase. The crystal structure of the K12C-E286C mutant at 1.99 Å resolution confirmed the formation of a distinct disulfide bond between the two subunits in the dimer. Structural analysis and biochemical data revealed that the C-terminal loop of AgUricase was flexible, and its interaction with neighboring subunits was required for the stability of the enzyme. We introduced an additional intersubunit K244C-C302 disulfide bond based on the crystal structure of the K12C-E286C mutant and confirmed that this additional disulfide bond further stabilized the flexible C-terminal loop and improved the thermostability of the enzyme. Disulfide cross-linking also protected AgUricase from protease digestion. Our studies suggest that the introduction of disulfide bonds into proteins is a potential strategy for enhancing the thermostability of multimeric proteins for medical applications.

Tophi deposit in the peripheral bone joints or soft tissues are formed by uric acid and urate crystal. It does not only affect local appearance but also destroy the bone and joint structure, resulting in loss of function. Traditional medical treatment is an effective way to control the hyperuricemia, but it is ineffective to tophus. Surgery is a relatively effective method for the treatment of tophus. It can successfully reduces the content of uric acid in the body and improves the limbs function, but it causes surgical trauma and complications. As new medicine, Urcase is a hot spot in the present study. It can not only control the blood uric acid level but also dissolve part of tophus. But the cost is higher than traditional medicines. There is still dispute in treatment for advanced tophus., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© 2017 by the China Journal of Orthopaedics and Traumatology Press.)

Hyperuricemia and gout, the clinical manifestation of monosodium urate crystal deposition, are common in patients with chronic kidney disease (CKD). Although the presence of CKD poses additional challenges in gout management, effective urate lowering is possible for most patients with CKD. Initial doses of urate-lowering therapy are lower than in the non-CKD population, whereas incremental dose escalation is guided by regular monitoring of serum urate levels to reach the target level of <6mg/dL (or <5mg/dL for patients with tophi). Management of gout flares with presently available agents can be more challenging due to potential nephrotoxicity and/or contraindications in the setting of other common comorbid conditions. At present, asymptomatic hyperuricemia is not an indication for urate-lowering therapy, though emerging data may support a potential renoprotective effect., (Copyright © 2017 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Uricase after modification with monomethoxy poly(ethylene glycol) (mPEG) is currently the sole agent to treat refractory gout. For formulating Bacillus fastidious uricase, succinimidyl carbonate of mPEG-5000 (SC-mPEG5k) and succinimidyl succinate of mPEG-5000 (SS-mPEG5k) were compared. SC-mPEG5k possessed higher purity, comparable reaction rate constant with glycine but lower hydrolysis rate, and stronger effectiveness to modify amino groups. The uricase possessed two types of amino groups bearing a 25-fold difference in reactivity with SC-mPEG5k or SS-mPEG5k at pH 9.2. Oxonate and xanthine concentration-dependently protected the bacterial uricase from inactivation during PEGylation. With SC-mPEG5k at a molar ratio of 200 to uricase subunits and oxonate of 50 µM, the PEGylated uricase (1) retained about 73% of the original activity, (2) displayed about 10% reactivity to rabbit anti-sera recognizing the native uricase, (3) elicited IgG in rats accounting for about 5% of that by the native uricase, (4) exhibited circulation half-life time of about 25 H in cock plasma in vivo, and (5) concurrently maintained uric acid at lowered levels for over 20 H. Hence, PEGylation with SC-mPEG under the protection of a competitive inhibitor was a practical approach to formulation of the bacterial uricase; protection of enzymes by competitive inhibitors during PEGylation may have universal significance., (© 2014 International Union of Biochemistry and Molecular Biology, Inc.)

The uricase enzyme yields allantoin, hydrogen peroxide, and carbon dioxide by catalyzing the oxidative opening of the purine ring in the urate pathway. This enzyme is important for biochemical diagnosis and reduces toxic urate accumulation during various diseases (hyperuricemia, gout, and bedwetting). Direct urate oxidase injection is recommended in renal complications-associated gout and to prevent chemotherapy-linked hyperuricemia disorders. Thus, uricase is a promising enzyme with diverse applications in medicine. Microbial production of uricase is featured by high growth rates, cost-effective bioprocessing, and easy optimization of the medium. Microbes produce the enzyme extracellular or intracellular. Extracellular uricase is preferred for biotechnological applications as it minimizes time, effort, and purification processes. This review provides insights into uricase-producing microbes, bacterial uric acid degradation pathways, degrading enzymes, and uricase-encoding genes.

The uricase enzyme yields allantoin, hydrogen peroxide, and carbon dioxide by catalyzing the oxidative opening of the purine ring in the urate pathway. This enzyme is important for biochemical diagnosis and reduces toxic urate accumulation during various diseases (hyperuricemia, gout, and bedwetting). Direct urate oxidase injection is recommended in renal complications-associated gout and to prevent chemotherapy-linked hyperuricemia disorders. Thus, uricase is a promising enzyme with diverse applications in medicine. Microbial production of uricase is featured by high growth rates, cost-effective bioprocessing, and easy optimization of the medium. Microbes produce the enzyme extracellular or intracellular. Extracellular uricase is preferred for biotechnological applications as it minimizes time, effort, and purification processes. This review provides insights into uricase-producing microbes, bacterial uric acid degradation pathways, degrading enzymes, and uricase-encoding genes. Furthermore, aspects influencing the microorganisms' production of the uricase enzyme, its activity, and its purification procedure are also emphasized. Cell disruption is mandatory for intercellular uricase production, which elevates production costs. Therefore, extracellular uricase-producing microbial strains should be investigated, and production factors should be optimized. Future techniques for obtaining extracellular enzymes should feature reduced time and effort, as well as a simple purification methodology. Furthermore, uricase gene-carrying recombinant probiotic microorganisms could become an effective tool for gout treatment. [ABSTRACT FROM AUTHOR]

The past year generated significant change and advancement of the urate field with novel insights regarding the role of uric acid (UA) in multiple pathophysiologic processes from gout to COVID-19. While these contributions continue to move the field forward, the basic biochemistry and biology of UA is often overlooked, being lost in the shadow of clinical associations and omics. However, the seminal impact of UA begins with biochemistry and the associated interplay with cell biology. In these basic reactions and resultant impacts on physiology, UA mediates its influence on clinical outcomes. As such, this review focuses on published advances in UA biochemistry and biology in 2022 and associates these advances with downstream consequences. [ABSTRACT FROM AUTHOR]

Despite growing prevalence and incidence, the management of gout remains suboptimal. The intermittent nature of the gout makes the long-term urate-lowering therapy (ULT) particularly important for gout management. However, patients are reluctant to take medication day after day to manage incurable occasional gout flares, and suffer from possible long-term toxicity. Therefore, a safe and easy-to-operate drug delivery system with simple preparation for the long-term management of gout is very necessary. Here, a chitosan-containing sustained-release microneedle system co-loaded with colchicine and uricase liposomes were fabricated to achieve this goal. This microneedle system was confirmed to successfully deliver the drug to the skin and maintain a one-week drug retention. Furthermore, its powerful therapeutic potency to manage gout was investigated in both acute gouty and chronic gouty models. Besides, the drug co-delivery system could help avoid long-term daily oral colchicine, a drug with a narrow therapeutic index. This system also avoids mass injection of uricase by improving its stability, enhancing the clinical application value of uricase. In general, this two-drug system reduces the dosage of uricase and colchicine and improves the patient's compliance, which has a strong clinical translation. A chitosan-containing sustained-release microneedle system co-loaded with colchicine (Col) and uricase (UAO) liposomes was designed and fabricated to provide a simple and effective way for gout management. [Display omitted] [ABSTRACT FROM AUTHOR]

Herein, we describe a uricase immobilized Au/Ni/PANI microrocket for the motion-based potential hyperuricemia therapy platform. This study has aimed to investigate the use of uricase immobilized magnetically guided microrockets to lower the uric acid level. The outermost Au layer of the microrocket facilitates the immobilization of the uricase enzyme, while the Ni layer allows controlling the motion of the microrocket via an external magnetic field. The innermost layer of PANI was integrated to prevent Ni from leaking. Optimum parameters for enzymatic reactions were determined, and optimum pH for free and immobilized uricase was found to be as 8.5, while optimum temperature was 35 °C for both uricase preparations. Also, the kinetic parameters of both free and immobilized uricase were evaluated. Km value for free uricase was found to be as 0.1212 mM, while immobilized uricase demonstrated lower Km value of 0.017 mM. Vmax of free and immobilized uricase was found to be as 0.8613 µmol/min and 0.02685 µmol/min, respectively. The immobilization process caused uricase to exhibit advanced stability for medium pH, resistance against protease enzymes, improved storage stability, and reusability. It was also observed that the substrate affinity for uric acid increased significantly after immobilization. As a model experiment, activity experiments were performed in an artificial serum medium to demonstrate the potential of the proposed system for future in vivo use. Artificial tests indicated that uricase-modified microrockets showed higher enzymatic activity than that of free counterpart and enzyme-free control studies. [ABSTRACT FROM AUTHOR]

Nanozymes have advantages over natural enzymes in terms of efficiency, stability, and economy. MVSM (Mixed Valence State MOF) is a nano‐oxidase with uricase‐like activity that may catalyze uric acid (UA) in the body into allantoin and H2O2 to treat gout and hyperuricemia by substituting natural uricase. However, it cannot specifically identify and choose UA. To increase the selectivity and affinity of MVSM for UA, the composite material MVSM@MIP is innovatively synthesized using a new synthetic approach termed the "two‐step synthesis method," which may prevent UA from being oxidized by MVSM during manufacture in this study. At the same time, this study also provides experimental proof of the effective creation of the material, the advantages of the "two‐step synthesis approach," and the high selectivity and affinity of MVSM@MIP for UA. Based on these findings, the suggested technique may be used to effectively catalyze uric acid in human urine with high activity. [ABSTRACT FROM AUTHOR]

• First report on in-silico identification of epitopes, hot-spot residues of Uricase. • T159W, D169C, N264W and Y203D mutations reduce immunogenicity for Ag - Uricase. • S139V, K215W, G216F and I172P mutations reduce immunogenicity for Bf -Uricase. • Muteins are found to have increased affinity towards uric acid. • Further, 100 ns MD simulation was done to confirm structural stability of mutein. The clinical utilization of Uricase against gout is limited due to the immunogenicity. In the present article, we identified the antigenic determinants of Uricase and reduced their immunogenicity via in-silico mutagenesis. Multiple sequence alignment and motif analysis were carried out to identify the conserved residues in evolutionary process. Emini surface accessibility, Parker hydrophilicity, and Karplus & Schulz flexibility methods were employed to predict the linear B-cell epitopes of both Ag -Uricase and Bf -Uricase. Deimmunization approach identified T-cell epitopes and the hot spot residues. Reduced antigenic probability was obtained in case of T159W, D169C, N264W and Y203D mutations for Ag -Uricase, while S139 V, K215W, G216 F and I172 P mutations for Bf -Uricase. The binding affinity values of uric acid towards the catalytic pocket of Ag -Uricase and Bf -Uricase models were found to be -48.71 kcal/mol and -40.93 kcal/mol, respectively. This energy is further stabilized in the mutant model by -6.36 kcal/mol and -1.45 kcal/mol for Ag -Uricase and Bf -Uricase, respectively. About 100 ns molecular dynamics simulation was performed to evaluate the conformational stability of both native and mutated Uricase. Insights obtained from this study provide guidelines for experimental design of Uricase muteins with reduced antigenicity. [ABSTRACT FROM AUTHOR]

Gout is a common rheumatic condition caused due to increase in serum uric acid level (hyperuricemia). Uricase is for lowering the level of uric acid but unfortunately, it is not produced in humans due to evolutionary changes. Therefore, it is administered to humans from outside in case of the high uric acid level in blood. A different formulation of uricase from bacterial, fungal, and mammalian sources is present in the market for the treatment of hyperuricemia conditions. Uricase formulation showed immunogenic response due to the occurrence of hypersensitivity reaction during the treatment that results in poor patient compliance. The purpose of this study was to clarify the variation of Uricase immunogenicity from different sources. We have used some immunoinformatic approaches to analyze and understand some structural aspects of immunogenic and allergenic epitopes of Uricase by calculation of relative frequency for eleven global alleles. As per our knowledge, this is the first immunoinformatic study of Uricase (structural based immunogenicity prediction) that deciphered the high immunogenic nature of Uricase but no significant difference in immunogenicity was found among Uricase isolated from Aspergillus flavus, Bacillus subtillis, and mammalian source. This study gives a further lead to develop some methods (include bioengineering of less immunogenic version of the uricase or utilizing the homologous enzymes) for minimizing immune response or search new sources of uricase that could be less or non-immunogenic. [ABSTRACT FROM AUTHOR]

Gout is a common inflammatory arthritis that is caused by chronically-elevated serum uric acid (sUA) levels (hyperuricemia). In humans, sUA levels are predominantly controlled by a variety of transporters that mediate the elimination of uric acid through the kidneys and intestines, a process that is altered in most gout patients. In this review, we highlight our current understanding of uric acid handling in healthy individuals and gout patients, therapies for gout that target uric acid transporters, and the mechanism of other therapies that alter sUA levels through interactions with uric acid transporters.

A uric acid biosensor aimed at a simplified determination of salivary uric acid was fabricated and utilized for the measurement of the diurnal variation of salivary uric acid. The biosensor measures uric acid as the change in the amount of hydrogen peroxide produced in the uricase reaction. Because uric acid is oxidized as easily as hydrogen peroxide, the osmium-HRP redox reaction was employed. The sensitivity of the biosensor was 170 nA/mM, which was sufficient for salivary uric acid determination. For simplified measurement of a saliva sample, a paperbased saliva sampling device, which enables the sample collection of a regulated amount of saliva in 5 s, was used. As a test using an actual sample, the diurnal variation of salivary uric acid was measured. The result indicated that salivary lactic acid increased in the morning. The total measurement time for the saliva measurement was approximately 3 min, which was sufficiently fast for the purpose of daily health management. The proposed method is expected to be used not only in gout treatment but also possibly in the measurement of other substances contained in saliva. [ABSTRACT FROM AUTHOR]

The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C. [ABSTRACT FROM AUTHOR]

Uricase (urate oxidase EC 1.7.3.3) is a therapeutic enzyme that is widely used to catalyze the enzymatic oxidation of uric acid in the treatment of hyperuricemia and gout diseases. In this study, three bacterial species capable of producing extracellular uricase were isolated from a poultry source and screened based on the size of the clear zone using a uric acid agar plate. The bacterial species capable of producing uricase with the highest uricolytic activity was identified asBacillus cereusstrain DL3 using a 16SrRNA gene sequencing approach. The time-course study of uricase production was performed and the medium was optimized. Carboxymethylcellulose and asparagine were found to be the best carbon and nitrogen sources. Maximum uricolytic activity was observed at pH 7.0 with an inducer concentration of 2.0 g/L. Inoculum size of 5% gave maximum uricolytic activity. The maximum uricolytic activity of 15.43 U/mL was achieved at optimized conditions, which is 1.61 times more than the initial activity. Further, enzymatic stability was determined at different pH and temperature. [ABSTRACT FROM PUBLISHER]

Gout is an inflammatory arthritis characterized by sudden, painful inflammation. Gout can affect any joint in an asymmetric distribution. Gouty attacks may be isolated or can be followed by years of recurrent flares. Over time, elevated serum urate levels and tophaceous deposits can lead to deformity and disability from underlying bony erosion. The concept of 'treatmentfailure gout' describes a unique population that has been either unable to tolerate allopurinol or who have not experienced normalization of serum urate levels on allopurinol. It is estimated that approximately 1-1.5% of the estimated 3-8 million people with gout in the USA have treatment-failure gout. Pegloticase is an US FDA-approved intravenous medication that is a mammalian recombinant uricase conjugated to monomethoxy polyethylene glycol. Two recent Phase III trials have found pegloticase to be effective in the management of treatment-failure gout. These studies also highlight safety concerns regarding the drug's immunogenicity. [ABSTRACT FROM AUTHOR]

Abstract: Uricase (EC 1.7.3.3, UC) catalyzes the oxidation of uric acid (UA) to more soluble allantoin thereby lowering plasma UA levels. In humans, when concentration of UA exceeds >7mg/dl, it leads to hyperuricemia, gout, nephrolithiasis and urolithiasis. A new remedy to cure such metabolic diseases is the enzyme supplementation therapy by UC but with high degree of antigenic independence. Therefore screening of new uricase sources to expand its usefulness and reduced antigenecity is needed. Present study employed cheminformatics approach to construct models of reported UC from different sources viz. Bacillus megaterium, Streptomyces bingchenggensis BCW-1, Paenibacillus sp, Solibacter usitatus Ellin6076, Truepera radiovictrix DSM 17093 and Ktedonobacter racemifer DSM 4496 in order to study their structure–function relationship for enzyme mass production and modification for improved characteristics. BioMed CAChe version 6.1 was further used to study enzyme–substrate interactions of models with uric acid using docking approach. Results indicated that models for UC of Streptomyces bingchenggensis BCW-1 accounted for better regio-specificity towards UA, supporting the interested metabolism and thus may further be implicated in enzyme supplementation therapy for hyperuricemic associated disorders. [Copyright &y& Elsevier]

Abstract: Available recombinant uricases (rasburicase, pegloticase) are potent hypouricaemic agents for tophaceous gout, but their long-term use is in question. We have performed a literature review on uricases, using Scirus, PubMed, Science Direct, and several other search engines. We have also consulted the records of drug regulatory authorities and patents on uricases. Rasburicase (Fasturtec®) was approved in Europe for tumour lysis syndrome induced by chemotherapy, in a single daily infusion dose for a maximum of 7days. A retrospective study (n =10) conducted in patients with gout and three clinical cases have shown that infusions spaced over time, over several months, ensure the control of serum uric acid and help to eliminate or significantly reduce the size of tophi. However, repeated gout attacks (despite colchicine) and hypersensitivity reactions (despite corticosteroids) have dampened enthusiasm for its use in gout. Pegloticase was recently approved by the Food and Drug Administration (FDA) for patients with chronic gout, refractory or intolerant to conventional hypouricaemic therapy. A 6month study versus placebo showed that pegloticase (infused at 8mg every 2weeks), induced a significant decrease in plasma uric acid in about 40% of patients (associated with a tendency for tophi dissolution). However, the remaining patients were non-responders, which correlated with the formation of pegloticase antibodies and infusion reactions. Research efforts are needed to develop less immunogenic uricases. In conclusion, some uricases could have an important role in the treatment of gout, for instance as a first-line treatment and over a short period of several months in patients with severe and tophaceous gout to allow rapid tophi dissolution. [Copyright &y& Elsevier]

Résumé: Les uricases (rasburicase, pégloticase) sont des traitements hypo-uricémiants puissants pour le traitement des gouttes tophacées, mais leur utilisation à long terme n’est pas sans problèmes. Nous proposons une revue de la littérature sur le sujet, effectué avec Scirus, PubMed, Science Direct et plusieurs autres moteurs de recherche. Nous avons aussi consulté les registres des autorités de régulation du médicament et des brevets sur les uricases. La rasburicase (Fasturtec®) a été approuvée en Europe pour le traitement préventif du syndrome de lyse tumorale induit par les chimiothérapies, en perfusion quotidienne pendant sept jours au maximum. Une petite étude rétrospective (n =10) conduite chez des patients goutteux et trois cas cliniques ont démontré que des perfusions espacées dans le temps, pendant plusieurs mois, assurent le contrôle de l’uricémie et permettent d’éliminer ou réduire considérablement la taille des tophus. Cependant, des crises de goutte répétées (malgré la colchicine) et des réactions d’hypersensibilité (malgré les glucocorticoïdes) ont atténué l’enthousiasme pour son utilisation dans la goutte. La pégloticase a été récemment approuvée par la Food and Drug Administration (FDA, États-Unis) pour les patients souffrant de goutte chronique, réfractaires ou intolérants aux traitements hypo-uricémiants conventionnels. Des essais à six mois, versus placebo, ont montré que la pégloticase, administrée par voie intraveineuse aux doses de 8mg toutes les deux semaines, induit une diminution importante de l’uricémie associée à la dissolution du tophus, chez environ 40 % des patients. Toutefois, plus de la moitié des patients étaient non-répondeurs, ce qui est corrélé avec la formation d’anticorps anti-pégloticase et l’intolérance à la perfusion. Des efforts de recherche ont été mis en œuvre pour développer de nouvelles uricases (pegsiticase en phase 1), moins immunogènes. En conclusion, certaines uricases pourraient avoir un rôle important dans le traitement de la goutte, par exemple en première ligne et sur une courte période de quelques mois chez des patients sévères et tophacés, afin de permettre une dissolution rapide des tophus. Des efforts de recherche sont nécessaires afin de diminuer l’immunogénicité des uricases. [ABSTRACT FROM AUTHOR]

Gout is a debilitating disease secondary to chronic hyperuricemia, and the subsequent deposition of monosodium urate crystals is responsible for acute flare, gout arthropathies, tophi and renal lithiasis. Uric acid is the end product of purine metabolism in humans because the gene encoding uricase was lost during hominoid evolution. Pegloticase is a recombinant mammalian uricase conjugated to polyethylene glycol that catalyzes the oxidation of uric acid into allantoin, a more soluble end product. The use of this drug as urate-lowering therapy is a new approach in treating severe gout refractory to conventional therapy with xanthine oxidase inhibitors and uricosuric agents. Intravenous pegloticase has potent and long-lasting urate-lowering capacity with rapid efficacy on tophi resolution. However, pegloticase treatment is associated with infusion-related reactions despite prevention therapy with high-dose corticosteroids. Exacerbation of pre-existing cardiovascular diseases is another concern. The mechanisms of these events are unknown. Caution with long-term use of pegloticase is warranted, especially for patients with cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Uric acid (UA) is the end product of purine metabolism in humans due to the loss of uricase activity by various mutations of its gene during the Miocene epoch, which led to humans having higher UA levels than other mammals. Furthermore, 90% of UA filtered by the kidneys is reabsorbed, instead of being excreted. These facts suggest that evolution and physiology have not treated UA as a harmful waste product, but as something beneficial that has to be kept. This has led various researchers to think about the possible evolutionary advantages of the loss of uricase and the subsequent increase in UA levels. It has been argued that due to the powerful antioxidant activity of UA, the evolutionary benefit could be the increased life expectancy of hominids. For other authors, the loss of uricase and the increase in UA could be a mechanism to maintain blood pressure in times of very low salt ingestion. The oldest hypothesis associates the increase in UA with higher intelligence in humans. Finally, UA has protective effects against several neurodegenerative diseases, suggesting it could have interesting actions on neuronal development and function. These hypotheses are discussed from an evolutionary perspective and their clinical significance. UA has some obvious harmful effects, and some, not so well-known, beneficial effects as an antioxidant and neuroprotector. [ABSTRACT FROM PUBLISHER]

Abstract: PEGylation is a successful strategy for improving the biochemical and biopharmaceutical properties of proteins and peptides through the covalent attachment of polyethylene glycol chains. In this work, purified recombinant uricase from Candida sp. (UC-r) was modified by PEGylation with metoxypolyethilenoglycol-p-nitrophenyl-carbonate (mPEG-pNP) and metoxypolyethyleneglycol-4,6-dichloro-s-triazine (mPEG-CN). The UC-r-mPEG-pNP and UC-r-mPEG-CN conjugates retained 87% and 75% enzyme activity respectively. The K M values obtained 2.7×10−5 M (mPEG-pNP) or 3.0×10−5 M (mPEG-CN) for the conjugates as compared to 5.4×10−5 M for the native UC-r, suggesting enhancement in the substrate affinity of the enzyme attached. The effects of pH and temperature on PEGylated UC-r indicated that the conjugates were more active at close physiological pH and were stable up to 70°C. Spectroscopic study performed by circular dichroism at 20°C and 50°C did not show any relevant difference in protein structure between native and PEGylated UC-r. In rabbit and Balb/c mice, the native UC-r elicited an intense immune response being highly immunogenic. On the other hand, the PEGylated UC-r when injected chronically in mice did not induce any detectable antibody response. This indicates sufficient reduction of the immunogenicity this enzyme by mPEG-pNP or mPEG-CN conjugation, making it suitable for a possible therapeutical use. [Copyright &y& Elsevier]

Pegloticase is designed to convert urate into the easily excretable allantoin to treat hyperuricemia in gout. The aim of this analysis was to describe the pharmacokinetics and pharmacodynamics of pegloticase in 40 gout patients. Pegloticase was administered as intravenous infusions every 2 weeks at 4- and 8-mg doses or every 4 weeks at 8- or 12-mg doses for 12 weeks. Serum pegloticase concentrations, plasma urate, and serum antibody response were determined. Population pharmacokinetics and pharmacodynamics analyses were performed. Data were modeled simultaneously, and covariates were investigated (age, gender, race, body weight, ideal body weight, and antibody response). The dosing regimens to maintain uric acid levels below the therapeutic target of 6 mg/dL were then predicted by the model. The pharmacokinetics were best described by a 1-compartment linear model, while the pharmacodynamics model was fitted as a direct effect of pegloticase on uric acid concentrations with a suppressive maximum effect attributed to drug (Emax) function. Pegloticase suppressed uric acid levels up to 83%. Weight only affected clearance and volume of distribution. No covariates affected pharmacodynamics. Simulation suggests pegloticase administered at 8 mg every 2 or 4 weeks as 2-hour intravenous infusions will maintain uric acid levels well under 6 mg/dL. [ABSTRACT FROM AUTHOR]