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.

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.

Development of a new LC-MS/MS based enzyme activity assay for recombinant urate oxidase in plasma and its application to pharmacokinetics in human.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of recombinant urate oxidase in human plasma. This assay was based on the determination of enzyme reaction product, (15)N-allantoin, and phenacetin was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase of 30% water (containing 0.5% formic acid) and 70% methanol. Quantification was done using multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 161 → m/z 118 for (15)N-allantoin and m/z 180 → m/z 110.1 for IS at positive ionization mode. The calibration curve was established over the range of 2.077-42.06 U/l and the correlation coefficient was larger than 0.99. The intra-day and inter-day relative standard deviations were less than 10.6%. Accuracy determined at three concentrations ranged between 98.6% and 109.2%. This method was successfully applied to a pharmacokinetic study of intravenous recombinant urate oxidase produced from Escherichia coli in Chinese healthy volunteers.

Refrigeration is not necessary for measurement of uric acid in patients treated with rasburicase.

BACKGROUND: Rasburicase, used for hyperuricemia of tumor lysis syndrome, retains activity at room temperature (RT) in in vitro studies. Cold-temperature handling is recommended for uric acid measurements in patients receiving rasburicase: collection in prechilled tubes, transportation on ice, and 4°C centrifugation. We performed a prospective study of these requirements. METHODS: A total of 65 pairs of blood samples were collected from 34 patients, 12-24 h after receiving rasburicase. The effect of temperature on uric acid concentration was tested on paired samples handled either at RT or when cold: centrifugation (18 sample pairs), collection tube (14 pairs), transportation (24 pairs), and nine pairs were retested after 1 h at RT. RESULTS: No significant temperature effect was seen on the uric acid measurements for any of the cold-handling steps: proportional, absolute biases were -1.4%, -0.06 mg/dL (centrifugation), -1.5%, +0.02 mg/dL (tube temperature), and -2.2%, -0.01 mg/dL (transportation). A 20% negative bias was seen in samples retested after 1 h at RT. CONCLUSIONS: Cold handling (prechilled tubes, iced transportation, 4°C centrifugation) was equivalent to RT for immediate measurement. An additional 1 h delay at RT led to a 20% decrease in uric acid. The cold handling measures required by the manufacturer are not necessary for uric acid testing of patients receiving rasburicase treatment, if testing is performed without delay.

Impact of large aggregated uricases and PEG diol on accelerated blood clearance of PEGylated canine uricase.

BACKGROUND: Uricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated. METHODS AND FINDINGS: Recombinant canine uricase was first expressed and purified to homogeneity. Source 15Q anion-exchange chromatography was used to separate tetrameric and aggregated uricase prior to pegylation, while DEAE anion-exchange chromatography was used to remove Di-acid PEG (precursor of PEG diol) from unfractionated 5 kDa mPEG-propionic acid. Tetrameric and aggregated uricases were separately modified with the purified mPEG-SPA. In addition, tetrameric uricases was modified with unfractionated mPEG-SPA, resulting in three types of 5 kDa mPEG-SPA modified uricase. The conjugate size was evaluated by dynamic light scattering and transmission electron microscope. The influence of differently PEGylated uricases on pharmacokinetics and immunogenicity were evaluated in vivo. The accelerated blood clearance (ABC) phenomenon previously identified for PEGylated liposomes occurred in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies, rather than neutralizing antibodies, were found to mediate the ABC. CONCLUSIONS: The size of conjugates is important for triggering such phenomena and we speculate that 40-60 nm is the lower size limit that can trigger ABC. Removal of the uricase aggregates and the PEG diol contaminant and modifying with small PEG reagents enabled ABC to be successfully avoided and sufficient reduction in the immunogenicity of 5 kDa mPEG-modified tetrameric canine uricase.

Characterization, efficacy, pharmacokinetics, and biodistribution of 5kDa mPEG modified tetrameric canine uricase variant.

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ɛ amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated. Approximately 9.4 5 kDa mPEG chains were coupled to each monomeric uricase and the main conjugates contained 7-11 mPEG chains per subunit. mPEG-UHC showed significantly therapeutic or preventive effect on uric acid nephropathy and acute urate arthritis based on three different animal models. The clearance rate from an intravenous injection of mPEG-UHC varied significantly between species, at 2.61 mL/h/kg for rats and 0.21 mL/h/kg for monkeys. The long elimination half-life of mPEG-UHC in non-human primate (191.48 h, intravenous injection) indicated the long-term effects in humans. Moreover, the acceptable bioavailability of mPEG-UHC after subcutaneous administration in monkeys (94.21%) suggested that subcutaneous injection may be regarded as a candidate administration route in clinical trails. Non-specific tissue distribution was observed after administration of (125)I-labeled mPEG-UHC in rats, and elimination by the kidneys into the urine is the primary excretion route.

Low sensitivity for the metabolic syndrome to detect uric acid elevations in females and non-Hispanic-black male adolescents: an analysis of NHANES 1999-2006.

BACKGROUND: Uric acid is tightly linked to the metabolic syndrome (MetS) and among adults higher uric acid levels are associated with future risk for diabetes, cardiovascular disease, hypertension and renal disease. OBJECTIVE: Evaluate the sensitivity of MetS to identify adolescents with elevated uric acid levels on a race/ethnicity and gender-specific basis. METHODS: We evaluated 3296 male and female adolescents 12-19 y participating in the National Health and Nutrition Evaluation Survey 1999-06, comprised of 67.6% non-Hispanic whites, 15.1% non-Hispanic blacks, and 17.3% Hispanics. We used a definition of MetS modified for use in adolescents and evaluated the sensitivity of a diagnosis of MetS to identify individuals with uric acid elevations (approximately the 95th percentile of uric acid by gender among normal-weight adolescents). RESULTS: When used as a screening test to identify individuals with uric acid elevations MetS performed more poorly among females (18.0%) than among males (37.0%) (p<0.001). Among males, MetS exhibited a lower sensitivity among non-Hispanic blacks (17.8%) compared to Hispanics (45.9%) (p<0.01) and non-Hispanic whites (37.4%) (p<0.05). There were no race/ethnicity differences in detecting elevated uric acid levels among females (non-Hispanic-white 15.5%, non-Hispanic-black 19.4%, Hispanic 26.5%, p>0.05). CONCLUSION: Current criteria to diagnose MetS exhibit racial/ethnic and gender differences in the ability to identify adolescents with elevated uric acid levels, performing poorly among non-Hispanic-black males and among females. Given emerging data regarding the ability of uric acid elevations for predicting future disease, these data may have implications regarding the use of MetS as a marker of risk among all gender and racial/ethnic groups.

A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia.

The use of uricase-deficient mammals to screen formulations of engineered uricases as potential drugs for hyperuricemia involves heavy costs and presents a technical bottleneck. Herein, a new practical system was investigated to evaluate the pharmacological significance of a bacterial uricase based on its ability to eliminate uric acid in plasma in vitro, its pharmacokinetics in vivo in healthy rats, and the modeled pharmacodynamics in vivo. This uricase, before and after modification with the monomethyl ether of poly(ethylene glycol)-5000, effectively eliminated uric acid in vitro in rabbit plasma, but its action was susceptible to xanthine inhibition. After intravenous injection of the modified uricase without purification, a bi-exponential model fit well to uricase activities in vivo in the plasma of healthy rats; the half-life of the modified uricase was estimated without interference from the unmodified uricase leftover in the sample and was nearly 100-fold longer than that of the unmodified uricase. Using a model of the elimination of uric acid in vivo taking into account of uricase pharmacokinetics and xanthine inhibition, modeled pharmacodynamics supported that the half-life of uricase and its susceptibility to xanthine are crucial for the pharmacological significance of uricase. Hence, this practical system is desirable for doing preliminary screening of formulations of engineered uricases as potential drugs for hyperuricemia.

Effects of modification of amino groups with poly(ethylene glycol) on a recombinant uricase from Bacillus fastidiosus.

After modification with monomethoxyl-poly(ethylene glycol)-5000, a recombinant intracellular uricase from Bacillus fastidiosus ATCC 29604 showed residual activity of about 65%, a thermo-inactivation half-life >85 h, a circulating half-life about 20 h in rats in vivo, consistent effects of common cations, and consistent optima for reaction temperature and pH. Thus, this uricase can be formulated via modification with monomethoxyl-poly(ethylene glycol).

A study of rasburicase for the management of hyperuricemia in pediatric patients with newly diagnosed hematologic malignancies at high risk for tumor lysis syndrome.

Tumor lysis syndrome (TLS), including hyperuricemia, is a frequent serious complication in patients with hematologic malignancies. This study in Japanese patients evaluated the efficacy, safety, and pharmacokinetic profile of rasburicase in pediatric patients with hematologic malignancies. Patients aged <18 years at high risk for TLS, with newly diagnosed hematologic malignancies, were randomized to intravenous rasburicase 0.15 mg/kg/day (n = 15) or 0.20 mg/kg/day (n = 15) for 5 days. Chemotherapy was started 4-24 h after the first rasburicase dose. Response was defined as a reduction in plasma uric acid to < or = 6.5 mg/dL (patients <13 years) or < or = 7.5 mg/dL (patients > or = 13 years) by 48 h after the first administration, lasting until 24 h after the final administration. Response rates were 93.3 and 100% with rasburicase 0.15 and 0.20 mg/kg/day, respectively. Uric acid levels declined rapidly within 4 h of starting rasburicase administration in both groups. Most adverse events were related to the underlying chemotherapy regimens. Two hypersensitivity reactions, including grade 1/2 pruritus, were considered to be related to rasburicase. Rasburicase is effective and well tolerated for the management of hyperuricemia in Japanese pediatric patients at high risk of developing TLS.

Resultado del tratamiento hipouricemiante de la gota en ancianos / Results of hypouricemic treatment of gout in the elderly

Algunos autores recomiendan un abordaje conservador del tratamiento de la gota en ancianos. Sin embargo, en la bibliografía no se dispone de series publicadas al respecto.Se han revisado los datos procedentes de una cohorte de pacientes con gota. Se seleccionó a 52 de 242 pacientes (21 por ciento) con edad igual o superior a los 70 años. Evaluamos las reducciones de urato sérico, número de ataques y tofos, el porcentaje de pacientes con urato inferior a 6 mg/dl y la variación de la función renal. Se recogieron datos sobre comorbilidad y efectos adversos previos por tratamiento con antiinflamatorios no esteroides y durante el tratamiento hipouricemiante. Asimismo, se les remitió una encuesta anónima sencilla de cumplimentar.De los 49 pacientes vivos, contestaron al cuestionario 35 (71,4 por ciento), cuyos datos fueron analizados. El 87 por ciento presentaba comorbilidad. La uricemia se redujo de 8,84 ñ 1,41 a 4,70 ñ 0,91 mg/dl (p < 0,001), un 97 por ciento con cifras de urato inferiores a 6 mg/dl, y el número de ataques por paciente y año, de 3,14 ñ 1,35 a 0,17 ñ 0,3 (p < 0,001). Once de 13 (85 por ciento) de los pacientes con tofos no los tenían al final del seguimiento. El aclaramiento de creatinina pasó de 63 ñ 27 a 70 ñ 26 ml (p = 0,037). Diez pacientes habían presentado efectos adversos graves previos al tratamiento, mientras que sólo cuatro presentaron efectos adversos leves durante el tratamiento. El 66 por ciento refería encontrarse mucho mejor, el 31 por ciento mejor y sólo el 3 por ciento igual o peor. El 97 por ciento creía que su problema había sido mejor controlado desde la consulta especializada.Concluimos que la gota en ancianos, más compleja por la presencia de comorbilidad, debe ser abordada por especialistas, y los resultados, desde los puntos de vista objetivo y de los pacientes, fueron muy satisfactorios (AU)

Recombinant urate oxidase for the prophylaxis or treatment of hyperuricemia in patients With leukemia or lymphoma.

PURPOSE: To improve the control of hyperuricemia in patients with leukemia or lymphoma, we tested a newly developed uricolytic agent, recombinant urate oxidase (SR29142; Rasburicase; Sanofi-Synthelabo, Inc, Paris, France), which catalyzes the oxidation of uric acid to allantoin, a highly water-soluble metabolite readily excreted by the kidneys. PATIENTS AND METHODS: We administered Rasburicase intravenously, at 0.15 or 0.20 mg/kg, for 5 to 7 consecutive days to 131 children, adolescents, and young adults with newly diagnosed leukemia or lymphoma, who either presented with abnormally high plasma uric acid concentrations or had large tumor cell burdens. Blood levels of uric acid, creatinine, phosphorus, and potassium were measured daily. The pharmacokinetics of Rasburicase, the urinary excretion rate of allantoin, and antibodies to Rasburicase were also studied. RESULTS: At either dosage, the recombinant enzyme produced a rapid and sharp decrease in plasma uric acid concentrations in all patients. The median level decreased by 4 hours after treatment, from 9.7 to 1 mg/dL (P =.0001), in the 65 patients who presented with hyperuricemia, and from 4.3 to 0.5 mg/dL (P =.0001) in the remaining 66 patients. Despite cytoreductive chemotherapy, plasma uric acid concentrations remained low throughout the treatment (daily median level, 0.5 mg/dL). The urinary excretion rate of allantoin increased during Rasburicase treatment, peaking on day 3. Serum phosphorus concentrations did not change significantly during the first 3 days of treatment, decreased significantly by day 4 in patients presenting with hyperuricemia (P =.0003), and fell within the normal range in all patients by 48 hours after treatment. Serum creatinine levels decreased significantly after 1 day of treatment in patients with or without hyperuricemia at diagnosis (P =.0003 and P =.02, respectively) and returned to normal range in all patients by day 6 of treatment. Toxicity was negligible, and none of the patients required dialysis. The mean plasma half-lives of the agent were 16.0 +/- 6.3 (SD) hours and 21.1 +/- 12.0 hours, respectively, in patients treated at dosages of 0.15 or 0.20 mg/kg. Seventeen of the 121 assessable patients developed antibodies to the enzyme. CONCLUSION: Rasburicase is safe and highly effective for the prophylaxis or treatment of hyperuricemia in patients with leukemia or lymphoma.

Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry.

A method for the determination of 15N enrichment and concentration of allantoin and uric acid simultaneously in urine using gas chromatography/mass spectrometry (GC/MS) is described. The urine samples contained [1,3-15N2] uric acid and its oxidation product allantoin. The uric acid and allantoin were isolated using an AG1-X8 (Cl-form) anion-exchange column and heated with a mixture containing 1:1 dimethylformamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). The tert-butyldimethylsilyl (TBDMS) derivatives of allantoin and uric acid formed were injected into a gas chromatograph interfaced with a mass spectrometer operated under electron impact ionization conditions. Isotope ratio measurements were made from the abundance of the M-57 ions at m/z 398, 399 and 400 for allantoin and at m/z 567 and 569 for uric acid. 15N2 allantoin (99 at.%) was produced from [1,3-15N2] uric acid by treatment with uricase and used as a standard. Quantitation of allantoin and uric acid was based on isotopic dilution by spiking the urine sample with known quantities of 99 at.% [15N] uric acid and allantoin internal standards. The observed isotope ratio measurements from the prepared standards matched the theoretical values. Coefficients of variation in measurements of isotope ratio and concentration were 0.2 and 0.5%, respectively. The method was applied in a study to measure the urinary recovery of [1,3-15N2] uric acid continuously infused for 8-10 h into the blood of four sheep each on two occasions. Within 24 h, 65.9 +/- 9.1% of the tracer was excreted in the urine unchanged. Little was converted into allantoin (approximately 7% of the dose). The total recovery (5 days) of the infused tracer averaged 69.5 +/- 7.6% as uric acid and 76.8 +/- 9.3% as the sum of uric acid and allantoin. Uricase activities in plasma, liver and kidney of sheep were also measured using [1,3-15N2] uric acid as a substrate. Uricase activity was estimated to be 0.6 mU g-1 wet tissue in the liver and there appeared to be none in plasma and kidney. The low uricase activities in sheep tissues appeared to explain the limited conversion of the intravenously administered [15N] uric acid to allantoin but did not explain the large quantities of allantoin excreted in urine (8.96 +/- 0.86 and 1.36 +/- 0.25 mmol d-1 for allantoin and uric acid, respectively). The GC/MS method for the determination of 15N enrichment and concentration of allantoin and uric acid in urine is accurate and precise and provides a useful tool for studies on uric acid and allantoin metabolism.

Development of an assay method for purine catabolic enzymes in the mouse and its adaptation for use on an autoanalyzer.

An assay method has been developed for the purine catabolic enzymes adenosylhomocysteinase, adenosine deaminase (ADA), purine-nucleoside phosphorylase (PNP), and urate oxidase in mice. The assay links H2O2 produced during purine catabolism to the production of a dye complex. The assay method has been developed for ADA and PNP in erythrocytes and for all four enzymes in liver. The assay is cheap, sensitive, and easy to perform. The dye complex absorbs in the visible range, negating the need for an expensive ultraviolet spectrophotometer and allowing the use of an autoanalyzer.

Influence of triglycerides and urate on methods for determination of fructosamine.

The original fructosamine assay, based on reduction of NBT, has been modified and marketed as a kit. The development includes addition of detergents, uricase, change of the buffer and NBT concentrations, and use of a new calibrator. We report the effects of these changes on the measured values of fructosamine in lipemic and uricemic sera. A secondary calibrator produced from pooled serum and stabilized by removal of glucose was used to calibrate the original fructosamine assay and enhance transferability of results. There was no correlation between the difference between the results obtained by the two procedures and the concentration of urate or triglycerides. The development of colour was faster with the original method. It is suggested that measurements of fructosamine by either method are practically equivalent. However, the use of polylysine as a calibrator is advantageous. It is suggested that the generic term S-Glycated proteins is used for the measurand rather than S-Fructosamine which in effect has become a commercial term.

Adapting the uricase/peroxidase procedure for plasma urate to reduce interference due to haemolysis, icterus or lipaemia.

The estimation of serum urate by uricase/peroxidase procedures involving single point bichromatic absorbance monitoring gives inaccurate results with samples which are haemolysed, icteric or lipaemic. These inaccuracies can be greatly minimized by reducing the rate of chromogen formation and applying the assay in a kinetic mode.

Excretion of purine derivatives by ruminants: endogenous excretion, differences between cattle and sheep.

The endogenous urinary excretion of the purine derivatives allantoin, uric acid and xanthine plus hypoxanthine were measured in twenty-nine lambs, ten cattle (six steers, one cow and three preruminant calves) and four pigs. The sheep and mature cattle were nourished by intragastric infusion and the calves were given a milk-substitute. The pigs were fed on a purine-free diet. The excretion of total purine derivatives was substantially greater by the cattle, being 514 (SE 20.6) mumol/kg live weight (W)0.75 per d compared with 168 (SE 5.0) mumol/kg W0.75 per d by the sheep and 166 (SE 2.6) mumol/kg W0.75 per d by the pigs. Plasma from normally fed sheep, cows and pigs was incubated with either xanthine or uric acid. Sheep and pig plasma had no xanthine oxidase (EC 1.2.3.2) activity whereas plasma from cattle did. Uricase (EC 1.7.3.3) was not present in plasma of cattle and pigs and appeared to be present in trace amounts only in sheep plasma. It is suggested that the species differences in endogenous purine derivative excretion were probably due to the different profiles of xanthine oxidase activity in tissues and particularly in the blood. This is because a high xanthine oxidase activity would reduce the chance to recycle purines, by increasing the probability of degradation to compounds which could not be salvaged.

A radiochemical-high-performance liquid chromatographic assay for urate oxidase in human plasma.

Polyethylene glycol-modified urate oxidase (PEG-uricase) holds promise as a hypouricemic agent for treating gout and as an adjunct to cytolytic therapy of hematologic malignancies. Spectrophotometric assays of urate oxidase are not sensitive enough for pharmacokinetic evaluation of PEG-uricase in clinical trials. We have therefore developed a more sensitive radiochemical-HPLC assay for urate oxidase activity in untreated plasma, in which 14C in urate and in the reaction product, allantoin, is monitored in the uv detector effluent with a flow-through scintillation counter. The assay is linear with amount of enzyme and time of incubation and can detect less than 1 x 10(-5) U/ml uricase in plasma. The assay accounts for plasma samples of widely differing urate content.