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Overwhelming neutrophilic inflammation is a leading cause of lung damage in many pulmonary diseases, including cystic fibrosis (CF). The heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway mediates the resolution of inflammation and is defective in CF-affected macrophages (MΦs). Here, we provide evidence that systemic administration of PP-007, a CO releasing/O2 transfer agent, induces the expression of HO-1 in a myeloid differentiation factor 88 (MyD88) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)-dependent manner. It also rescues the reduced HO-1 levels in CF-affected cells induced in response to lipopolysaccharides (LPS) or Pseudomonas aeruginosa (PA). Treatment of CF and muco-obstructive lung disease mouse models with a single clinically relevant dose of PP-007 leads to effective resolution of lung neutrophilia and to decreased levels of proinflammatory cytokines in response to LPS. Using HO-1 conditional knockout mice, we show that the beneficial effect of PP-007 is due to the priming of circulating monocytes trafficking to the lungs in response to infection to express high levels of HO-1. Finally, we show that PP-007 does not compromise the clearance of PA in the setting of chronic airway infection. Overall, we reveal the mechanism of action of PP-007 responsible for the immunomodulatory function observed in clinical trials for a wide range of diseases and demonstrate the potential use of PP-007 in controlling neutrophilic pulmonary inflammation by promoting the expression of HO-1 in monocytes/macrophages.

Patients receiving pegfilgrastim (Neulasta®) for the treatment of neutropenia can experience bone pain following the injections required to achieve effective neutrophil levels. The safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of ANF-RHO™, a novel pegylated granulocyte colony stimulating factor, were assessed in a randomized, controlled, double-blind Phase 1 clinical study in healthy volunteers. Subjects received a single subcutaneous dose of ANF-RHO over a range of 6 doses (5-50 μg/kg), placebo (saline), or the recommended clinical dose of pegfilgrastim administered at the labeled fixed 6 mg dosage (equivalent to 80-100 μg/kg). The primary outcome measure was safety and tolerability. Secondary outcomes included PK and PD effects on absolute neutrophil count (ANC) and number of CD34+ progenitor cells. Severity of bone pain was also assessed. In healthy volunteers, ANF-RHO was administered at ascending doses up to 50 μg/kg without significant adverse effects; appeared to be better (5 to 30 μg/kg) or equally well (50 μg/kg) tolerated, and had lower mean bone pain scores as compared to pegfilgrastim. ANF-RHO achieved CD34+ and ANC numbers at significantly lower doses, and had a significantly longer circulating half-life than pegfilgrastim. These results suggest that ANF-RHO can be provided less frequently, at a lower dose, and with fewer side effects. ANF-RHO had unique, prolonged PK/PD attributes as compared to marketed pegfilgrastim, suggesting that it may provide an improved clinical benefit in further clinical studies in patients with chemotherapy-induced or chronic idiopathic neutropenia.

Historically, blood substitutes were under development that would provide oxygen carrying capacity as well as fluid replacement for both trauma and surgical indications. Their development was halted by the inability of the products to deliver therapeutic amounts of oxygen targeted to hypoxic tissue as well as from the inherent toxicity of the molecules. This led to the concept of an oxygen therapeutic that would be targeted for indications caused by anemia/ischemia/hypoxia but would not exhibit the toxicity that plagued earlier products. The complex pathophysiology of diseases such as sickle cell and hemorrhagic stroke not only has hypoxia as a pivotal event but also includes inflammation and vasoconstriction that perpetuate the oxygen deprivation. There is a need for an effective therapeutic that addresses the multiple events of inflammation and oxygen deprivation. SANGUINATE acts as a dual mode carbon monoxide (CO) and oxygen delivery therapeutic. SANGUINATE is designed not only to treat hypoxia but also to act on concurrent pathologies such as inflammation and reperfusion injury. This expands the potential therapeutic utility of SANGUINATE beyond anemia into indications such as early brain injury and delayed kidney graft function, where inflammation plays a pivotal pathological role as well as in indications such as sickle cell disease where the inflammation and hypoxia contribute to the development of comorbidities such as vaso-occlusive crisis. Clinical trials in multiple indications are underway.

PEGylated carboxyhemoglobin bovine (SANGUINATE) is a dual action carbon monoxide releasing (CO)/oxygen (O2 ) transfer agent for the treatment of hypoxia. Its components inhibit vasoconstriction, decrease extravasation, limit reactive oxygen species production, enhance blood rheology, and deliver oxygen to the tissues. Animal models of cerebral ischemia, peripheral ischemia, and myocardial ischemia demonstrated SANGUINATE's efficacy in reducing myocardial infarct size, limiting necrosis from cerebral ischemia, and promoting more rapid recovery from hind limb ischemia. In a Phase I trial, three cohorts of eight healthy volunteers received single ascending doses of 80, 120, or 160 mg/kg of SANGUINATE. Two volunteers within each cohort served as a saline control. There were no serious adverse events. Serum haptoglobin decreased, but did not appear to be dose related. The T1/2 was dose dependent and ranged from 7.9 to 13.8 h. In addition to the Phase I trial, SANGUINATE was used under an expanded access emergency Investigational New Drug. SANGUINATE was found to be safe and well tolerated in a Phase I clinical trial, and therefore it will advance into further clinical trials in patients.

Historically, blood substitutes were under development that would provide oxygen carrying capacity as well as fluid replacement for both trauma and surgical indications. Their development was halted by the inability of the products to deliver therapeutic amounts of oxygen targeted to hypoxic tissue as well as from the inherent toxicity of the molecules. This led to the concept of an oxygen therapeutic that would be targeted for indications caused by anemia/ischemia/hypoxia but would not exhibit the toxicity that plagued earlier products. The complex pathophysiology of diseases such as sickle cell and hemorrhagic stroke not only has hypoxia as a pivotal event but also includes inflammation and vasoconstriction that perpetuate the oxygen deprivation. There is a need for an effective therapeutic that addresses the multiple events of inflammation and oxygen deprivation. SANGUINATE acts as a dual mode carbon monoxide (CO) and oxygen delivery therapeutic. SANGUINATE is designed not only to treat hypoxia but also to act on concurrent pathologies such as inflammation and reperfusion injury. This expands the potential therapeutic utility of SANGUINATE beyond anemia into indications such as early brain injury and delayed kidney graft function, where inflammation plays a pivotal pathological role as well as in indications such as sickle cell disease where the inflammation and hypoxia contribute to the development of comorbidities such as vaso-occlusive crisis. Clinical trials in multiple indications are underway.

Background: Pain during Vaso-Occlusive Crisis (VOC) is a hallmark of sickle cell disease (SCD) as deoxygenated (sickled) red blood cells (RBC) aggregate in the microvasculature due to Hemoglobin S (HbS) polymerization. PEGylated carboxyhemoglobin bovine (PEG-COHb; SANGUINATE®) was designed as a gas transfer agent to depolymerize HbS resulting from hypoxia. Prior in vitro studies have shown PEG-COHb capability to transfer carbon monoxide (CO) and oxygen (O2) to SCD hypoxic RBCs, reversing sickling. A prospective, randomized, single-dose Phase II clinical study is in progress (NCT02411708) evaluating the safety and efficacy of PEG-COHb in the treatment of severe VOC including pain reduction in the ambulatory setting. This study is also evaluating the levels of RBC sickling following treatment and association with pain scoring. Methods: Participants were randomized to a PEG-COHb (320 mg/kg) or saline (placebo) arm, in addition to standard treatment and IV opioid per institutional practice. Pain was assessed using a 10-point pain scale at screening, pre-infusion, hourly until discharge, 24-hours, 72-hours, and Day 7 post-infusion. Blood was collected at pre-infusion, time for readiness to discharge and 72 hours post-infusion to assess RBC shape morphology changes using image-based flow cytometry. Results: Interim results showed PEG-COHb treated subjects experienced significant reduction at discharge in pain score of 4.3 (±0.3) compared to 2.4 (±0.6) reduction in the placebo arm (p Conclusions: PEG-COHb appeared to be safe, well tolerated, and associated with significantly reduced pain scores. The flow cytometry analysis of blood samples revealed PEG-COHb treatment reverted RBC to a more normal round morphology when compared to the placebo group. Individual patient data showed a trend between RBC unsickling (roundness) and pain score reductions. These results are consistent with depolymerization of HbS resulting from transfer of CO and/or O2 to sickled RBCs and O2 delivery to hypoxic tissue. This study supports advanced clinical trials of PEG-COHb as a potential therapy to reduce the pain associated with VOC. Disclosures Buontempo: Prolong Pharmaceuticals: Employment. Jubin: Prolong Pharmaceuticals: Employment. Buontempo: Prolong Pharmaceuticals: Employment. Real: Prolong Pharmaceuticals: Employment. Kazo: Prolong Pharmaceuticals: Employment. O'Brien: Prolong Pharmaceuticals: Employment. Adeel: Prolong Pharmaceuticals: Employment. Abuchowski: Prolong Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

The endeavor to study desensitization in kidney transplantation hasn't been matched by an effort to investigate strategies to prevent sensitization. In this study (NCT02437422), we investigated the safety, impact on sensitization and pharmacokinetics of SANGUINATE (SG), a hemoglobin-based oxygen carrier, as a potential alternative to packed red blood cells (PRBC) in transplant candidates with end-stage renal disease (ESRD). Ten ESRD subjects meeting inclusion/exclusion (I/E) criteria were planned to receive 3 weekly infusions of SG (320mg/Kg). The study was stopped after 5 subjects were enrolled and their data was analyzed after completing a follow up period of 90 days. Two subjects had elevated troponin I levels in setting of SG infusion, one of which was interpreted as a non-ST elevation myocardial infarction. All other adverse events were transient. SG pharmacokinetic analysis showed mean(sd) Cmax, Tmax, AUC and half-life of 4.39(0.69)mg/ml, 2.42(0.91)hours, 171.86(52.35)mg.hour/ml and 40.60(11.96)hours respectively. None of the subjects developed new anti-HLA antibodies following SG infusion and throughout the study period. In conclusion, SG is a potential alternative to PRBCs in ESRD patients considered for kidney transplantation as it was not associated with humoral sensitization. Larger studies in highly sensitized patients are required to further evaluate for potential safety signals. This article is protected by copyright. All rights reserved.

We investigated the pre-clinical utility of carbon monoxide form of PEGylated hemoglobin (PEG-Hb also named SANGUINATE(™)) in myocardial infarction (MI) and in particular the response of diabetic tissues to superimposed ischemia/reperfusion injury. SANGUINATE(™) was evaluated in diabetic and normal mice subjected to 30 min of coronary artery ligation followed by either 48 h or 28 days of reperfusion. Our results demonstrate that SANGUINATE(™) was effective in reducing infarct size when administered either prior to left anterior descending coronary artery (LAD) occlusion or during reperfusion. This finding is an important step in exploring the efficacy of a pharmacoinvasive strategy using SANGUINATE(™) in patients with acute coronary syndromes.

e14085Background: Anti-Neutropenia Factor - RHO (ANF-RHO) is a novel pegylated version of native human recombinant G-CSF protein. ANF-RHO has distinct biophysical and biological properties that pro...

The effect of transfusion of PEGylated hemoglobin (PEG-Hb) was evaluated in anesthetized rats subjected to 2 hours of focal cerebral ischemia and 1 day of reperfusion. PEG-Hb was stored in the carboxy state (PEG-COHb) to reduce autooxidation and increase the shelf life. Transfusion of 10 ml/kg of PEG-COHb at 20 minutes of ischemia did not alter arterial blood pressure or increase red cell flux in the ischemic core. Plasma hemoglobin increased to only 0.6 g/dL, yet infarct volume was markedly decreased and neurological deficits were improved. We conclude that early topload transfusion of PEG-COHb protects the brain from ischemic stroke.

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.

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.

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.

Background: Patients receiving pegfilgrastim (Neulasta) for the treatment of neutropenia can experience bone pain following the frequent injections required to achieve effective neutrophil levels. Bone pain is thought to be caused by the rapid expansion of bone marrow hematopoietic cells and accompanying secretion of certain cytokines. ANF-Rho (ANF) is a novel modified granulocyte colony stimulating factor that was found in preclinical animal studies to be approximately 2-4 times more active than pegfilgrastim. A Phase I study was conducted to determine its safety profile and the potential to reduce the occurrence of side effects such as bone pain as well as its dose-response relationship in healthy adults. Methods: This was a first-in-human, double-blind, randomized, placebo-controlled, single ascending dose Phase 1 study in 76 healthy subjects. Subjects received a single subcutaneous (sc) dose of ANF over a range of 6 doses (5-50 ug/kg), placebo (saline) or the recommended clinical dose of pegfilgrastim (100μg/kg). The primary outcome measure was safety and tolerability. Secondary outcomes included pharmacokinetics (PK) and pharmacodynamic (PD) effect on absolute neutrophil count (ANC) and number of CD34+ progenitor cells. The Bond and Lader Visual Analogue Scale (VAS) was used to assess the severity of bone pain. Results: ANF was well to moderately-well tolerated up to a dose level of 50 µg/kg and appeared to be better tolerated than pegfilgrastim. There were no deaths or withdrawals due to adverse events (AEs) during this study. A total of 354 AEs were reported by 71 (93%) of the subjects. The majority of the AEs were of mild intensity (87%), and 13% of the AEs were of moderate intensity. The most frequently reported were musculoskeletal and connective tissue disorders. Mean bone pain scores were lower in the 5 to 30 µg/kg ANF groups compared to the pegfilgrastim group and were similar for the 50 µg/kg ANF and pegfilgrastim group. There were no clinically significant findings for ANF with respect to clinical laboratory, vital signs, ECG, Holter monitoring, physical examination, and local tolerability. One patient was positive for a low level of anti-drug antibody to ANF. The t1/2 of ANF ranged between 38.5 and 51 hours (hr) and is longer compared to pegfilgrastim (28hr). The tmax of ANF is 36 hr and is dose-independent and occurs later than for the pegfilgrastim (16 hr). ANF-Rho has a Cmax and AUC0-t and AUC0-inf values that increase with dose without a dose-linear relationship, a Cmax approximately 1.8-fold lower for the 50 µg/kg dose and a mean AUC0-t and AUC0-inf that are approximately 1.5-fold higher compared to treatment with 100μg/kg pegfilgrastim. There was a more gradual and prolonged increase of ANC and CD34+ cell by ANF as compared to pegfilgrastim. A maximum mean ANC (8.6 x109/L (5 µg/kg) and 45x109/L (50 µg/kg) was reached between Day 6 and Day 10 as compared to Day 4 for pegfilgrastim (21.5x109/L) (Fig A, B). A maximum number of CD34+ cells (10.74 (5 µg/kg) and 71.4 (50 µg/kg) cells/µL) was reached on Day 7 as compared to Day 5 for pegfilgrastim (66.98 cells/µL). The 20 µg/kg ANF dose showed a PD effect (ANC and CD34+) that was comparable to treatment with 100μg/kg of pegfilgrastim, whereas Cmax and AUC values were 4.3-fold and 1.7-fold, respectively, lower for the 20 µg/kg ANF dose compared to the pegfilgrastim. This PD effect appeared to be more sustained in the ANF-Rho dose groups compared to pegfilgrastim. Summary: In healthy volunteers, ANF was administered at a dose of 50µg/kg without significant adverse effects. ANF appeared to be better (5 to 30 µg/kg) or equally well (50 µg/kg) tolerated and had lower mean bone pain scores as compared to pegfilgrastim. ANF achieved CD34+and ANC numbers at significantly lower doses and had a significantly longer circulating half-life than pegfilgrastim. These results suggest that ANF can be provided less frequently at a lower dose and with fewer side effects. Phase 2 trials of ANF are planned to begin shortly in patients. Figure 1. Figure 1. Disclosures Parmar: Prolong Pharmaceuticals: Employment. Byczkowski:Prolong Pharmaceuticals: Employment. Jubin:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Gitlin:Prolong Pharmaceuticals: Employment. Boro:Prolong Pharmaceuticals: Employment. Misra:Prolong Pharmaceuticals: Employment.

Background: In Sickle Cell disease (SCD), a single amino acid substitution in the beta globin chain converts HbA to sickle genotype HbS. This genetic change promotes HbS polymerization upon deoxygenation that can promote occlusion of small blood vessels that is often associated with increased blood viscosity, and circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-COHb; SANGUINATE) was designed as a novel therapeutic agent to initially release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. Delivery of either CO and/or O2 to hypoxic, sickled red blood cells (RBCs) should return cells to a more normal cell morphology and help re-establish normal blood flow and rheology. PEG-COHb was shown to mediate transfer of either a CO or O2/CO mixture and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying or slowing sickling, which should maintain normal blood flow through hypoxic microvasculature. Unsickling is expected to be expedited by O2 transfer by PEG-COHb. To examine these potential therapeutic effects, current in vitro studies examined the effects of time and dose of PEG-COHb to not only reverse, but also prevent or delay sickling by transferring CO as well as expedite atmospheric O2 transfer to the sickled RBCs. Methods: Reversal of sickling studies were conducted by deoxygenating RBCs from healthy (control) and SCD volunteers in followed by treatment with either PEG-COHb, fully oxygenated PEG-Hb (PEG-O2Hb) or PEG-BSA for 2 hours. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-COHb and then subjected to hypoxia for 3 hours. Time-dose effects were quantified by area under the curve (AUC) analysis. O2 transfer studies were conducted by treating hypoxic, sickled RBCs to increasing concentrations of PEG-COHb and raising the pO2 from 3.8mm to 40mm. In all studies, the fractions of CO-Hb, O2-Hb and reduced Hb were determined by co-oximetry and sickled RBCs were quantified by imaging flow cytometry of fixed RBC specimens. Results: PEG-COHb-mediated delivery of either CO or O2 can unsickle hypoxic SCD RBCs. Controls exhibited gas exchange similar to SCD RBCs. Interestingly, sickle reversion time-course studies showed differential kinetics between the CO and O2 capacity to cause unsickling. AUC analysis at 20 minutes demonstrated that both CO and O2 reversed sickling by 41% and 42%, respectively. PEG-O2Hb was able to exert substantial unsickling by 5 minutes, where PEG-COHb showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-COHb prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline (PBS). In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that although, not completly inhibited, sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-COHb increased the rate of unsickling as measured by AUC by 50% and 15% at 4 and 2 mg per mL, respectively. These levels are within the expected therapeutic dosage of SANGUINATE. Summary: RBCs from patients with SCD undergo a conformational shift upon deoxygenation resulting in HbS polymerization and morphological changes of the RBCs. The occlusive and fragile properties of sickled RBCs are responsible for the development of the numerous comorbidities associated with SCD. It is only when the fraction of oxygenated or carboxylated HbS reaches a sufficient level that reversion to normal cell morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-COHb ability to deliver both O2 and CO to sickled RBC. These data suggested that PEG-COHb is a promising gas transfer agent that has the potential to improve sickle cell morphology by reversing sickling; the underlying pathology of sickle cell disease co-morbidities. Disclosures Buontempo: Prolong Pharmaceuticals: Employment. Yglesias:Prolong Pharmaceuticals: Employment. Chen:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Jubin:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment.

e18086 Background: Patients who cannot receive blood transfusions in the presence of severe anemia can sustain significant morbidity and mortality. Religious beliefs and hemolytic reactions are the...

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.

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-α- 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The in vitro blastogenic response of rat splenocytes to concana-valin 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 blasto-genesis are not likely to be correct and propose that asparaginase interacts with a mitogenic factor.

There are many clinical disorders which, in theory might be treated by the use of appropriate enzymes. However, there are serious problems associated with enzyme therapy. First, inadequate amounts of human enzymes are available; and most foreign enzymes are immunogenic in humans. They can be administered only a few times. Second, the circulating life in the blood may be very short even on the first injection (often a matter of a few minutes). And third, the cost of highly purified enzymes may be too great to allow general use.

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.

Uricase from Candida utilis was covalently attached to polyethyleneglycol (PEG). The modification of 71% of the amino groups in the uricase molecule produced a preparation retaining 11% of activity which did not elicit an antibody response in mice or man. 5 men with advanced haematological malignancies received a single intravenous injection of PEG-uricase which resulted in the rapid disappearance of serum uric acid for over 32 hours. An identical injection thirty days later gave similar results. No toxic effects were seen.