Abraham Abuchowski

The therapeutic value of poly(ethylene glycol)-modified proteins

Abstract The field of protein therapy has flourished in the past few years, mainly due to the advent of recombinant DNA techniques and large-scale manufacturing processes. It was believed that human-derived proteins would eliminate the problems of short circulating lives, low stability and immunogenicity associated with the use of non-human proteins as drugs. Unfortunately, it has been demonstrated that many human proteins still suffer from short circulating lives and low stability, and often require the use of large doses to maintain therapeutic efficacy. This in turn increases the chances for the development of an adverse immune response, or at least for the development of clearing antibodies. The discovery that the modification of proteins with poly(ethylene glycol) (PEGNOLOGYSM) results in an adduct with decreased to non-existent immunogenicity increased circulating life, increased stability and opened the way for the development of protein pharmaceuticals that would realize the promise of protein therapy. In this paper, we will review the clinical value of PEGNOLOGYSM as researched by Enzon Inc. and discuss the future for this technology.

The clinical efficacy of poly(ethylene glycol)-modified proteins

Abstract Modification of proteins with poly(ethylene glycol) (PEG) creates a conjugate with a dramatically increased circulating life and reduced immunogenicity and antigenicity. The increase in circulating life is attributed to the masking of ligands or antigenic determinants recognized by cellular receptors. Steric hindrance of the antigenic determinants on the protein is believed to reduce the immunogenicity and antigenicity. Currently four PEG-modified enzymes are undergoing clinical trials. PEG- l -asparaginase is in Phase III studies for treatment of acute lymphoblastic leukemia; PEG-adenosine deaminase (PEG-ADA) is in phase III trials and is used in ADA-deficient Severe Combined Immunodeficiency Syndrome; PEG-superoxide dismutase (PEG-SOD) is in Phase III studies for treatment of reperfusion injury associated with kidney transplantations; PEG-uricase is a uricolytic agent in Phase I trials for treatment of hyperuricemia associated with chemotherapy. PEG-catalase has completed preclinical trials and shows potential therapeutic use in trauma, burns and hydrogen peroxide-mediated injuries. All these enzymes demonstrate increased circulating lives and decreased immunogenicity. Clinical studies have shown these modified enzymes to be safe and have therapeutic value.

Safety evaluation of free radical scavengers peg-catalase and peg-superoxide dismutase

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.

Immunogenicity of polyethylene glycol-modified superoxide dismutase and catalase.

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.

Prevention of oxygen toxicity with superoxide dismutase and catalase in premature lambs

Abstract 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 PaO 2 was > 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.

A preliminary study on the evaluation of asparaginase. Polyethylene glycol conjugate against canine malignant lymphoma

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‐aspnraginase 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 dogs with spontaneously occurring malignant lymphoma.

Mitigation of Pulmonary Oxygen Toxicity in Premature Lambs with Intravenous Antioxidants

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.

PEG-Modified Hemoglobin as an Oxygen Carrier

A need is strongly recognized for a safe red cell substitute that can carry oxygen to the hypoxic tissues of an anemic body. Banked blood is in short supply because of mounting fears of donors and limited storage life. A safe, stable, oxygen carrier would eliminate many problems such as cross-matching of blood types, danger of virus infections, short shelf life, and availability. Not only could this be used as an oxygen-carrying plasma expander for trauma victims and patients in surgery, such as cardiac bypass and angioplasty, this product could also be used as a perfusate for the preservation of isolated organs for transplantation. Cold storage is the popular choice for preservation at present, but there is an increasing demand for a safe organ perfusate that would promote the use of perfusion preservation to obtain a longer period of preservation.

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.