Multivalent Albumin-Neonatal Fc Receptor Interactions Mediate a Prominent Extension of the Serum Half-Life of a Therapeutic Protein.

Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.

Polypeptides with High Zwitterion Density for Safe and Effective Therapeutics.

The commonly used "stealth material" poly(ethylene glycol) (PEG) effectively promotes the pharmacokinetics of therapeutic cargos while reducing their immune response. However, recent studies have suggested that PEG could induce adverse reactions, including the emergence of anti-PEG antibodies and tissue histologic changes. An alternative stealth material with no or less immunogenicity and organ toxicity is thus urgently needed. We designed a polypeptide with high zwitterion density (PepCB) as a stealth material for therapeutics. Neither tissue histological changes in liver, kidney, or spleen, nor abnormal behavior, sickness or death was induced by the synthesized polymer after high-dosage administration for three months in rats. When conjugated to a therapeutic protein uricase, the uricase-PepCB bioconjugate showed significantly improved pharmacokinetics and immunological properties compared with uricase-PEG conjugates.

Prediction of Therapeutic Effect of Rasburicase on Hyperuricemia Associated with Chemotherapy Based on Theoretical Model.

Rasburicase has a strong and fast effect for reducing blood levels of uric acid. However, there have been no reports of theoretical analysis for the rational dose and interval of administration. Thus we constructed a pharmacokinetic and pharmacodynamic model to determine changes in uric acid level after rasburicase administration at various doses and regimens. The time courses of uric acid level predicted using our model were in good agreement with observed data, indicating adequate performance for our model. The therapeutic effects after a single infusion at various rates of generation of uric acid were predicted. The maximum effect was not a large difference, in spite of the generation rate. Then, the therapeutic effects of repeated administrations were predicted. The effect did not change when rasburicase was administered at more than the usual dose. Besides, as the administration interval increased, the difference between minimum and maximum level of uric acid became greater. However, in all doses and regimens, adequate therapeutic effects were obtained. In conclusion, the model was found useful for predicting therapeutic effect of rasburicase and individually determining rational dosage regimen of rasburicase.

Pharmacokinetics and pharmacodynamics of pegloticase in patients with end-stage renal failure receiving hemodialysis.

AIMS: Phase 3 trial data indicate that treatment of chronic tophaceous gout with pegloticase, a recombinant uricase conjugated to polyethylene glycol, does not reduce estimated glomerular filtration rate in chronic kidney disease (CKD) patients and that pegloticase therapeutics are independent of CKD stages 1 - 4. We determined the pharmacokinetics/pharmacodynamics of pegloticase after a single-dose in non-gout subjects with stage 5 CKD receiving hemodialysis. METHODS: In this open-label phase 1 study, 12 subjects received a single intravenous dose of pegloticase 8 mg 3 hours prior to hemodialysis. Blood samples for determination of serum pegloticase concentrations and serum uric acid (SUA) levels were collected immediately predose and at regular intervals before, during, and after hemodialysis. RESULTS: Mean serum pegloticase concentrations remained stable and were unaffected by dialysis sessions. Mean SUA fell to undetectable levels within 3 hours and remained undetected for up to 72 hours postdose. CONCLUSION: Our findings indicate no significant effect of hemodialysis on either the stability of serum pegloticase concentrations after a single dose or the capacity of pegloticase to lower SUA. No new safety signals were detected. Administration of pegloticase in patients with comorbid chronic tophaceous gout and endstage renal failure requiring hemodialysis appears feasible.

[The Pharmacokinetics and Pharmacodynamics of Intravenous Uricase Multivesicular Liposomes].

OBJECTIVE: To determine and compare the pharmacokinetics and pharmacodynamics of uricase-multivesicular liposomes (UOMVLs) with free uricase (UOX) in rats. METHODS: UOMVLs were prepared by the double emulsion method and confirmed with its entrapment efficiency, size and Zeta potential. Twelve healthy rats were randomly divided into two groups: one with i. v. injection of UOMVLs, and the other with i. v. injection of UOX. Their serum activity of uricase was assayed. The pharmacokinetic parameters were calculated using software DAS 2. 1. 1. Another 24 male SD rats were enrolled, the rat model of hyperuricemia was established with hypoxanthine and potassium oxonate, while normal group (n=6) was set as control. Injection of UOMVLs (1 mL, 0. 47 U/mL), UOX (1 mL, 0. 47 U/mL) and nothiy were given 1 h later in UOMVLs group (n=6), UOX group (n=6) and model group (n=6), and their serum uric acid levels were determined 1, 2, 3, 5, 7, 9, 12, 24, 36, 48 h after the establishment of hyperuricemia model. RESULTS: The entrapment efficiency of UOMVLs was (63. 75 ± 3. 65) %, with an average particle size of (22. 56 ± 1. 70) µm and Zeta potential of (-41. 81±6. 59) mV. The pharmacokinetic parameters of UOMVLs and UOX were as follows, respectively: area under time-concentration curve from 0 to infinity time (AUC0-∞) (498. 83 ± 58. 85) U/L . h and (28. 49 ± 9. 95) U/L . h; time to peak concentration (Tmax) (1. 00±0. 00) h and (0. 00±0. 00) h; peak concentration (Cmax) (73. 04±6. 35) U/L and (31. 00±6. 03) U/L; elimination half-life (t1/2) (3. 49±0. 80) h and (1. 17±0. 33) h. The relative bioavailability of UOMVLs was (1 750. 90±206. 56) %. UOMVLs decreased serum uric to normal in 9 h; whereas it took 48 h for the UOX group and the model group to return to normal. CONCLUSION: UOMVLs can prolong tmax and t1/2 and improve the relative bioavailability. UOMVLs decrease serum uric acid levels in rats with hyperuricemia more effectively than UOX.

Pharmacodynamic analysis of intravenous recombinant urate oxidase using an indirect pharmacological response model in healthy subjects.

AIM: Pharmacodynamic analysis of intravenous recombinant urate oxidase produced by Escherichia coli was performed in healthy subjects using a pharmacokinetic/pharmacodynamic (PK/PD) model. METHODS: A randomized, single-blind, placebo-controlled study was performed in 40 healthy Chinese subjects (4 groups of 10 subjects each, placebo 4:1 ratio) who received infusions of uricase (single doses of 0.1, 0.2, and 0.3 mg/kg; multiple doses of 0.2 mg·kg(-1)·d(-1) for 7 d). PK profiles were determined through plasma uricase activity, and PD profiles were established using uric acid levels in plasma and urine. The plasma PD parameter was estimated as changes in plasma uric acid levels as the effect in the indirect response model. Adverse events were also monitored. RESULTS: A two-compartment PK model with constant iv input and first-order output was used to describe the kinetic process of plasma uricase. The low value (2.8 U/L) of drug concentration that achieved 50% of maximum effect (EC50) indicated that low plasma uricase concentrations were sufficient to produce pharmacological effects. A strong relationship (r(2)=0.9991) between the mean uric acid concentration in blood and the mean uric acid excretion rate in urine in the range of 11 to 30 h after single dosing was found. Infusions of uricase were well tolerated in all subjects. CONCLUSION: The PK/PD model predicted the effective dose to be 0.1 mg/kg in healthy subjects. The excretion rate of uric acid in urine may be used as a new index for pharmacological effects in further clinical trials.

Co-delivery of indomethacin and uricase as a new strategy for inflammatory diseases associated with high uric acid.

Uric acid is the final metabolite in humans. High level of uric acid chronically induces urate deposition, aggravates kidney damage, and concomitantly causes an increase in inflammatory factors. Alleviating acute inflammation and decreasing uric acid levels are the key points in the treatment of inflammatory diseases associated with high uric acid. However, a drug delivery system that combines anti-inflammatory and uric acid reduction functions at the same time remains a challenge to be settled. Here, we designed a nanocrystal-based co-delivery platform, IND Nplex, characterized by loading of indomethacin (IND) and uricase. Compared with free IND or uricase, IND Nplex possessed a better anti-inflammatory effect by restraining the release of inflammation-related factors in vitro. In addition, pharmacokinetic and biodistribution studies revealed that IND Nplex significantly prolonged the retention time in vivo and was more concentrated in the kidney. In acute gouty arthritis model rats, IND Nplex markedly relieved ankle joint swelling and mitigated synovial inflammation. In acute kidney injury model rats, IND Nplex indicated better biocompatibility and significant amelioration of renal fibrosis. Moreover, IND Nplex showed the effect of anti-inflammatory and improved renal function via determination of inflammatory factors and biochemical markers in the serum and kidney. In conclusion, these results indicate that IND Nplex exerts anti-inflammatory activity and uric acid-lowering effect and could become a promising candidate for the treatment of uric acid-related diseases.

Pegloticase: a novel agent for treatment-refractory gout.

OBJECTIVE: To evaluate efficacy and safety of pegloticase, approved by the Food and Drug Administration in September 2010 for treatment of patients with chronic treatment-refractory gout. DATA SOURCES: Literature searches were conducted using PubMed (1948-January 2012), TOXLINE, International Pharmaceutical Abstracts (1970-January 2012), and Google Scholar using the terms pegloticase, puricase, PEG-uricase, gout, uricase, and Krystexxa. Results were limited to English-language publications. References from selected articles were reviewed to identify additional citations. STUDY SELECTION AND DATA EXTRACTION: Studies evaluating the pharmacology, pharmacokinetics, safety, and efficacy of pegloticase for the treatment of chronic treatment-refractory gout were included. DATA SYNTHESIS: Pegloticase represents a novel intravenous treatment option for patients who have chronic gout refractory to other available treatments. Pegloticase is a recombinant uricase and achieves therapeutic effects by catalyzing oxidation of uric acid to allantoin, resulting in decreased uric acid concentrations. Results of published trials demonstrate the ability of pegloticase to maintain uric acid concentrations below 7 mg/dL in patients with chronic gout. Data supporting reduction of gout flares are limited. Pegloticase is well tolerated but associated with gout flares and infusion reactions. Other adverse events include nausea, dizziness, and back pain. During Phase 3 trials, 2 patients in the pegloticase biweekly group and 1 in the monthly group experienced heart failure exacerbation; another patient in the monthly group experienced a nonfatal myocardial infarction. Providers should exercise caution before administering pegloticase to patients with cardiovascular disease. The cost burden and safety profile may limit its use in practice, in addition to limited data available to support decreases in patient-centered outcomes (eg, gouty attacks). CONCLUSIONS: Pegloticase is an effective option for patients with symptomatic gout for whom current uric acid-lowering therapies are ineffective or contraindicated.

Polyethylene Glycol-Like Brush Polymer Conjugate of a Protein Drug Does Not Induce an Antipolymer Immune Response and Has Enhanced Pharmacokinetics than Its Polyethylene Glycol Counterpart.

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.

Pharmacokinetics of Polyethylene Glycol-Modified Canine Uricase Following Single and Multiple Intravenous Injections in Cynomolgus Monkeys.

BACKGROUND AND OBJECTIVE: Polyethylene glycol-modified canine uricase (PEG-UHC) prepared with a lower-molecular-weight (5 kDa) PEG is used to treat gout. This study investigated the comparative pharmacokinetics of single and multiple doses of PEG-UHC administered intravenously and a single dose of uricase (UHC) administered intravenously in cynomolgus monkeys. METHODS: A noncompartmental model was used to fit the plasma drug concentration-time curve and calculate the pharmacokinetic parameters of PEG-UHC, which were compared with those obtained for UHC at the equivalent dose (2 mg/kg). To study the pharmacokinetics after multiple dose administration, cynomolgus monkeys were administered five intravenous injections of PEG-UHC (0.5 mg/kg), with one injection performed every 15 days. RESULTS: The area under the curve (AUC) and the maximum plasma concentration (Cmax) of PEG-UHC were positively correlated with dose, whereas plasma half-life (t1/2) and clearance (CL) did not change significantly with increasing dose, suggesting that these pharmacokinetic characteristics are linear. Intravenous PEG-UHC exhibited an average t1/2 that was 125.79 times longer and an AUC0-t that was 64.45 times larger than the corresponding values for UHC at the same dose (2 mg/kg), while the CL of PEG-UHC was 1/72.73 times the CL of intravenous UHC. The plasma drug concentration reached a steady state after five injections, and the t1/2 values following the first and last drug administration did not differ significantly. CONCLUSION: Our data show that PEG-UHC is markedly superior to UHC in terms of duration of action, and that the pharmacokinetics of PEG-UHC in cynomolgus monkeys are linear. Sequential administration of PEG-UHC did not accelerate drug clearance. Our findings provide the basis for future clinical studies of PEG-UHC.

Safety and efficacy of rasburicase (SR29142) in a Japanese phase II study.

The purpose of this study was to investigate the safety profile of SR29142 when administered as a single agent both prior to chemotherapy and during treatment, and to compare the efficacy of SR29142 administered at two dose levels in adult Japanese patients with leukemia or lymphoma. During this open-label, multicenter, phase II study, patients received SR29142 for 5 days, administered at either 0.15 or 0.20 mg/kg per day. Chemotherapy was started 4­24 h after the first infusion of SR29142. The primary end-point was overall response rate, defined as the normalization of plasma uric acid to 7.5 mg/dL or less, from 48 h after the first infusion to 24 h after the last infusion of SR29142. SR29142-related adverse events including hypersensitivity (allergic) reactions were assessed. Overall, 50 patients received SR29142 at either 0.15 mg/kg per day (n = 25) or 0.20 mg/kg per day (n = 25) followed by chemotherapy. The overall response rate was 100.0% (95% confidence interval, 86.3­100.0%) with 0.15 mg/kg and 96.0% (95% confidence interval, 79.6­99.9%) with 0.20 mg/kg. Both dose levels of SR29142 were equally effective at reducing plasma uric acid levels. In six patients, seven drug-related adverse events of grade 1/2 occurred before chemotherapy. SR29142-related, hypersensitivity-associated reactions occurred in three patients, and rash, anorexia, application site pain and pyrexia occurred in one patient each; only five patients (10%) showed anti-SR29142 antibodies by day 29. In conclusion, SR29142 is effective at reducing plasma uric acid levels with a tolerable safety profile as a single agent both prior to chemotherapy and during treatment.

Reduction of plasma urate levels following treatment with multiple doses of pegloticase (polyethylene glycol-conjugated uricase) in patients with treatment-failure gout: results of a phase II randomized study.

OBJECTIVE: To assess the efficacy of pegloticase in achieving and maintaining plasma urate levels of <6 mg/dl in gout patients in whom other treatments have failed, and to assess the pharmacokinetics and safety of pegloticase. METHODS: Forty-one patients were randomized to undergo 12-14 weeks of treatment with pegloticase at 1 of 4 dosage levels: 4 mg every 2 weeks, 8 mg every 2 weeks, 8 mg every 4 weeks, or 12 mg every 4 weeks. Plasma uricase activity, plasma urate, and antipegloticase antibodies were measured, pharmacokinetic parameters were assessed, and adverse events were recorded. RESULTS: The mean plasma urate level was reduced to

Pegloticase, a polyethylene glycol conjugate of uricase for the potential intravenous treatment of gout.

Savient Pharmaceuticals Inc (formerly Bio-Technology General Corp), under license from Duke University, is developing pegloticase, PEG conjugates of uricase (urate oxidase), for the potential treatment of gout. The in-life portion of the phase III trials have been completed.

Population pharmacokinetic and pharmacodynamic analysis of pegloticase in subjects with hyperuricemia and treatment-failure gout.

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 (E(max)) 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.

Efficacy and (pharmaco)kinetics of one single dose of rasburicase in patients with chronic kidney disease.

AIMS: Hyperuricemia is a risk factor associated with cardiovascular and renal disease. Recently, rasburicase, a recombinant urate oxidase, has been developed for the treatment of hyperuricemia in patients with primarily hematological malignancies. We studied the pharmacokinetics and metabolism of rasburicase in the treatment of chronic asymptomatic hyperuricemia in chronic kidney disease (CKD) patients. MATERIALS AND METHODS: We studied 9 CKD patients with hyperuricemia, whose mean serum acid concentration was 10.2 (range 8.3-15.8) mg/dl. No study subject was taking allopurinol (3/9 are allopurinol intolerant). Patients were treated with rasburicase (0.2 mg/kg/day) in single dose by intravenous infusion over a 30-min period. Serum samples were collected after 1, 4, 8, 24, 48 and 72 h, after 1 week, and after 1 month. To evaluate the efficacy of rasburicase, plasma and urinary concentrations of uric acid were determined by the standard method; the plasma activity of rasburicase was determined using a new assay developed by our laboratory (chromatography-mass method, a colorimetric 96-well microtiter plate assay). RESULTS: All the treated patients experienced a rapid reduction in their plasma uric acid concentration. Data showed an undetectable value within 1 h of treatment. The rasburicase effect ended after 50 h, with a slow increase in the plasma level of uric acid. CONCLUSION: A single dose of rasburicase is highly effective and well tolerated in the treatment of hyperuricemia in selected CKD patients.

A minimal physiologically based pharmacokinetic model that predicts anti-PEG IgG-mediated clearance of PEGylated drugs in human and mouse.

Circulating antibodies that specifically bind polyethylene glycol (PEG), a polymer routinely used in protein and nanoparticle therapeutics, have been associated with reduced efficacy and increased adverse reactions to some PEGylated therapeutics. In addition to acute induction of anti-PEG antibodies (APA) by PEGylated drugs, typically low but detectable levels of APA are also found in up to 70% of the general population. Despite the broad implications of APA, the dynamics of APA-mediated clearance of PEGylated drugs, and why many patients continue to respond to PEGylated drugs despite the presence of pre-existing APA, remains not well understood. Here, we developed a minimal physiologically based pharmacokinetic (mPBPK) model that incorporates various properties of APA and PEGylated drugs. Our mPBPK model reproduced clinical PK data of APA-mediated accelerated blood clearance of pegloticase, as well as APA-dependent elimination of PEGyated liposomes in mice. Our model predicts that the prolonged circulation of PEGylated drugs will be compromised only at APA concentrations greater than ~500 ng/mL, providing a quantitative explanation to why the effects of APA on PEGylated treatments appear to be limited in most patients. This mPBPK model is readily adaptable to other PEGylated drugs and particles to predict the precise levels of APA that could render them ineffective, providing a powerful tool to support the development and interpretation of preclinical and clinical studies of various PEGylated therapeutics.

Rasburicase represents a new tool for hyperuricemia in tumor lysis syndrome and in gout.

Hyperuricemia is a feature of several pathologies and requires an appropriate and often early treatment, owing to the severe consequences that it may cause. A rapid and massive raise of uric acid, during tumor lysis syndrome (TLS), and also a lower and chronic hyperuricemia, as in gout, mainly damage the kidney. To prevent or treat these consequences, a new therapeutic option is represented by rasburicase, a recombinant form of an enzyme, urate oxidase. This enzyme converts hypoxanthine and xanthine into allantoin, a more soluble molecule, easily cleared by kidney. The several types of urate oxidase have followed each other, with progressive reduction of adverse reactions. The most important among them are allergenicity and the development of antibodies which compromise their effectiveness. Nevertheless, a limit of rasburicase's use remains its cost, which obliges to a judicious choice to prevent TLS in high risk patients with cancer and in case of allergy or impossibility to take allopurinol orally both in TLS and in gout. A large body of evidence confirms the efficacy and safety of rasburicase, even in comparison to the standard drugs used in the aforementioned pathologies.

Nanocapsules of therapeutic proteins with enhanced stability and long blood circulation for hyperuricemia management.

Among a broad spectrum of medical treatments, protein therapeutics holds tremendous opportunities for the treatment of metabolic disorders, cancer, autoimmune diseases and etc. Broad adaption of protein therapeutics, however, still remain challenging, not only because of poor protein stability, but they also experience fast clearance after administrated and elicit immune responses, resulting in undesirable biodistribution and short blood residence time. In this study, we demonstrate a novel protein delivery method via encapsulating therapeutic proteins within thin shells of poly(N-vinylpyrrolidone) (PVP), which leads to significantly improved protein stability, reduced macrophage uptake, prolonged circulation time and reduced immunogenicity. Exemplified with urate oxidase (UOx), the enzyme used for hyperuricemia treatment, as-formed UOx nanocapsules, n(UOx), exhibits enhanced stability, more significant therapeutic effects, and a more than 10-fold improvement in circulation time when compared with native UOx. This technology not only demonstrates the use of UOx nanocapsules for hyperuricemia management, but also provides a general approach for a broad spectrum of therapeutic proteins for in vivo applications.

Bioengineered robust hybrid hydrogels enrich the stability and efficacy of biological drugs.

Biological drugs are exquisitely tailored components offering the advantages of high specificity and efficacy that are considered safe for treating diseases. Nevertheless, the effectiveness of biological drugs is limited by their inherent short biological half-life and poor stability in vivo. Herein, we engineered a novel delivery platform based on hybrid injectable hydrogels, in which pH- and temperature-responsive biodegradable copolymers were site-specifically coupled to the sulfhydryl group of human serum albumin, which effectively enhances the stability and circulation half-life of the biological drug, recombinant uricase enzyme (Uox). The albumin ligand conjugated to the Uox allowed specific-binding of the enzyme within the protein shell, and the synthetic polymers effectively shield the protein-enzyme complex. Such close confinement exhibits strong resistance towards various physical, chemical and therapeutically relevant stressors such as temperature, pH and proteases. Subcutaneous administration of Uox-loaded bioengineered hybrid hydrogel improved the pharmacokinetics by prolonging its circulation half-life. As a consequence, the bioengineered hybrid hydrogel normalized the serum uric acid level in hypoxanthine/potassium oxonate-induced hyperuricemia mice, and no obvious side effects were observed in the major organs. The characteristic of the bioengineered hydrogel networks applicable to a variety of biological drugs by simple mixing that unlock the possibility of adapting biological drugs to therapeutic applications.

Sampling on ice will not yield reliable uric acid monitoring in rasburicase-treated patients.

OBJECTIVES: Rasburicase is administered to prevent hyperuricemia and counteract the consequences of tumour lysis syndrome (TLS). The benefit of monitoring uric acid (UA) concentrations in rasburicase-treated patients is questionable as spuriously low values are most frequently encountered. The manufacturer recommends a cold sample handling procedure to arrest ex vivo uricolysis. Contrariwise, it was recently considered that the temperature does not significantly affects rasburicase uricolysis. We here present a thorough investigation on rasburicase kinetics in clinical samples. DESIGN AND METHODS: UA was spiked in sera from rasburicase-treated patients at varying concentrations, divided in three fractions for incubation at 4°C, 22°C or 37°C and measured at fix time points. The Michaelis-Menten constant (Km) and activation energy (Eact) were estimated by linear regression and the Arrhenius equation, respectively. Additionally, UA concentrations retrieved in sera of rasburicase-treated patients were retrospectively studied (3.5years period). RESULTS: Although uricolysis increased at a higher temperature, incubation at 4°C did not arrest uricolysis entirely. The yielded Km of 128µmol/L highlights that maximum uricolytic activity is reached at UA concentrations lower than those observed for TLS patients. Furthermore, the Eact of 27kJ/mol corresponds to only a modest logarithmic decrease of the uricolytic capacity by 4-5% per -1°C. In routine practise, 'negative' UA concentrations were observed during 88.5% of the rasburicase therapy episodes, even when samples were stored at 4°C. CONCLUSION: In contrast to manufacturer's guidelines, simple cooling of the sample will not arrest the temperature-dependent uricolysis provoked by rasburicase and therefore not yield reliable UA monitoring.