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Leslie Z Benet - One of the best experts on this subject based on the ideXlab platform.
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DMD #13334 1 Studies on the Metabolism of Tolmetin to the Chemically Reactive Acyl-Coenzyme A Thioester Intermediate in Rats
2014Co-Authors: Jørgen Olsen, Leslie Z Benet, Christian Skonberg, Inga Bjørnsdottir, Ulrik Sidenius, Steen Honoré Hansen, Novo Nordisk A/sAbstract:2 Running Title: Metabolic Activation of Tolmetin Corresponding author: Jørgen Olsen, Ph.D
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studies on the metabolism of Tolmetin to the chemically reactive acyl coenzyme a thioester intermediate in rats
Drug Metabolism and Disposition, 2007Co-Authors: Jørgen Olsen, Leslie Z Benet, Christian Skonberg, Inga Bjørnsdottir, Ulrik Sidenius, Steen Honoré HansenAbstract:Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of Tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), Tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites Tolmetin-taurine conjugate (Tol-Tau) and Tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid-treated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.
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Studies on the metabolism of Tolmetin to the chemically reactive acyl-coenzyme A thioester intermediate in rats. Drug Metab Dispos 35:758–764.
2007Co-Authors: Jørgen Olsen, Leslie Z Benet, Christian Skonberg, Inga Bjørnsdottir, Ulrik Sidenius, Steen Honoré HansenAbstract:ABSTRACT: Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of Tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), Tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites Tolmetin-taurine conjugate (Tol-Tau) and Tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acidtreated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins
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synthesis and mass spectrometric characterization of human serum albumins modified by covalent binding of two non steroidal anti inflammatory drugs Tolmetin and zomepirac
Biochemical Journal, 1995Co-Authors: Parnian Ziaamirhosseini, Antony F Mcdonagh, A Ding, Alma L Burlingame, Leslie Z BenetAbstract:Human serum albumins modified by covalently bound Tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS. Three binding sites were common to both products, i.e. lysine-195, -199 and -351. Three further modified residues were identified for the Tolmetin-albumin product, i.e. aspartic acid 1, and lysine-524 and -536.
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reactivity of Tolmetin glucuronide with human serum albumin identification of binding sites and mechanisms of reaction by tandem mass spectrometry
Drug Metabolism and Disposition, 1995Co-Authors: A Ding, Antony F Mcdonagh, Parnian Ziaamirhosseini, Alma L Burlingame, Leslie Z BenetAbstract:The structures of adducts formed from in vitro incubation of a drug (Tolmetin) glucuronide (TG) and human serum albumin (HSA), and the preferred binding sites on this protein were determined by mass spectrometry. In addition, the concentration dependence of covalent modification of HSA by TG was studied at three different concentration ratios of TG to HSA. Protein adducts were enzymatically digested and peptide fragments were separated by HPLC. Tolmetin-containing peptides (indicated by absorbance at 313 nm) were analyzed by liquid secondary-ion mass spectrometry, continuous flow-fast atom bombardment mass spectrometry, and collision-induced dissociation using a four-sector tandem mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, and in selected cases by Edman sequencing. The identified peptides contained Tolmetin linked covalently via a glucuronic acid to a protein lysine group (lysine 199 and to a lesser extent lysines 195 and 525) or Tolmetin directly linked to lysines (lysines 199 and 541), serines (serines 220, 232, and 480), or arginines (arginine 222). In addition, there was indirect evidence for binding of TG to lysine 541, and binding of Tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Our data suggest, however, that the former, in which the glucuronic acid moiety of the acyl glucuronide is retained within the adducts, is favored at lower (closer to physiological) metabolite concentrations.
Santy Daya - One of the best experts on this subject based on the ideXlab platform.
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non steroidal anti inflammatory agents Tolmetin and sulindac attenuate quinolinic acid qa induced oxidative stress in primary hippocampal neurons and reduce qa induced spatial reference memory deficits in male wistar rats
Life Sciences, 2007Co-Authors: Amichand Dairam, Adrienne C Muller, Santy DayaAbstract:Abstract Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodegenerative disorders. In the present study, the possible neuroprotective properties of Tolmetin and sulindac were investigated using quinolinic acid (QA)-induced neurotoxicity as well as behavioral studies. QA, a metabolite of the tryptophan–kynurenine pathway, significantly induces lipid peroxidation, superoxide anion generation and decreases cell viability in primary hippocampal neurons established from one day old rat pups. However, co-incubation of the neurons with Tolmetin or sulindac markedly reduces oxidative stress and enhances cell viability. Animals were trained in a Morris water maze for four consecutive days and thereafter received 0.6 μmol of QA intrahippocampally. The animals were divided into groups and were treated with either Tolmetin or sulindac (5 mg/kg twice a day for five days). During test trials, the time taken for each rat to find the submerged platform was recorded over a period of two weeks. Animals were thereafter sacrificed and the hippocampi analyzed for protein carbonyl and glutathione content. The results show that both sulindac and Tolmetin reduce the QA-induced spatial memory deficit and sulindac treated animals respond better in the water maze compared to the Tolmetin treated animals. Both agents also reduce protein oxidation in rat hippocampus and attenuate the decrease in hippocampal glutathione content induced by QA. This study indicates that the antioxidant properties of Tolmetin and sulindac may be beneficial in the treatment of neurodegenerative disorders such as Alzheimer's disease.
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non steroidal anti inflammatory agents Tolmetin and sulindac inhibit liver tryptophan 2 3 dioxygenase activity and alter brain neurotransmitter levels
Life Sciences, 2006Co-Authors: Amichand Dairam, Edith Antunes, K S Saravanan, Santy DayaAbstract:Hepatic tryptophan 2,3-dioxygenase (TDO) is one of the rate-limiting enzymes in tryptophan catabolism and plays an important role in regulating the physiological flux of tryptophan into relevant metabolic pathways. In this study, we determined the effect of the non-steroidal anti-inflammatory agents, Tolmetin and sulindac, on rat liver TDO activity and the subsequent changes in the hippocampal and striatal neurotransmitter levels. The amount of melatonin produced by the pineal gland was also measured using high performance liquid chromatography (HPLC). Treatment of rats with Tolmetin or sulindac (5 mg/kg/bd for 5 days) significantly inhibited liver TDO activity. The results show that whilst Tolmetin and sulindac increase serotonin levels in the hippocampus, these agents also significantly reduce dopamine levels in the striatum. Tolmetin, but not sulindac, increased the amount of melatonin produced by the pineal gland. The results of this study suggest that whilst Tolmetin and sulindac may be beneficial for patients suffering from depression, these agents also have the potential to induce adverse effects in patients suffering with neurological disorders such as Parkinson's disease.
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non steroidal anti inflammatory agents Tolmetin and sulindac attenuate oxidative stress in rat brain homogenate and reduce quinolinic acid induced neurodegeneration in rat hippocampal neurons
Metabolic Brain Disease, 2006Co-Authors: Amichand Dairam, Prakash Chetty, Santy DayaAbstract:Alzheimer's disease (AD) is the most common form of neurodegenerative disease in the elderly. Anti-inflammatory agents have been shown to be beneficial in preventing neurodegenerative disorders such as AD. In this study we investigated the possible antioxidant and neuroprotective properties of two non-steroidal anti-inflammatory drugs (NSAIDS), Tolmetin and sulindac, using quinolinic acid (QA)-induced neurotoxicity as a model. We used the thiobarbituric acid assay to measure the extent of lipid peroxidation and the nitroblue tetrazolium assay to measure the superoxide anion generated in rat brain homogenate. QA (1 mM) induced lipid peroxidation in rat brain homogenate was significantly curtailed by co-treatment of the homogenate with Tolmetin and/or sulindac. Tolmetin and sulindac both reduced the generation of superoxide anions by the known neurotoxin, potassium cyanide (KCN). Intrahippocampal injections of QA induced neurotoxicity in rat hippocampus. N-Methyl-D-Aspartate (NMDA) receptor counts were conducted do give an indication of the amount protection offered by the NSAIDS. QA drastically reduced the number of NMDA binding sites by approximately 37%. This sharp decrease was considerably attenuated by the pre-treatment of the rats with Tolmetin and sulindac (5 mg/kg/bd for five days). This study shows the antioxidant and neuroprotective properties of Tolmetin and sulindac and hereby postulates that these drugs have important implications in the prevention or treatment of neurodegenerative diseases such as AD.
Amichand Dairam - One of the best experts on this subject based on the ideXlab platform.
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non steroidal anti inflammatory agents Tolmetin and sulindac attenuate quinolinic acid qa induced oxidative stress in primary hippocampal neurons and reduce qa induced spatial reference memory deficits in male wistar rats
Life Sciences, 2007Co-Authors: Amichand Dairam, Adrienne C Muller, Santy DayaAbstract:Abstract Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodegenerative disorders. In the present study, the possible neuroprotective properties of Tolmetin and sulindac were investigated using quinolinic acid (QA)-induced neurotoxicity as well as behavioral studies. QA, a metabolite of the tryptophan–kynurenine pathway, significantly induces lipid peroxidation, superoxide anion generation and decreases cell viability in primary hippocampal neurons established from one day old rat pups. However, co-incubation of the neurons with Tolmetin or sulindac markedly reduces oxidative stress and enhances cell viability. Animals were trained in a Morris water maze for four consecutive days and thereafter received 0.6 μmol of QA intrahippocampally. The animals were divided into groups and were treated with either Tolmetin or sulindac (5 mg/kg twice a day for five days). During test trials, the time taken for each rat to find the submerged platform was recorded over a period of two weeks. Animals were thereafter sacrificed and the hippocampi analyzed for protein carbonyl and glutathione content. The results show that both sulindac and Tolmetin reduce the QA-induced spatial memory deficit and sulindac treated animals respond better in the water maze compared to the Tolmetin treated animals. Both agents also reduce protein oxidation in rat hippocampus and attenuate the decrease in hippocampal glutathione content induced by QA. This study indicates that the antioxidant properties of Tolmetin and sulindac may be beneficial in the treatment of neurodegenerative disorders such as Alzheimer's disease.
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non steroidal anti inflammatory agents Tolmetin and sulindac inhibit liver tryptophan 2 3 dioxygenase activity and alter brain neurotransmitter levels
Life Sciences, 2006Co-Authors: Amichand Dairam, Edith Antunes, K S Saravanan, Santy DayaAbstract:Hepatic tryptophan 2,3-dioxygenase (TDO) is one of the rate-limiting enzymes in tryptophan catabolism and plays an important role in regulating the physiological flux of tryptophan into relevant metabolic pathways. In this study, we determined the effect of the non-steroidal anti-inflammatory agents, Tolmetin and sulindac, on rat liver TDO activity and the subsequent changes in the hippocampal and striatal neurotransmitter levels. The amount of melatonin produced by the pineal gland was also measured using high performance liquid chromatography (HPLC). Treatment of rats with Tolmetin or sulindac (5 mg/kg/bd for 5 days) significantly inhibited liver TDO activity. The results show that whilst Tolmetin and sulindac increase serotonin levels in the hippocampus, these agents also significantly reduce dopamine levels in the striatum. Tolmetin, but not sulindac, increased the amount of melatonin produced by the pineal gland. The results of this study suggest that whilst Tolmetin and sulindac may be beneficial for patients suffering from depression, these agents also have the potential to induce adverse effects in patients suffering with neurological disorders such as Parkinson's disease.
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non steroidal anti inflammatory agents Tolmetin and sulindac attenuate oxidative stress in rat brain homogenate and reduce quinolinic acid induced neurodegeneration in rat hippocampal neurons
Metabolic Brain Disease, 2006Co-Authors: Amichand Dairam, Prakash Chetty, Santy DayaAbstract:Alzheimer's disease (AD) is the most common form of neurodegenerative disease in the elderly. Anti-inflammatory agents have been shown to be beneficial in preventing neurodegenerative disorders such as AD. In this study we investigated the possible antioxidant and neuroprotective properties of two non-steroidal anti-inflammatory drugs (NSAIDS), Tolmetin and sulindac, using quinolinic acid (QA)-induced neurotoxicity as a model. We used the thiobarbituric acid assay to measure the extent of lipid peroxidation and the nitroblue tetrazolium assay to measure the superoxide anion generated in rat brain homogenate. QA (1 mM) induced lipid peroxidation in rat brain homogenate was significantly curtailed by co-treatment of the homogenate with Tolmetin and/or sulindac. Tolmetin and sulindac both reduced the generation of superoxide anions by the known neurotoxin, potassium cyanide (KCN). Intrahippocampal injections of QA induced neurotoxicity in rat hippocampus. N-Methyl-D-Aspartate (NMDA) receptor counts were conducted do give an indication of the amount protection offered by the NSAIDS. QA drastically reduced the number of NMDA binding sites by approximately 37%. This sharp decrease was considerably attenuated by the pre-treatment of the rats with Tolmetin and sulindac (5 mg/kg/bd for five days). This study shows the antioxidant and neuroprotective properties of Tolmetin and sulindac and hereby postulates that these drugs have important implications in the prevention or treatment of neurodegenerative diseases such as AD.
Gere S Dizerega - One of the best experts on this subject based on the ideXlab platform.
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Prevention of Adhesion Formation with Intraperitoneal Administration of Tolmetin and Hyaluronic Acid
Journal of investigative surgery : the official journal of the Academy of Surgical Research, 1997Co-Authors: Kathleen E. Rodgers, Wefki Girgis, Douglas B. Johns, Gere S DizeregaAbstract:Adhesion formation after peritoneal surgery is a major source of postoperative complications and pain. Previous studies showed that intraperitoneal administration of the nonsteroidal anti-inflammatory drug Tolmetin reduced adhesion formation after two types of peritoneal surgery. The effect of Tolmetin combined with hyaluronic acid (HA), a high-molecular-weight glucosaminoglycan found in the extracellular matrix, on the formation of adhesions was examined. In this study, the effect of Tolmetin in HA on adhesion formation was evaluated in a standardized rabbit model. The medicament was administered intraperitoneally at the end of surgery. One week after surgery, a second laparotomy was performed and the extent of adhesion formation was determined. A range of molecular weights (7.5 x 10(5)-2 x 10(6) Da) and viscosities (1000-25,000 centapoise) of HA in combination with Tolmetin was effective in reducing adhesion formation. However, low viscosity HA solutions in combination with Tolmetin, 0.5-2.0 mg/mL, were most efficacious in reducing adhesion formation. These data suggest that HA, in combination with Tolmetin, acts as an effective carrier to reduce adhesion formation in the abdominal cavity after surgery.
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evaluation of models methodologies devices and new surgical concepts time dependent effect of Tolmetin sodium in a rabbit uterine adhesion model
Journal of Investigative Surgery, 1994Co-Authors: David M Wiseman, Douglas B. Johns, Kathleen E. Rodgers, James W Huang, Gere S DizeregaAbstract:Tolmetin is a nonsteroidal anti-inflammatory drug (NSAID) that reduces adhesion formation in several animal models after a single intraperitoneal (i.p.) dose delivered at the time of surgery. We set out to determine the period during which Tolmetin could prevent adhesions. Adhesions were induced in New Zealand White rabbits (2-3 kg) by abrading the uterine horns and removing their mesouterine vasculature. Tolmetin sodium (1 mg/5 ml saline) was given at various times relative to the start of surgery as a single dose i.p. One week later adhesions were assessed using a standard scoring system (0 = no adhesions; 1 = light adhesions involving both uterine horns: 2 = more tenacious adhesions to bowel or bladder; 3 = tenacious adhesions to bowel and bladder partly immobilizing the uterus; 4 = completely fixed horns adherent to bowel and bladder). Scores were arranged in ascending rank order. Mean rank positions were calculated for each group and compared against controls (Dunnett's multiple comparison). Tolmetin...
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modulation of postsurgical cell infiltration and fibrinolytic activity by Tolmetin in two species
Journal of Surgical Research, 1994Co-Authors: Kathleen E. Rodgers, Wefki Girgis, Dolph D Ellefson, Gere S DizeregaAbstract:Previous studies showed that Tolmetin administered as a single instillate intraperitoneally at the end of surgery can reduce adhesion formation. In this report, studies on the mechanism by which this occurs were conducted. The effects of Tolmetin administered intraperitoneally on red (RBC) and white blood cell (WBC) number, macrophage and polymorphonuclear neutrophil infiltration, protease activity in lavage fluid, and the fibrinolytic activity of a biopsy of nonsurgical and traumatized peritoneal sidewall were examined. Tolmetin was shown to increase the number of RBC at one postoperative time point in rabbits, but not in rats. In addition, Tolmetin administration elevated the number of WBC harvested from the peritoneum predominantly through an increase in macrophage number. Administration of Tolmetin also modulated the level of protease and protease inhibitor activity in the lavage fluid harvested from the peritoneum. The most pronounced change was a decrease in the level of plasminogen activator inhibitor activity. In addition, acute administration of Tolmetin to rats elevated the level of fibrinolytic activity at the site of trauma as measured by in vitro cultures. In summary, intraperitoneal administration of Tolmetin, a nonsteroidal anti-inflammatory drug which reduces adhesion formation in both rats and rabbits, at the end of surgery modulated the number of WBCs in the peritoneal cavity and the protease and protease inhibitory activities present in the peritoneal lavage fluid and peritoneum after surgery.
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efficacy of Tolmetin sodium for adhesion prevention in rabbit and rat models
Journal of Surgical Research, 1994Co-Authors: Edmund K Legrand, Gere S Dizerega, Kathleen E. Rodgers, Wefki Girgis, Joseph D Campeau, Karen Struck, Timothy C KiorpesAbstract:Tolmetin sodium's efficacy in preventing primary adhesions was evaluated in two adhesion models each in two species: (1) rabbit uterine horn, (2) rat uterine horn, (3) rabbit peritoneal side wall, and (4) rat peritoneal side wall. In each model a single instillation of Tolmetin sodium solution into the peritoneal cavity at the time of surgery effectively reduced adhesion formation. This efficacy extended over a wide range of concentrations, volumes, and total dosages, and was similar in rabbits and rats. An aqueous solution of 1 mg/ml Tolmetin sodium in 5-15 ml in rabbits and in 3 ml in rats was consistently efficacious in reducing postoperative adhesion formation.
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peritoneal lavage fluid protease levels after in vivo administration of Tolmetin in hyaluronic acid
Journal of Surgical Research, 1993Co-Authors: Hiromasa Abe, Kathleen E. Rodgers, Wefki Girgis, Joseph D Campeau, Dolph D Ellefson, Gere S DizeregaAbstract:Tolmetin sodium in a hyaluronic acid carrier (Tolmetin-HA) was previously shown to reduce adhesion formation and alter the rate and extent to which wound repair cells enter the peritoneal cavity after surgery. In this study, the effect of Tolmetin-HA on the levels of protease activities present in lavage fluid from the peritoneal cavity was determined at various postsurgical times. Collagenase activity in peritoneal lavage fluid was suppressed at 6, 12, and 24 hr after administration of Tolmetin-HA in comparison to control. Elastase activity levels were biphasic with peak levels at 6 and 72 hr in lavage fluid from controls compared to peak levels at 6 and 48 hr in lavage fluid from treated rabbits. Plasminogen activator activity present in lavage fluid was significantly decreased at 48 hr after surgery in the Tolmetin-HA-treated rabbits compared to controls. However, no alteration in the level of plasminogen activator inhibitor activity was observed in either the Tolmetin-HA-treated or control rabbits. These data suggest that Tolmetin-HA treatment altered the levels of neutral protease activity present in the peritoneal cavity and may therefore effect the proteolytic potential in the peritoneal cavity after surgery.
Parnian Ziaamirhosseini - One of the best experts on this subject based on the ideXlab platform.
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synthesis and mass spectrometric characterization of human serum albumins modified by covalent binding of two non steroidal anti inflammatory drugs Tolmetin and zomepirac
Biochemical Journal, 1995Co-Authors: Parnian Ziaamirhosseini, Antony F Mcdonagh, A Ding, Alma L Burlingame, Leslie Z BenetAbstract:Human serum albumins modified by covalently bound Tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS. Three binding sites were common to both products, i.e. lysine-195, -199 and -351. Three further modified residues were identified for the Tolmetin-albumin product, i.e. aspartic acid 1, and lysine-524 and -536.
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reactivity of Tolmetin glucuronide with human serum albumin identification of binding sites and mechanisms of reaction by tandem mass spectrometry
Drug Metabolism and Disposition, 1995Co-Authors: A Ding, Antony F Mcdonagh, Parnian Ziaamirhosseini, Alma L Burlingame, Leslie Z BenetAbstract:The structures of adducts formed from in vitro incubation of a drug (Tolmetin) glucuronide (TG) and human serum albumin (HSA), and the preferred binding sites on this protein were determined by mass spectrometry. In addition, the concentration dependence of covalent modification of HSA by TG was studied at three different concentration ratios of TG to HSA. Protein adducts were enzymatically digested and peptide fragments were separated by HPLC. Tolmetin-containing peptides (indicated by absorbance at 313 nm) were analyzed by liquid secondary-ion mass spectrometry, continuous flow-fast atom bombardment mass spectrometry, and collision-induced dissociation using a four-sector tandem mass spectrometer, matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, and in selected cases by Edman sequencing. The identified peptides contained Tolmetin linked covalently via a glucuronic acid to a protein lysine group (lysine 199 and to a lesser extent lysines 195 and 525) or Tolmetin directly linked to lysines (lysines 199 and 541), serines (serines 220, 232, and 480), or arginines (arginine 222). In addition, there was indirect evidence for binding of TG to lysine 541, and binding of Tolmetin to arginine 521. Our results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Our data suggest, however, that the former, in which the glucuronic acid moiety of the acyl glucuronide is retained within the adducts, is favored at lower (closer to physiological) metabolite concentrations.
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enhanced covalent binding of Tolmetin to proteins in humans after multiple dosing
Clinical Pharmacology & Therapeutics, 1994Co-Authors: Parnian Ziaamirhosseini, Antony F Mcdonagh, Joseph C Ojingwa, Hildegard Spahnlangguth, Leslie Z BenetAbstract:The in vivo stability of Tolmetin-plasma protein adducts was characterized in six healthy human volunteers after a 400 mg single dose and after a multiple-dose regimen of 400 mg Tolmetin every 12 hours for 10 days. Although the mean +/- SD maximum bound concentration was only 2.72 +/- 0.98 ng drug/mg protein after a single dose, it was almost an order of magnitude higher after multiple dosing. The protein adduct exhibited an average half-life of 4.8 +/- 0.9 days in contrast to the much shorter 5-hour half-lives for Tolmetin and its glucuronide.
