Biomedical subjects
Publications and source records attributed to A Abuchowski.
An initial evaluation of PEG-bHb was performed using a modified hypovolemic shock model. PEG-bHb had a substantially longer intravascular half-life than native Hb and no measurable hemoglobinuria was observed in the canine. PEG-bHb allowed successful resuscitation with an oxygen carrying capacity of 14-22% over that of lactated Ringer's solution.
When exposed continuously to hyperoxia (100% O2, 760 Torr barometric pressure), rats pretreated with polyethylene glycol (PEG)-attached superoxide dismutase and catalase (PEG-SOD + PEG-CAT) lived longer (79.1 + 7.6 h) than rats pretreated with saline (60.7 +/- 2.1 h) or PEG-inactivated-SOD + PEG-inactivated-CAT (62.3 +/- 1.6 h). Rats pretreated with PEG-SOD + PEG-CAT also had less hyperoxia-induced acute oxidative edematous lung injury, as assessed by increases in lung oxidized glutathione (GSSG) contents, pleural effusions, and lung lavage albumin concentrations than saline-pretreated rats. Rats pretreated with the long-lived conjugates PEG-inactivated-SOD + PEG-inactivated-CAT or PEG-albumin also had decreased acute oxidative edematous lung injury compared with rats pretreated with PEG, SOD + CAT + PEG, SOD + CAT, or saline. In vitro studies suggested that PEG itself may have contributed to protection by scavenging hydroxyl radical (.OH) but not superoxide (O2-.) or H2O2. Compared with more effective endogenous (via preexposure to hypoxia) or exogenous (via liposomes) means for increasing lung antioxidant enzymes, PEG enzymes are less protective against lung injury from continuous hyperoxia.
Thirty-seven dogs with malignant lymphoma were treated with either polyethylene glycol conjugated (PEG) asparaginase alone (10-30 IU/kg intraperitoneally [IP] weekly--20 dogs) or PEG-asparaginase combined with one cycle of chemotherapy (vincristine, cyclophosphamide, methotrexate, and prednisone), followed by maintenance PEG-asparaginase (30 IU/kg, IP weekly--17 dogs). In the 20 dogs (eight were chemotherapy resistant) treated with PEG-asparaginase alone, seven had a complete response (CR), seven had a partial response (PR), five had no response (NR), and one was not evaluable (NE). The duration of response (CR + PR) ranged from 14 to 102 days (median, 48 days). In the eight chemotherapy-resistant dogs (seven were previously resistant to L-asparaginase) four had responses (one CR and three PR). In the 17 dogs treated with combined PEG-asparaginase and chemotherapy, 13 had a CR, two had a PR, and two had NR. None of the dogs had had prior chemotherapy, and the duration of response (CR + PR) ranged from 7 to 840+ days, with a median of 126+ days. Four dogs are still on maintenance PEG-asparaginase at 16+, 21+, 26+, and 28+ months. Toxicity consisted of death due to massive tumor breakdown (two dogs), disseminated intravascular coagulation (DIC--one dog), hypersensitivity reaction (one dog), vomiting (three dogs) and soft stools (three dogs). Four normal dogs were given very high doses of PEG-asparaginase (200 IU/kg and 1200 IU/kg) once weekly for two treatments without any significant toxicity. These results indicate that PEG-asparaginase has antitumor activity in dog with spontaneously occurring malignant lymphoma.
Treatment with catalase and SOD (superoxide dismutase) could diminish the damage due to oxygen free radical formation, but these enzymes are rapidly removed from circulation. The covalent attachment of monomethoxypolyethylene glycol (PEG) to catalase and SOD extended their plasma half-lives. Toxicity of PEG-catalase and PEG-SOD was evaluated in mice and rats prior to their use as free radical scavengers. Rodents used in acute, subacute, and subchronic toxicologic studies could tolerate large doses of PEG-catalase and PEG-SOD without developing toxic signs. The conjugates did not affect survival rate, appearance, behavior, food intake, blood chemistry, hematology, or urinalysis. In general, body weight gains, organ weights, and histomorphology were also unaffected. Massive doses of PEG-catalase caused slight weight loss, splenic hypertrophy, and generalized splenic stimulation in mice. Massive doses of PEG-SOD resulted in vacuolation in splenic macrophages in rats. PEG-catalase and PEG-SOD circulated for 3 days and 8 days, respectively, in mice following i.v. or i.m. administration.
The use of high oxygen concentrations and high mean airway pressures during mechanical ventilation of premature newborn infants with respiratory distress syndrome leads in 20%-30% of the survivors to chronic lung disease. This study explores if exogenous polyethylene glycol conjugated superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) mitigate oxygen toxicity in premature lambs with respiratory distress syndrome. Six pairs of premature lambs were delivered by cesarean section and treated by tracheal instillation of 60 mg natural sheep surfactant/kg/body weight. After birth, all lambs were ventilated with 100% oxygen, and one of each pair received a single intravenous injection of 1 million U/kg PEG-CAT and 50,000 U/kg PEG-SOD. At 8 h of age or after respiratory failure was established, the lambs were killed and the lungs were removed intact. Lung damage was assessed by microscopy. The arterial blood gases, pH, and mean airway pressures of the lambs treated with PEG-SOD/PEG-CAT did not differ from those of the controls. Mean PaO2 was greater than 140 mmHg during the first 4 h of the experiments. In the lambs treated with PEG-SOD/PEG-CAT, SOD and CAT levels were very high during the study period and less bronchiolar epithelial damage and lung hemorrhages were found at microscopy.
Modification of proteins with polyethylene glycol (PEG) has been shown to result in a decrease in immunogenicity. Superoxide dismutase (SOD) and catalase were modified with PEG and used to immunize mice. Antibody titers against the antigens were determined by ELISA. Mice immunized with PEG-SOD had antibody titers 0.03%-0.07% of that seen in mice with SOD, while mice immunized with PEG-catalase developed titers 0.02%-0.09% of that seen in mice with catalase. The modified enzymes retained the ability to react with preformed antibodies to the unmodified antigens. Antibodies to SOD reacted equally well with the PEG-SOD or SOD antigen. Antibodies to catalase reacted to PEG-catalase but at only 0.02% of the reaction with catalase antigen. In reciprocal studies, antisera against the PEG-proteins failed to react to an appreciable level with the corresponding unmodified protein. Modification with PEG resulted in a decrease in immunogenicity of both SOD and catalase.
A conjugate of asparaginase and monomethoxypolyethylene glycol was evaluated in acute, subacute, and subchronic toxicologic studies in mice, rats, and dogs. The drug induced low-grade toxicosis. The appearance and behavior of rats and dogs were not affected by the treatment. Only large doses produced inactivity, loss of appetite, and loss of weight. The LD50 could not be established. The drug retarded slightly body weight gains in dogs and female rats and produced mild anemia in 30% of the female rats. Urinalysis and blood chemical determinations in rats and dogs were generally not affected by the treatment. Monomethoxypolyethylene glycol-asparaginase was detectable in the plasma of mice 13 days after IV, intraperitoneal, or IM administration, and in dogs for 3 to 4 weeks.
A man with hyperasparaginemia, presumably due to chronic deficiency of asparaginase activity, had been schizophrenic and unresponsive to antipsychotic drugs for at least 22 years. He was given repeated injections of bacterial L-asparaginase rendered relatively nonimmunogenic by covalent binding to polyethylene glycol (PEG). PEG-asparaginase lowered plasma asparagine concentrations from 4 to 5 SD above normal down to undetectable levels, and eliminated asparagine from the cerebrospinal fluid. Despite biochemical correction lasting at least 55 days, the patient did not improve psychiatrically. Experience limited to this single patient suggests that PEG-asparaginase therapy is relatively innocuous, but does not clarify whether there is an etiological relationship between hyperasparaginemia and psychiatric illness.
The covalent attachment of polyethylene glycol of 5000 daltons to non-essential groups on trypsin produces an adduct that no longer precipitates with anti-trypsin antibody. In comparison with trypsin, polyethylene glycol-trypsin preparations show equal or greater activity against N-alpha-benzoyl-L-arginine ethyl ester, about one-fourth activity against angiotensin II, and little activity against bovine liver catalase. The polyethylene glycol-trypsin adduct dissolves soft blood clots at one-fourth the rate of trypsin. Soybean trypsin inhibitor produces two-thirds inhibition of the adduct under conditions that cause complete inhibition of trypsin.
Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.
A simple procedure has been developed for the purification of L-asparaginase from Vibrio succinogenes. Only two steps of ion-exchange chromatography are required. A higher yield and higher specific activity are obtained than previously reported.
Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.
An L-glutaminase-L-asparaginase from Achromobacter has been rendered nonimmunogenic by the covalent attachment of polyethylene glycol (PEG) to nonessential amine groups of the enzyme. PEG-L-glutaminase-L-asparaginase exhibits a greatly enhanced half-life in the bloodstream compared to the unmodified enzyme in normal mice, and is effective in prolonging the survival of BDF1 mice inoculated ip with L5178Y cells. PEG-L-glutaminase-L-asparaginase appears rapidly in the blood following ip injection.
Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.
Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.
Deficiencies of antioxidants and increased free radical generation may explain the high incidence of bronchopulmonary dysplasia in premature infants. Long-acting antioxidants such as polyethylene glycol (PEG) conjugated superoxide dismutase (SOD), and catalase might modify this process. We delivered 32 premature lambs, 16 pairs of twins, by cesarean section at 125-141 days of gestation (term 146 days) and stabilized them on ventilators in normocapnic hyperoxia for a period of 8 h. One lamb of each twin pair received an intravenous dose of 7,500-50,000 IU/kg of PEG-SOD and of 37,500-1,000,000 IU/kg of PEG-catalase at birth. Their siblings acted as controls. Mean airway pressure, arterial pressure, and heart rate were recorded continuously. Arterial blood gases and pH were obtained every 30 min. After sacrifice, standardized lung biopsies were prepared for quantitative morphometrics and electron microscopy. Administration of PEG antioxidants at birth reduced the influx of neutrophils and macrophages into the lung and damage to arterioles, bronchiolar mucosa, and type II pneumocytes without major changes in alveolar surface area or pulmonary function. These effects were dose-related and detectable even at the lowest doses of PEG antioxidants administered.
Polyethylene glycol (PEG)-L-asparaginase, at doses ranging from 500 to 8000 units/m2, was infused iv over 60 min in 31 patients of whom 27 were evaluable pharmacokinetically. The plasma disappearance of PEG-L-asparaginase is described by a monophasic curve with a mean half-life of 357 +/- 243 hr which is much longer than that of the unconjugated enzyme (half-life of approximately 20 hr). The rate of total clearance (128 +/- 74 ml/m2 X day) is much slower than that of L-asparaginase (2196 +/- 1098 ml/m2 X day). The volume of distribution is 2093 +/- 643 ml/m2, which is similar to that of L-asparaginase, indicating that PEG-L-asparaginase is mainly localized in the plasma. No enzyme could be measured in urine samples taken from nine patients for a period of up to 4 days. Additionally, no enzyme was measurable in one patient's pleural fluid obtained at the end of infusion and 6 days after infusion of a 1000-unit/m2 dose; the corresponding concentrations in plasma were 0.64 and 0.62 units/ml, respectively. In general, the plasma enzyme concentrations at the end of the 1-hr infusion and at 14 days after drug administration were proportional to the dose given. However, in two patients, a sudden disappearance of enzyme levels occurred which preceded anaphylactic reactions during subsequent treatment. A third patient developed severe bronchospasm 30 min after the first dose, but his enzyme levels were within the normal range.