Safety and Pharmacokinetics of Multiple Doses of Recombinant Human CuZn Superoxide Dismutase Administered Intratracheally to Premature Neonates With Respiratory Distress Syndrome
Objectives. To examine the safety and pharmacokinetics of multiple intratracheal (IT) doses of recombinant human CuZn superoxide dismutase (rhSOD) in premature infants with respiratory distress syndrome who are at risk for developing bronchopulmonary dysplasia (BPD).
Methods. Thirty-three infants (700 to 1300 g) were randomized and blindly received saline, 2.5 mg/kg or 5 mg/kg rhSOD IT within 2 hours of surfactant administration. Infants were treated every 48 hours (as long as endotracheal intubation was required) up to 7 doses. Serial blood and urine studies, chest radiographs, neurosonograms, SOD concentration and activity measurements, and tracheal aspirate (TA) inflammatory markers were assessed throughout the 28-day study.
Results. SOD concentrations in serum (0.1 [0.05/0.15] μg/mL–geometric mean with lower/upper confidence intervals), tracheal aspirates (TA) (0.2 [0.1/0.3] μg/mL) and urine (0.3 [0.2/0.4] μg/mL) were similar at baseline in all 3 groups and did not change significantly in the placebo group. In the rhSOD treatment groups, SOD concentrations were increased on day 3 and did not change significantly thereafter over the 14-day dosing period (also measured on days 5, 7, and 13). SOD concentrations averaged 0.4 [0.3/0.5] μg/mL in serum, 0.8 [0.6/1.2] μg/mL in TA and 1.1 [1.0/1.3] μg/mL in urine for the low-dose group and 0.6 [0.5/0.7] μg/mL in serum, 1.1 [0.9/1.5] μg/mL in TA, and 2.2 [1.6/2.9] μg/mL in urine for the high-dose group over the 14-day dosing period. Enzyme activity directly correlated with SOD concentration and rhSOD was active even when excreted in urine. TA markers of acute lung injury (neutrophil chemotactic activity, albumin concentration) were lower in the rhSOD agroups compared with placebo. No significant differences in any clinical outcome variable were noted between groups.
Conclusions. These data indicate that multiple IT doses of rhSOD increase the concentration and activity of the enzyme in serum, TA and urine, reduce TA lung injury markers and are well-tolerated. Further clinical trials examining the efficacy of rhSOD in the prevention of BPD are warranted.
The benefits of surfactant replacement therapy for the treatment of respiratory distress syndrome (RDS) in premature infants have now been well documented.1-4 However, reductions in oxygen and ventilator requirements after surfactant treatment do not appear to have substantially affected the incidence of bronchopulmonary dysplasia (BPD). In fact, with improvements in survival of many very low birth weight infants, the total number of infants developing BPD may actually be increasing.5
The pathogenesis of BPD is complex. BPD has been hypothesized to begin as acute inflammatory changes secondary to cell injury from toxic oxygen-derived free radicals that then evolve into acute and chronic lung disease.5 Premature infants, who may be particularly sensitive to the damaging effects of these oxygen radicals, are relatively deficient in endogenous antioxidant enzymes at birth.6,7 The superoxide dismutases (SODs) are ubiquitous antioxidant enzymes that have been shown in several animal studies to have a role in reducing lung injury caused by exposure to hyperoxia and mechanical ventilation.8-10 We have previously shown that a single intratracheal (IT) dose of recombinant human CuZn superoxide dismutase (rhSOD) is well-tolerated and significantly increases SOD concentration and activity in serum, tracheal aspirates (TA) and urine for 2 to 3 days in premature infants with RDS.11 In addition, TA markers of inflammation were significantly reduced over the first week of life in rhSOD treated infants compared with placebo controls, suggesting that less severe acute lung injury had occurred. However, it is unlikely that a single dose of rhSOD administered soon after birth would prevent the development of BPD.
It was the purpose of the present study to examine the safety and pharmacokinetics of multiple doses of rhSOD given to premature infants with RDS over the course of 14-days. Pharmacokinetics of rhSOD were evaluated as well as adverse effects and TA indicators of acute inflammation and lung injury.
Thirty-three infants were enrolled in this randomized, placebo controlled, blinded, dose ranging study at the six participating hospitals from November 1994 to June 1995. Neonates received either placebo (saline, n = 11), 2.5 mg/kg of rhSOD (n = 11) or 5.0 mg/kg of rhSOD (n = 11) dissolved in saline. Infants were eligible for enrollment if they were ≤24 hours of age, weighed 700 to 1300 g at birth, required intubation and mechanical ventilation for treatment of RDS (clinical and radiographic criteria) and had received surfactant therapy (Survanta, Ross Laboratories, Columbus, OH) within the first 24 hours of life. Infants were stratified by both center and birth weight (700 to 1000 g and 1001 to 1300 g). Infants were eligible to receive additional doses of surfactant if clinically indicated after rhSOD administration. Parental informed consent was obtained before study entry. The study was approved by the institutional review boards at all hospitals. Patients were excluded from the study if evidence of congenital infection, major congenital anomalies (chromosomal, cardiac, pulmonary, renal), or perinatal asphyxia was present. The study period included the first 28- days of the infant's life, although all adverse events were recorded until the time of hospital discharge.
The rhSOD used in this study was supplied by Bio-Technology General Corporation (Iselin, NJ) under Food and Drug Administration approval (IND 28 225). The rhSOD was produced in Escherichia coli utilizing recombinant DNA technology. The amino acid composition and sequence of rhSOD is identical to human SOD, although it lacks an N-terminal acetyl group, and is therefore defined as an analog. The rhSOD preparation has 4000 U of activity per mg of enzyme.
The placebo group received IT saline (1 mL/kg total volume), the low-dose group received 2.5 mg/kg rhSOD IT (rhSOD is dissolved in 1 mL/kg saline) and the high dose group received 5 mg/kg (1 mL/kg) of rhSOD. The initial drug or placebo was administered in two aliquots over a 1-minute period within 30 to 120 minutes after surfactant administration (because of potential drug-drug interactions when combining the two agents, the surfactant and rhSOD were given separately). Infants were placed in 30° of Trendelenberg and received the first aliquot in the right lateral decubitus position and the second aliquot in the left lateral decubitus position. Previous animal studies have shown that this technique optimizes pulmonary distribution of rhSOD after IT administration, allowing the drug to reach terminal airways and alveoli in a relatively homogeneous fashion.12Repeat doses were administered IT every 48 hours (suspended in 2 mL/kg of saline to improve pulmonary distribution) for up to 7 doses, as long as the infant continued to require intubation and mechanical ventilation. This dosing interval was chosen after initial pharmacokinetic studies demonstrated that a single IT dose significantly increased SOD concentration and activity in premature neonates for 48 to 72 hours.11 If infants were extubated before 14-days of age, then the study drug was discontinued. However, if infants were reintubated before 14-days of age, additional doses were administered every 48 hours until 14-days of life. rhSOD or placebo was never administered after 14-days of life. Laboratory evaluations obtained for safety and pharmacokinetics are listed in Table 1. Chest radiographs and cranial ultrasounds were interpreted by a single pediatric radiologist who was also blinded to treatment assignment.
Collection of Blood and TA Samples
Before drug administration, umbilical cord blood (0.3 mL) was obtained for the measurement of serum concentration and activity of endogenous SOD. Total serum SOD (rhSOD and endogenous SOD) was assayed on days 3, 5, 7, and 13 just before the administration of placebo or rhSOD. If the study drug was discontinued, final samples were obtained 48 to 72 hours after the last dose.
TA samples were obtained by instilling 1 mL of saline into the endotracheal tube and suctioning the fluid into a leukens trap. The catheter was then rinsed with an additional 1.5 mL of saline. The first TA was collected before surfactant administration (baseline). To prevent premature removal of drug, initial postdrug TA collections were performed 48 hours after placebo or rhSOD administration (if clinically possible). TA samples were subsequently collected just before study drug administration only if infants continued to require intubation and mechanical ventilation. The TA was then centrifuged at 350 × g for 10 minutes to pellet the cells. The supernatant was removed, transferred to clean microfuge tubes and recentrifuged at 5000 × g for 2 minutes, then divided into 4 aliquots and frozen at −70°C for SOD concentration and activity analyses as well as for assays of pulmonary inflammation. The cell pellet was resuspended in 100 μL of saline and live cells (exclusion of trypan blue) were counted using a hemacytometer. Cell differentials were determined using cytocentrifugation and staining with Diff-Quik (Baxter Scientific, Megaw Park, IL).
Urine Sample Collection
Each urine sample consisted of the total urine voided over a specified collection interval. Urine samples were collected and pooled for the first 24 hours from all infants after initial placebo or rhSOD administration. Subsequent samples were collected for 24 hours before the next scheduled dose.
rhSOD Concentration and Activity Measurements
Serum, TA, and urine samples were analyzed by radioimmunoassay using a monoclonal antibody specific for rhSOD as previously reported in our first clinical trial.11 Enzymatic activity of SOD was assessed by the reduction in cytochrome C.13 SOD activity gels and Western blots were also performed on urine samples.14 SOD activity in urine was measured using a 10% nonreducing polyacrylamide gel. Approximately 20 μg of protein from each sample was run in each lane, with one lane of molecular weight marker proteins and one lane for rhSOD. After electrophoresis, the gel was immersed in a solution of 2 mg/mL nitroblue tetrazolium in 100 μM riboflavin diluted 1:5 with water. After incubation (with 1% tetramethylethylenediamine) and shaking, water was added and the gel was exposed to fluorescent light for staining. The Western blot was run on a 4 to 20% gradient gel under denaturing conditions. Twenty μg of sample protein, molecular weight markers or rhSOD were loaded in each well and then transferred to polyvinyl membranes. Blotting was performed with a 1:1000 dilution of anti-rhSOD antibody and a 1:3000 dilution of antimouse immunoglobulin G (IgG) (Bio-Rad, Richmond, CA) conjugated with alkaline phosphatase. After washing, the color was developed with nitroblue tetrazolium (5-bromo-4-chloro-3-indolyl-phosphate; Boehringer Mannheim, Mannheim, Germany).
Biochemical Assessments of Lung Inflammation
In vitro neutrophil chemotaxis assays were used as an indicator of lung inflammation as previously described in our last trial.11 The chemotactic activity of the TA was calculated as the percent of the positive control (zymosan-activated serum). Background was determined with buffer alone. As a measure of the integrity of the alveolar-capillary barrier, serum albumin in TA was assayed by enzyme-linked immunosorbent assay.15
To determine if any infants had developed antibodies to rhSOD, serum was also obtained at baseline and at 28-days of life and tested by enzyme-linked immunosorbent assay. A 96-well MaxiSorp microtiter plate (Nunc, Denmark) was coated with rhSOD and incubated overnight. After blocking and rinsing, test sera and negative controls (1/100 and 1/500 dilutions) were added to the wells. The positive controls (mouse anti-rhSOD, BTG, Iselin, NJ) were then added at 5 dilutions from 1/100 to 1/1000. After incubation and rinsing, horseradish peroxidase conjugated antihuman IgG (Sigma, St Louis, MO) at 1/2000 dilution was added to the test wells and the negative controls. Horseradish peroxidase conjugated antimouse IgG was added at the same dilution to the positive controls. After incubation and rinsing, ABTS[2,2-Azino-di-(3-ethyl-benzthiaolin)] (Boehringer Mannheim, Indianapolis, IN) substrate solution was added and the plate read at 405 nm in a microplate reader.
Concentrations and activity of SOD and analyses of inflammatory markers in TA specimens over time were examined using analysis of variance (ANOVA). Log transformation was used to normalize the data (values expressed as geometric means with upper and lower confidence limits that are 1 standard error of the mean). The incidence of various complications was analyzed using χ2 and Fisher's Exact Test. Duration of ventilatory support and chest radiograph scores were analyzed with ANOVA and Student's t tests.
Thirty-three infants were enrolled in the trial. One infant receiving placebo died from overwhelming E coli sepsis and one infant in the 5.0 mg/kg rhSOD group died from pulmonary hypoplasia at a few hours of life, both shortly after receiving a single dose of the study medication. Data from these infants were included in the final analysis even though they met exclusion criteria before study entry. There were no statistical differences between the placebo and treatment groups with respect to sex, race, gestational age, birth weight, the use of antenatal steroids or tocolytics, mode of delivery, presence of bleeding or chorioamnionitis in the mothers, or 1- and 5-minute Apgar scores (Table 2).
Serum, TA, and Urine SOD Concentrations
Initial pretreatment concentrations of human SOD in serum [0.1 (0.05/(0.15)], TA [0.2 (0.1/(0.3)] and urine [0.3 (0.2/0.4)] μg/mL; geometric mean with lower–upper confidence limits that are 1 standard error of the mean were similar in all 3 groups at baseline. Infants in the placebo group received 3.5 ± 2.7 doses of study medication, that was similar to the 2 rhSOD treatment groups. The placebo group showed no increases in SOD during the first 14-days of life. In sharp contrast, total SOD concentrations increased significantly in both treatment groups when first measured on day 3 and did not change significantly thereafter over the 14-day dosing period. SOD concentrations in serum, TA, and urine were averaged over the 14-day dosing period. Data are presented in Fig 1(panels A,B,C). The 5.0 mg/kg rhSOD group had significantly higher SOD levels compared with the 2.5 mg/kg and placebo groups when measured by radioimmunoassay (P < .05, ANOVA). In addition, the highest concentrations of SOD were detected in urine, followed by TA and then serum. Because it was somewhat surprising to find such relatively high levels of immunoreactive SOD in urine, we determined whether the antibody had recognized a proteolytic fragment or intact SOD. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotted and incubated with anti-SOD antibody. Fig 2 (A) shows that the antibody reacted with intact SOD and not with peptides as small as 8.0 kDa (the lower limit of gel resolution). These data indicate that the rhSOD is freely filtered by the kidney of the premature infant.
To determine if the immunoreactive protein was enzymatically active, samples were also analyzed for SOD activity in serum, TA, and urine. SOD concentration correlated directly with activity of the enzyme (P < .05). Serum SOD activity was 2.5 (1.5/4.3) U/mL in the placebo group, 8.3 (7.6/9.1) U/mL in the 2.5 mg/kg group, and 8.5 (7.5/9.7) U/mL in the 5.0 mg/kg group. TA SOD activity was 7.8 (6.7/9.1) U/mL in the placebo group, 17.0 (12.0/23.9) U/mL in the 2.5 mg/kg group, and 21.1 (8.6/51.8) U/mL in the 5.0 mg/kg group. These differences were not statistically significant due to increased variability. To determine if the enzyme was limited to intact SOD and not to a proteolytic fragment that was not immunoreactive, proteins were analyzed by polyacrylamide gel zymography. Fig 2B shows that enzyme activity was limited to protein migrating with intact rhSOD. These data indicate that enzymatically active and immunodetectable SOD remains undegraded, even in urine.
TA Markers of Acute Inflammation
The presence of any chemotactic stimulus present in TA was analyzed using a neutrophil chemotaxis assay. Activity was low and similar for all 3 groups at baseline. However, neutrophil chemotactic activity was significantly lower than the placebo group when data from the rhSOD treatment groups were combined and analyzed over the first 2 weeks of life (P < .05; Fig 1D). Albumin concentrations in TA were assayed as a measurement of macromolecular edema. Albumin levels were similar in all 3 groups at baseline. Albumin concentrations averaged 108.9 (92.3/128.5) μg/mL in the placebo group, 102.0 (82.1/126.7) μg/mL in the 2.5 mg/kg group, and 61.4 (36.6/103.1) μg/mL in the 5.0 mg/kg rhSOD treatment group over the 14-day treatment period. These differences did not reach statistical significance due to increased variability. Samples were obtained and data analyzed over the first 2 weeks of life because most infants are weaned from mechanical ventilation by that time and relatively little data would be available at later timepoints for statistical analyses.
The sera of infants were tested for the presence of anti-SOD antibodies that might have been produced in response to therapy. There were no anti-rhSOD antibodies detected in the serum from any placebo or rhSOD treated infant at baseline. At 28-days of life, an extremely low, although detectable antibody level was found in one infant in the 2.5 mg/kg group who had received 2 doses of rhSOD. The observed antibody level was well below the linear range of the assay (less than one third of lowest standard concentration).
Clinical Outcome Variables
No specific side effects of rhSOD treatment were noted. One infant died from necrotizing enterocolitis 39-days after receiving a single dose of rhSOD. Another infant died from respiratory failure 39 days after receiving the fourth dose of rhSOD. Both deaths occurred in the 2.5 mg/kg group after the 28-day study period ended. Neither death was attributed to study drug.
To assess initial disease severity, chest radiographs were examined to confirm the diagnosis of RDS, with severity assigned by a score of 1 (mild) to 5 (severe). The placebo group had a score of 1.8 ± 0.4, the 2.5 mg/kg rhSOD group scored 2.6 ± 0.5, and the 5.0 mg/kg group scored 2.3 ± 0.5 (P = NS, ANOVA). BPD was defined as oxygen dependency at 28-days of life and an abnormal chest radiograph (Edwards Score ≥4).16 Three infants developed BPD, 2 in the low-dose and 1 in the high dose rhSOD group. When an alternative definition of BPD was used (O2dependency at 36 weeks postconceptual age), then one infant from each of the three groups developed BPD.17
Intraventricular hemorrhage occurred in 4 infants: 2 in the placebo group and 1 each in the low and the high dose rhSOD groups. One infant (5.0 mg/kg rhSOD group) had a clinically significant (more than grade II) intraventricular hemorrhage that was detected on a baseline cerebral ultrasound before rhSOD administration. No significant differences were found among any of the groups in any other adverse event (Table 3). There were 5 cases of sepsis in the low-dose, 2 in the high dose group and 1 in the placebo group. Coagulase-negative staphylococcal infections caused 60% of cases in the low-dose and both cases in the high dose groups. These infants had significantly more invasive procedures performed (ie, chest tubes, central venous catheters) compared with placebo treated infants. Even when the 2 rhSOD treatment groups were combined and compared with placebo (Fisher's exact test), the incidence of sepsis was still not significantly increased.
There were no significant differences among the 3 groups with regard to total days in oxygen or the duration of mechanical ventilatory support (ventilation and continuous positive airway pressure). When all laboratory parameters were analyzed (Table 1), no significant differences between groups were identified.
A popular hypothesis to explain the pathogenesis of BPD is that hyperoxia and mechanical ventilation damage cells in the lung.18 Under normoxic conditions, the production of oxygen-derived free radicals is counter-balanced by antioxidant defenses. However, under conditions of hyperoxia (even 21% O2 is supraphysiologic with a premature infant) or inflammation, oxy-radicals are not adequately scavenged. Premature infants appear to be especially susceptible because concentrations of antioxidant enzymes may be inadequate and the enzymes may not be inducible in response to hyperoxia.7 Damage caused by oxy-radicals can be extensive, including cell membrane destruction and modification of macromolecules.19 Oxy-radicals can also directly cause cell death either by apoptosis or necrosis, which can markedly interfere with normal lung development during a particularly important period of lung growth.19
A rational strategy for the prevention of hyperoxic lung disease (BPD) is to augment antioxidant enzyme activity. In particular, the use of rhSOD is based on extensive cell culture, animal experiments and limited experience in human subjects. In genetically engineered mice with disrupted extracellular SOD genes and no functional extracellular SOD, exposure to 100% O2 resulted in significantly more lung injury and reduced survival compared with normal diploid controls.20 In a different study, mice overexpressing manganese superoxide dismutase in type II cells were able to survive much longer in a hyperoxic environment compared with control animals, that had relatively low levels of manganese superoxide dismutase in type II cells.21 Several other animal studies using intravenous, intraperitoneal, or IT administration of SOD have shown significant improvements in lung morphology and survival from prolonged hyperoxia.7-10 We have recently demonstrated that rhSOD is rapidly incorporated into a variety of cell types in the lung after IT administration and prevents the inflammatory changes and acute lung injury from 48 hours of hyperoxia and mechanical ventilation in newborn piglets.8,22 Taken together, these studies demonstrate that SOD can be efficacious in preventing hyperoxic lung injury and provided the impetus for clinical trials in premature infants.
In our first clinical trial, 26 premature infants with RDS received a single IT dose of either placebo or rhSOD immediately after surfactant treatment.11 We demonstrated that rhSOD administration appeared to be safe and resulted in increased SOD concentration and activity in serum, TA, and urine for 2 to 3 days, compared with placebo controls. However, it seemed unlikely that a single dose of rhSOD at birth might completely prevent lung damage from prolonged hyperoxia and mechanical ventilation and the subsequent development of BPD.
The dosing strategies used in the present study resulted in an average four- to sevenfold increase in SOD concentration in serum, TA and urine over the 14-day dosing period. Interestingly, the rhSOD appears to be freely filtered by the kidney because it remains largely intact and active in urine. The molecular weight of the rhSOD is sufficiently small (31 kDa) so that it can be filtered in the glomerulus and most likely not reabsorbed in the distal tubule.
rhSOD appeared to be well-tolerated and was not associated with any obvious toxicity. However, our concern was aroused by the increased number of infants in the low-dose rhSOD group who developed late onset sepsis. In principle, the rhSOD could interfere with superoxide generation by neutrophils that might impair bacterial killing and increase infections. However, available data suggest that rhSOD does not affect neutrophil chemotaxis or bacterial killing in vivo.8,23 Moreover, because the majority of infections were seen in the low-dose group, it seems unlikely to be the result of the study drug. It is important to note that the infections in the rhSOD treatment groups were primarily caused by coagulase negative staphylococci, the most common organism found in late onset neonatal sepsis.24 Other potential problems with a low incidence may not have been observed, due to the small numbers of patients studied. Similarly, there were too few patients studied to permit an accurate assessment of efficacy. However, the rhSOD treated infants did have significantly reduced markers of acute inflammation in their TA over the first 2 weeks of life. Similar findings were seen both in our animal studies and earlier clinical trials.8,11 This is notable, because the early inflammatory changes seem to be an important determinant in the pathogenesis of BPD.5,13,25 Future studies involving larger number of infants are needed to ensure safety and demonstrate efficacy. The incidence of BPD (approximately 10%) found here is significantly lower than the 30% to 60% reported in the literature for infants of this gestational age and birth weight.5 This is most likely the result of small numbers of infants in the present study, but could influence the number of infants enrolled in future clinical trials of the efficacy of rhSOD in preventing BPD.
In summary, BPD continues to be a major sequelae of neonatal intensive care. Both the human and financial consequences of BPD can be overwhelming. To date, attempts to prevent BPD have not proven to be efficacious. A new direction in treatment—augmentation of antioxidant defenses—needs to be further evaluated. This study provides a stepping stone for further investigations for the prevention of BPD and other disease processes of premature infants involving oxygen-free radical injury.26
The authors wish to thank Drs T. Allen Merritt, Jacob V. Aranda, Ivan D. Frantz III, and Sylvan Wallenstein and Lisa Salerno for their contributions to the study. We would also like to acknowledge the support of the neonatal intensive care unit staffs at all the participating hospitals.
- Received September 23, 1996.
- Accepted November 18, 1996.
Reprint requests to (J.M.D.) Department of Pediatrics, Winthrop-University Hospital, SUNY Stony Brook School of Medicine, 259 First St, Mineola, NY 11501.
- RDS =
- respiratory distress syndrome •
- BPD =
- bronchopulmonary dysplasia •
- SOD =
- superoxide dismutase •
- IT =
- intratracheal •
- rhSOD =
- recombinant human CuZn superoxide dismutase •
- TA =
- tracheal aspirates •
- IgG =
- immunoglobulin G •
- ANOVA =
- analysis of variance
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- Copyright © 1997 American Academy of Pediatrics