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 monomethoxypolyethylene glycol (PEG) to asparaginases from Escherichia coli and Vibrio succinogenes by new coupling methodology produced conjugates that are active, stable, without significant immune response, and with greatly extended plasma half-lives in mice. Therapeutic efficacies were greater for the PEG-asparaginases than for the unmodified asparaginases in mice infected with the L5178Y lymphosarcoma or the 6C3HED tumor. Large single doses of native or modified enzymes were more effective against tumors than the same amount of enzyme given in smaller doses over several days.
The in vitro blastogenic response of rat splenocytes to concanavalin A stimulation is inhibited by inclusion of asparaginase in the culture medium. The glutaminase-free asparaginase from Vibrio succinogenes is as potent an inhibitor as the Escherichia coli enzyme which has 2% glutaminase activity. The polyethylene glycol-modified forms of both enzymes are also inhibitory. We suggest that previously proposed explanations for the ability of asparaginases to inhibit blastogenesis are not likely to be correct and propose that asparaginase interacts with a mitogenic factor.
The immunosuppressive effects of polyethylene glycol-modified asparaginases from Vibrio succinogenes (PEG-asparaginase VS) and Escherichia coli (PEG-asparaginase EC) have been investigated in mice. Measurements of the mitogen-induced blastogenic responses of splenocytes, harvested 5 days after in vivo administration of the PEG-enzymes, show that PEG-asparaginase VS is not immunosuppressive, whereas PEG-asparaginase EC does cause immunosuppression. Both enzymes cause the spleen to be smaller than the control mice. In mice carrying the L5178Y tumour and its associated LDH-elevating virus, which causes the circulation life of asparaginase VS to be comparable to that of PEG-asparaginase VS, tumour regression and its attendant immunological changes are identical in animals treated with either the native or the modified enzyme. The data presented in this paper, along with independent immunological evidence presented by other workers strongly suggest that PEG-asparaginase VS may be the enzyme of choice for clinical use.
Poly(ethylene glycol) of 5 000 daltons has been attached covalently to preparations of urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from hog liver and Candida utilis. Attachment of sufficient poly(ethylene glycol) to either urate oxidase renders the enzyme incapable of eliciting antibody production in mice, or of reacting with antibodies to the unmodified enzyme. The poly(ethylene glycol) : urate oxidase conjugates exhibit higher Km and lower V values than the unmodified urate oxidases. Optimal pH values are increased for the poly(ethylene glycol) : urate oxidases, and optimal temperatures are decreased. The blood circulating lives of the modified urate oxidases following intravenous injection are much longer than those of the unmodified urate oxidases: repetitive injections over a period of 90 days dd not alter the blood circulating lives of the poly(ethylene glycol) : urate oxidases. The unmodified enzymes, on the other hand, were cleared from the blood with extreme rapidity after a few intravenous injections.
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The polyethylene glycol (PEG) adduct of Escherichia coli L-asparaginase was administered intravenously to 4 patients with chemotherapy refractory cancers. The PEG-enzyme in plasma exhibited a half-life of 16-25 days. Doses of 250IU/m2 or greater reduced plasma asparagine to undetectable levels for as long as enzyme was detectable in plasma. All doses of enzyme administered (250-1000 IU/m2) caused similar increases in plasma aspartate, i.e. no dose-response relationship. Pleural fluid and ascites contained detectable enzyme but at a value 10-15% of simultaneously drawn plasma levels. Toxicity in this small group of patients was minimal; nausea and transient fever predominated. There were no clinical signs of PEG-asparaginase-induced pancreatitis, renal dysfunction, hypocalcemia and hyperglycemia. No patient developed evidence of a PEG-asparaginase allergic reaction; no patient formed antibodies to asparaginase or PEG-asparaginase. Two patients with large cell lymphoma showed a partial response to treatment.
Uricase from C. utilis and uricase to which polyethylene glycol had been attached were injected into leghorn cockerels in an attempt to lower plasma urate levels. Twenty units of either enzyme reduced urate levels to near zero for 24 hr on initial injection, whereas 10 U were effective for less than 6 hr. After injection with four-weekly doses of enzyme, unmodified uricase was ineffective in lowering plasma urate levels. Polyethylene glycol-uricase, however, was as effective as on the first injection. Both enzyme preparations appear in the lymph shortly after injection into the rat, although polyethylene glycol-uricase appears slightly sooner and maintains a more constant level than uricase.
Polyethylene glycol was attached covalently to adenosine deaminase (ADA) using cyanuric chloride as the coupling agent. The modified adenosine deaminase (PEG-ADA) appears to lose its immunogenicity in mice following multiple intravenous injections. PEG-ADA does not react with antibodies raised against native ADA. The circulating half-life (T1/2) of PEG-ADA was increased to 28 hr. The lack of detectable antibody formation and long circulating life may make PEG-ADA suitable for treating human ADA deficiency.
Methoxypolyethylene glycol (PEG) of 5000 daltons was attached covalently to 19 of the 20 available lysine residues of bovine erythrocyte superoxide dismutase. The adduct (PEG-superoxide dismutase) has 51% of the enzymatic activity of superoxide distmutase. PEG-superoxide dismutase exhibits a sharply enhanced serum circulating life during repetitive intravenous injections compared to the native enzyme. While no evidence of an immune response to repetitive injections of PEG-superoxide dismutase is observed, the unmodified enzyme appears to produce slight sensitization in the form of quicker removal from the serum after 13 injections over a period of 30 days. Antisera to superoxide dismutase and PEG-superoxide dismutase, however, produce no detectable antibodies as determined by the Ouchterlony method. Intraperitoneally injected PEG-superoxide dismutase enters the bloodstream more readily than superoxide dismutase. PEG modification slightly improves the enzyme's anti-inflammatory activity, which was observed in rats over a period of eight days.
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.