Preparation and properties of polyethylene glycol-trypsin adducts.

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.

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

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.

Properties of two urate oxidases modified by the covalent attachment of poly(ethylene glycol).

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.

Use of the polyethylene glycol adduct of L-asparaginase for the treatment of hyperasparaginemia in a schizophrenic patient.

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.

Toxicologic studies of a conjugate of asparaginase and polyethylene glycol in mice, rats, and dogs.

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.

Enzyme therapy: II. Effect of covalent attachment of polyethylene glycol on biochemical parameters and immunological determinants of beta-glucosidase and alpha-galactosidase.

The covalent attachment of polyethylene glycol (PEG) to beta-glucosidase from sweet almonds and alpha-galactosidase from green coffee beans results in alterations of their catalytic properties and masking of specific determinant sites on the enzymes. Both enzymes have increased Km and decreased Vmax values against their respective p-nitrophenyl substrate analogs after PEG attachment. When PEG is attached to 30% of alpha-galactosidase epsilon-amino groups, 12% activity remains against ceramide trihexoside, while its ability to convert type B erythrocytes to type H specificity is lost. However, it still is able to cleave terminal galactose residues from human saliva blood group substance B. PEG-beta-glucosidase (38%) did not elicit the production of complement-fixing antibodies, nor did it react with antibodies produced against the native enzyme. Antibody and lectin-specific binding were lost from both modified enzymes (PEG-beta-glucosidase and PEG-alpha-galactosidase). After conjugation with PEG, beta-glucosidase lost its ability to bind to concanavalin A-Sepharose. Antibodies directed against native alpha-galactosidase blocked its enzyme activity, but lost their ability to inhibit activity in progressively higher modified preparations of the enzyme. Antisera against PEG-alpha-galactosidase (53%) did not inhibit enzyme activity in any alpha-galactosidase or PEG-alpha-galactosidase preparation. These results indicate that PEG tends to cover lectin-specific carbohydrate moieties and antigenic determinants and that these sites probably remain cryptic during in vivo processing of PEG-enzymes.

Induction of tolerance in mice by uricase and monomethoxypolyethylene glycol-modified uricase.

The ability to induce tolerance to uricase by the administration of native uricase, and uricase modified by the covalent attachment of monomethoxypolyethylene glycol (PEG) was examined. Uricase, and uricase with PEG attached to 35% (PEG-uricase 35%) and 70% (PEG-uricase 70%) of available amino groups were found to induce tolerance in mice not previously sensitized to uricase. There was a dampening of the IgG, IgE and IgM antibody response to uricase which persisted even after a second sensitizing dose of uricase was administered to these animals. PEG-uricases were found to have little or no immunogenicity when injected into mice and a reduced immunogenicity and antigenicity when tested in rabbits. Native uricase, however, was found to be immunogenic and antigenic in mice and rabbits. Mice sensitized to native uricase were injected with uricase, PEG-uricase 35% or 70% to induce tolerance. After a second sensitizing injection of uricase, circulating levels of IgE, IgG and IgM were measured. All three enzymes induced tolerance in the IgE class of antibody but there was no significant change in the hemagglutinating antibody levels of the mice. Mice injected with 1 mg of uricase died from anaphylaxis. PEG-uricase 35% was found to induce the most effective tolerance in both unsensitized and sensitized mice.

Preparation of a polyethylene glycol: superoxide dismutase adduct, and an examination of its blood circulation life and anti-inflammatory activity.

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.

A rapid purification procedure for L-asparaginase from Vibrio succinogenes.

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.

Reduction of plasma urate levels in the cockerel with polyethylene glycol-uricase.

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.

Immunosuppressive properties and circulating life of Achromobacter glutaminase-asparaginase covalently attached to polyethylene glycol in man.

The immunosuppressive effects and circulating life of Achromobacter glutaminase-asparaginase (GA) covalently attached to polyethylene glycol (PEG) were examined in human subjects following a single iv dose of 1000 IU/m2. Plasma half-life of PEG-GA was 72 hours. Skin test reactivity to recall antigens (mumps and tuberculin) was lost in all four patients tested. In vitro phytohemagglutinin-induced blastogenesis, "natural killing," and phytohemagglutinin-induced cell cytotoxicity was diminished as long as enzyme levels were detectable. In vivo and in vitro activities returned to normal following total plasma clearance of enzyme.

Immunological effects of native and polyethylene glycol-modified asparaginases from Vibrio succinogenes and Escherichia coli in normal and tumour-bearing mice.

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.

Inhibition of blastogenesis by native and polyethylene glycol-modified asparaginases from Escherichia coli and Vibrio succinogenes.

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.

Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase.

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.

Some properties of polyethylene glycol:phenylalanine ammonia-lyase adducts.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to phenylalanine ammonia-lyase from Rhodotorula glutinis. Attachment of sufficient quantities of PEG to phenylalanine ammonia-lyase substantially reduces immunological recognition and clearance of the conjugated enzyme in mice. The modified enzyme demonstrates altered catalytic properties such as shifts in the pH and temperature optima, an increase in the Michaelis-Menten constant, and a lowered Vmax in comparison with the native enzyme. PEG-phenylalanine ammonia-lyase has increased resistance to proteolytic digestion, particularly when in the presence of cinnamate, a competitive inhibitor, while the native enzyme is rapidly inactivated. In the ultracentrifuge PEG-phenylalanine ammonia-lyase exhibits a lower sedimentation rate than the unmodified enzyme, despite the fact that it is much larger. The electrophoretic mobility of PEG-phenylalanine ammonia-lyase is greatly decreased in comparison to the unmodified enzyme. PEG-phenylalanine ammonia-lyase had a much longer blood-circulating life in mice, both initially and after a number of injections, than did the native enzyme. PEG-phenylalanine ammonia-lyase was a good immunogen but a poor antigen in mice and rabbits, that is, it readily induced antibody formation, but reacted poorly in vitro with the antibodies that were formed against it.

Alteration of the circulating life and antigenic properties of bovine adenosine deaminase in mice by attachment of polyethylene glycol.

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.