The Experts below are selected from a list of 2007 Experts worldwide ranked by ideXlab platform
Michael S Hershfield - One of the best experts on this subject based on the ideXlab platform.
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treating gout with pegloticase a pegylated Urate Oxidase provides insight into the importance of uric acid as an antioxidant in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Michael S Hershfield, Nancy J Ganson, Susan J Kelly, Edna Scarlett, Jackson L Roberts, Ines Santisteban, Denise Jaggers, John S SundyAbstract:A high plasma Urate concentration (PUA), related to loss of Urate Oxidase in evolution, is postulated to protect humans from oxidative injury. This hypothesis has broad clinical relevance, but support rests largely on in vitro data and epidemiologic associations. Pegloticase therapy generates H2O2 while depleting Urate, offering an in vivo test of the antioxidant hypothesis. We show that erythrocytes can efficiently eliminate H2O2 derived from Urate oxidation to prevent cell injury in vitro; during therapy, disulfide-linked peroxiredoxin 2 dimer did not accumulate in red blood cells, indicating that their perOxidase capacity was not exceeded. To assess oxidative stress, we monitored F2-Isoprostanes (F2-IsoPs) and protein carbonyls (PC), products of arachidonic acid and protein oxidation, in plasma of 26 refractory gout patients receiving up to five infusions of pegloticase at 3-wk intervals. At baseline, PUA was markedly elevated in all patients, and plasma F2-IsoP concentration was elevated in most. Pegloticase infusion rapidly lowered mean PUA to ≤1 mg/dL in all patients, and PUA remained low in 16 of 21 patients who completed treatment. F2-IsoP levels did not correlate with PUA and did not increase during 15 wk of sustained Urate depletion. There also was no significant change in the levels of plasma PC. Because refractory gout is associated with high oxidative stress in spite of high PUA, and profoundly depleting uric acid did not increase lipid or protein oxidation, we conclude that Urate is not a major factor controlling oxidative stress in vivo.
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pharmacokinetics and pharmacodynamics of intravenous pegylated recombinant mammalian Urate Oxidase in patients with refractory gout
Arthritis & Rheumatism, 2007Co-Authors: John S Sundy, Nancy J Ganson, Susan J Kelly, Edna Scarlett, Claudia Rehrig, William Huang, Michael S HershfieldAbstract:Objective To evaluate the efficacy, immunogenicity, and tolerability of intravenous (IV) PEGylated recombinant mammalian Urate Oxidase (PEG-uricase) for the treatment of severe gout. Methods Single infusions of PEG-uricase (at doses ranging from 0.5 mg to 12 mg) were administered to 24 patients (6 cohorts of 4 patients each) in a phase I clinical trial. Plasma uricase activity (pUox), the plasma Urate concentration (pUAc), and the uric acid–to-creatinine ratio (UAc:Cr) in urine were monitored for 21 days after dosing. Adverse events and the IgG antibody response to PEG-uricase were followed up for 35 days. Results All patients completed the trial. Maximum pUox was linearly related to the IV dose of PEG-uricase, the area under the curve (AUC) value increased linearly (up to a dose of 8 mg), and the pUox half-life was 6.4–13.8 days. After doses of 4–12 mg, the pUAc fell within 24–72 hours, from a mean ± SD value of 11.1 ± 0.6 mg/dl to 1.0 ± 0.5 mg/dl; the AUC value for the pUAc was equivalent to maintaining the pUAc at 1.2–4.7 mg/dl for 21 days postinfusion. The UAc:Cr ratio in urine fell in parallel with the pUAc. IgG antibodies to PEG-uricase, mostly IgG2 and specific for PEG, developed in 9 patients, who had more rapid enzyme clearance but no allergic reactions. All adverse events were mild to moderate, with gout flares being most common. Conclusion The bioavailability, efficacy, and tolerability of IV PEG-uricase were greater than the bioavailability, efficacy, and tolerability observed in a previous phase I trial of subcutaneous PEG-uricase. Infusing 4–12 mg of PEG-uricase every 2–4 weeks should maintain the pUAc well below the therapeutic target of 6 mg/dl and greatly reduce renal uric acid excretion. This treatment could be effective in depleting expanded tissue Urate stores in patients with chronic or tophaceous gout.
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control of hyperuricemia in subjects with refractory gout and induction of antibody against poly ethylene glycol peg in a phase i trial of subcutaneous pegylated Urate Oxidase
Arthritis Research & Therapy, 2005Co-Authors: Nancy J Ganson, John S Sundy, Susan J Kelly, Edna Scarlett, Michael S HershfieldAbstract:PEG-modified recombinant mammalian Urate Oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.
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Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated Urate Oxidase
Arthritis Research & Therapy, 2005Co-Authors: Nancy J Ganson, John S Sundy, Susan J Kelly, Edna Scarlett, Michael S HershfieldAbstract:PEG-modified recombinant mammalian Urate Oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.
John S Sundy - One of the best experts on this subject based on the ideXlab platform.
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treating gout with pegloticase a pegylated Urate Oxidase provides insight into the importance of uric acid as an antioxidant in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Michael S Hershfield, Nancy J Ganson, Susan J Kelly, Edna Scarlett, Jackson L Roberts, Ines Santisteban, Denise Jaggers, John S SundyAbstract:A high plasma Urate concentration (PUA), related to loss of Urate Oxidase in evolution, is postulated to protect humans from oxidative injury. This hypothesis has broad clinical relevance, but support rests largely on in vitro data and epidemiologic associations. Pegloticase therapy generates H2O2 while depleting Urate, offering an in vivo test of the antioxidant hypothesis. We show that erythrocytes can efficiently eliminate H2O2 derived from Urate oxidation to prevent cell injury in vitro; during therapy, disulfide-linked peroxiredoxin 2 dimer did not accumulate in red blood cells, indicating that their perOxidase capacity was not exceeded. To assess oxidative stress, we monitored F2-Isoprostanes (F2-IsoPs) and protein carbonyls (PC), products of arachidonic acid and protein oxidation, in plasma of 26 refractory gout patients receiving up to five infusions of pegloticase at 3-wk intervals. At baseline, PUA was markedly elevated in all patients, and plasma F2-IsoP concentration was elevated in most. Pegloticase infusion rapidly lowered mean PUA to ≤1 mg/dL in all patients, and PUA remained low in 16 of 21 patients who completed treatment. F2-IsoP levels did not correlate with PUA and did not increase during 15 wk of sustained Urate depletion. There also was no significant change in the levels of plasma PC. Because refractory gout is associated with high oxidative stress in spite of high PUA, and profoundly depleting uric acid did not increase lipid or protein oxidation, we conclude that Urate is not a major factor controlling oxidative stress in vivo.
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pharmacokinetics and pharmacodynamics of intravenous pegylated recombinant mammalian Urate Oxidase in patients with refractory gout
Arthritis & Rheumatism, 2007Co-Authors: John S Sundy, Nancy J Ganson, Susan J Kelly, Edna Scarlett, Claudia Rehrig, William Huang, Michael S HershfieldAbstract:Objective To evaluate the efficacy, immunogenicity, and tolerability of intravenous (IV) PEGylated recombinant mammalian Urate Oxidase (PEG-uricase) for the treatment of severe gout. Methods Single infusions of PEG-uricase (at doses ranging from 0.5 mg to 12 mg) were administered to 24 patients (6 cohorts of 4 patients each) in a phase I clinical trial. Plasma uricase activity (pUox), the plasma Urate concentration (pUAc), and the uric acid–to-creatinine ratio (UAc:Cr) in urine were monitored for 21 days after dosing. Adverse events and the IgG antibody response to PEG-uricase were followed up for 35 days. Results All patients completed the trial. Maximum pUox was linearly related to the IV dose of PEG-uricase, the area under the curve (AUC) value increased linearly (up to a dose of 8 mg), and the pUox half-life was 6.4–13.8 days. After doses of 4–12 mg, the pUAc fell within 24–72 hours, from a mean ± SD value of 11.1 ± 0.6 mg/dl to 1.0 ± 0.5 mg/dl; the AUC value for the pUAc was equivalent to maintaining the pUAc at 1.2–4.7 mg/dl for 21 days postinfusion. The UAc:Cr ratio in urine fell in parallel with the pUAc. IgG antibodies to PEG-uricase, mostly IgG2 and specific for PEG, developed in 9 patients, who had more rapid enzyme clearance but no allergic reactions. All adverse events were mild to moderate, with gout flares being most common. Conclusion The bioavailability, efficacy, and tolerability of IV PEG-uricase were greater than the bioavailability, efficacy, and tolerability observed in a previous phase I trial of subcutaneous PEG-uricase. Infusing 4–12 mg of PEG-uricase every 2–4 weeks should maintain the pUAc well below the therapeutic target of 6 mg/dl and greatly reduce renal uric acid excretion. This treatment could be effective in depleting expanded tissue Urate stores in patients with chronic or tophaceous gout.
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control of hyperuricemia in subjects with refractory gout and induction of antibody against poly ethylene glycol peg in a phase i trial of subcutaneous pegylated Urate Oxidase
Arthritis Research & Therapy, 2005Co-Authors: Nancy J Ganson, John S Sundy, Susan J Kelly, Edna Scarlett, Michael S HershfieldAbstract:PEG-modified recombinant mammalian Urate Oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.
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Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated Urate Oxidase
Arthritis Research & Therapy, 2005Co-Authors: Nancy J Ganson, John S Sundy, Susan J Kelly, Edna Scarlett, Michael S HershfieldAbstract:PEG-modified recombinant mammalian Urate Oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.
Nancy J Ganson - One of the best experts on this subject based on the ideXlab platform.
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treating gout with pegloticase a pegylated Urate Oxidase provides insight into the importance of uric acid as an antioxidant in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Michael S Hershfield, Nancy J Ganson, Susan J Kelly, Edna Scarlett, Jackson L Roberts, Ines Santisteban, Denise Jaggers, John S SundyAbstract:A high plasma Urate concentration (PUA), related to loss of Urate Oxidase in evolution, is postulated to protect humans from oxidative injury. This hypothesis has broad clinical relevance, but support rests largely on in vitro data and epidemiologic associations. Pegloticase therapy generates H2O2 while depleting Urate, offering an in vivo test of the antioxidant hypothesis. We show that erythrocytes can efficiently eliminate H2O2 derived from Urate oxidation to prevent cell injury in vitro; during therapy, disulfide-linked peroxiredoxin 2 dimer did not accumulate in red blood cells, indicating that their perOxidase capacity was not exceeded. To assess oxidative stress, we monitored F2-Isoprostanes (F2-IsoPs) and protein carbonyls (PC), products of arachidonic acid and protein oxidation, in plasma of 26 refractory gout patients receiving up to five infusions of pegloticase at 3-wk intervals. At baseline, PUA was markedly elevated in all patients, and plasma F2-IsoP concentration was elevated in most. Pegloticase infusion rapidly lowered mean PUA to ≤1 mg/dL in all patients, and PUA remained low in 16 of 21 patients who completed treatment. F2-IsoP levels did not correlate with PUA and did not increase during 15 wk of sustained Urate depletion. There also was no significant change in the levels of plasma PC. Because refractory gout is associated with high oxidative stress in spite of high PUA, and profoundly depleting uric acid did not increase lipid or protein oxidation, we conclude that Urate is not a major factor controlling oxidative stress in vivo.
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pharmacokinetics and pharmacodynamics of intravenous pegylated recombinant mammalian Urate Oxidase in patients with refractory gout
Arthritis & Rheumatism, 2007Co-Authors: John S Sundy, Nancy J Ganson, Susan J Kelly, Edna Scarlett, Claudia Rehrig, William Huang, Michael S HershfieldAbstract:Objective To evaluate the efficacy, immunogenicity, and tolerability of intravenous (IV) PEGylated recombinant mammalian Urate Oxidase (PEG-uricase) for the treatment of severe gout. Methods Single infusions of PEG-uricase (at doses ranging from 0.5 mg to 12 mg) were administered to 24 patients (6 cohorts of 4 patients each) in a phase I clinical trial. Plasma uricase activity (pUox), the plasma Urate concentration (pUAc), and the uric acid–to-creatinine ratio (UAc:Cr) in urine were monitored for 21 days after dosing. Adverse events and the IgG antibody response to PEG-uricase were followed up for 35 days. Results All patients completed the trial. Maximum pUox was linearly related to the IV dose of PEG-uricase, the area under the curve (AUC) value increased linearly (up to a dose of 8 mg), and the pUox half-life was 6.4–13.8 days. After doses of 4–12 mg, the pUAc fell within 24–72 hours, from a mean ± SD value of 11.1 ± 0.6 mg/dl to 1.0 ± 0.5 mg/dl; the AUC value for the pUAc was equivalent to maintaining the pUAc at 1.2–4.7 mg/dl for 21 days postinfusion. The UAc:Cr ratio in urine fell in parallel with the pUAc. IgG antibodies to PEG-uricase, mostly IgG2 and specific for PEG, developed in 9 patients, who had more rapid enzyme clearance but no allergic reactions. All adverse events were mild to moderate, with gout flares being most common. Conclusion The bioavailability, efficacy, and tolerability of IV PEG-uricase were greater than the bioavailability, efficacy, and tolerability observed in a previous phase I trial of subcutaneous PEG-uricase. Infusing 4–12 mg of PEG-uricase every 2–4 weeks should maintain the pUAc well below the therapeutic target of 6 mg/dl and greatly reduce renal uric acid excretion. This treatment could be effective in depleting expanded tissue Urate stores in patients with chronic or tophaceous gout.
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control of hyperuricemia in subjects with refractory gout and induction of antibody against poly ethylene glycol peg in a phase i trial of subcutaneous pegylated Urate Oxidase
Arthritis Research & Therapy, 2005Co-Authors: Nancy J Ganson, John S Sundy, Susan J Kelly, Edna Scarlett, Michael S HershfieldAbstract:PEG-modified recombinant mammalian Urate Oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.
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Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated Urate Oxidase
Arthritis Research & Therapy, 2005Co-Authors: Nancy J Ganson, John S Sundy, Susan J Kelly, Edna Scarlett, Michael S HershfieldAbstract:PEG-modified recombinant mammalian Urate Oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.
Takafumi Itoh - One of the best experts on this subject based on the ideXlab platform.
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identification of quasi stable water molecules near the thr73 lys13 catalytic diad of bacillus sp tb 90 Urate Oxidase by x ray crystallography with controlled humidity
Journal of Biochemistry, 2021Co-Authors: Takao Hibi, Takafumi ItohAbstract:Urate Oxidases (UOs) catalyze the cofactor-independent oxidation of uric acid, and an extensive water network in the active site has been suggested to play an essential role in the catalysis. For our present analysis of the structure and function of the water network, the crystal qualities of Bacillus sp. TB-90 Urate Oxidase were improved by controlled dehydration using the humid air and glue-coating method. After the dehydration, the P21212 crystals were transformed into the I222 space group, leading to an extension of the maximum resolution to 1.42 A. The dehydration of the crystals revealed a significant change in the five-water-molecules' binding mode in the vicinity of the catalytic diad, indicating that these molecules are quasi-stable. The pH profile analysis of log(kcat) gave two pKa values: pKa1 at 6.07 ± 0.07 and pKa2 at 7.98 ± 0.13. The site-directed mutagenesis of K13, T73 and N276 involved in the formation of the active-site water network revealed that the activities of these mutant variants were significantly reduced. These structural and kinetic data suggest that the five quasi-stable water molecules play an essential role in the catalysis of the cofactor-independent Urate oxidation by reducing the energy penalty for the substrate-binding or an on-off switching for the proton-relay rectification.
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Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity
2016Co-Authors: Takao Hibi, Asami Kume, Akie Kawamura, Takafumi Itoh, Harumi Fukada, Yoshiaki NishiyaAbstract:Bacillus sp. TB-90 Urate Oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879–3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two β-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ∼20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cβ distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277–300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins
James A. Fraser - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the Complete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoformans
2016Co-Authors: Russel I. Lee, Gaseene Sebetso, Thi H. N. Doan, Carl A. Morrow, Rebecca Allen, Liting Yang, Ross Blundell, Edmund Y. L. Lui, James A. FraserAbstract:Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the Urate Oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (Urate Oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants ’ ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 wer
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Characterization of the complete uric acid degradation pathway in the fungal pathogen Cryptococcus neoformans
PLOS ONE, 2013Co-Authors: Liting Yang, Gaseene Sebetso, Thi H. N. Doan, Ross D. Blundell, Carl A. Morrow, Rebecca Allen, James A. FraserAbstract:Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the Urate Oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (Urate Oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed.
