Background:

Preterm neonates can develop chronic pulmonary insufficiency of prematurity (CPIP) later in infancy. Recombinant human CC10 protein (rhCC10) is an anti-inflammatory agent that could potentially prevent CPIP.

Methods:

The safety and efficacy of a single intratracheal dose of rhCC10 in reducing CPIP at 12 months corrected gestational age (CGA) was evaluated in a Phase II double-blind, randomized, placebo-controlled, multisite clinical trial. Eighty-eight neonates were randomized: 22 to placebo and 22 to 1.5 mg/kg rhCC10 in the first cohort and 21 to placebo and 23 to 5 mg/kg rhCC10 in the second cohort. Neonates were followed to 12 months CGA.

Results:

With CPIP defined as signs/symptoms, medical visits, hospital readmissions, and use of medications for respiratory complications at 12 months CGA, no significant differences were observed between rhCC10 or placebo groups. Only 5% of neonates had no evidence of CPIP at 12 months CGA.

Conclusions:

A single dose of rhCC10 was not effective in reducing CPIP at 12 CGA. Since most neonates had evidence of CPIP using these exploratory endpoints, it is essential to develop more robust outcome measures for clinical trials of respiratory medications in high risk premature neonates.

Table 1:

Study medication:

The rhCC10 study medication was supplied by Therabron Therapeutics, Inc. (Rockville, MD) in a 5.5 mg/ml formulation that was diluted to 1.5 or 5 mg/kg (volume was constant at 2 ml/kg). The placebo was normal saline. The pharmacist received a computer generated, randomized treatment assignment at enrollment. The rhCC10 study drug was a clear colorless liquid indistinguishable from placebo. All other hospital personnel were blinded to the treatment received.

Dose selection:

The Phase 1 clinical trial evaluated doses of 1.5 mg/kg and then 5.0 mg/kg (in 2 ml/kg) in premature neonates and was based on safety and efficacy analyses in several animal models (10,11). Similar escalating dosage approaches were used in the present trial, with an initial low-dose cohort enrolling 50% of the neonates followed by a second high-dose cohort that enrolled the remainder of the sample.

Drug administration:

The single dose of study drug was administered intratracheally within the first 24 hours of life and within 4 hours of the first dose of exogenous surfactant (Curosurf™). Doses were calculated based on the neonate’s birthweight. The drug was administered in an identical fashion to surfactant administration.

Study Design:

The study used a multicenter, double-blind, placebo-controlled, randomized, dose-escalation design. In the first cohort (n=44), neonates were randomized to either placebo or 1.5 mg/kg rhCC10. Following a safety assessment by the Data Safety Monitoring Board (DSMB), the second cohort (n=44) was randomized to either placebo or 5 mg/kg rhCC10.

Inclusion criteria:

1) ≤24 hours of age, 2) 24 0/7 – 29 0/7 weeks gestational age at birth based on best estimate using obstetrical sonography (first or second trimester), solid dating criteria, or Ballard examination, 3) birth weight 600–1200 g, 4) 5 minute Apgar score >5, 5) diagnosis of respiratory distress syndrome (clinical, radiographic criteria), 6) intubated and receiving positive pressure mechanical ventilation, 7) growth parameters at birth appropriate for gestational age; 8) received at least one dose of surfactant, and 9) parent or guardian has signed informed consent.

Exclusion criteria:

1) 5 minute Apgar score of ≤5, 2) major congenital anomaly (chromosomal, renal, cardiac, neurologic, or pulmonary malformations), 3) congenital infection, 4) use of postnatal corticosteroids before enrollment, 5) mother and/or neonate enrolled in another clinical trial of an investigational drug, 6) requirement for a major surgical procedure within the first 48 h of life, 7) use of inhaled nitric oxide, 8) mother known to be seropositive for HIV, and 9) parent or guardian unable or unwilling to complete study procedures after hospital discharge.

Enrollment/Randomization:

In each cohort, subjects were randomized to either placebo or rhCC10. Treatment was stratified by gestational age (24 0/7 – 27 0/7; 27 1/7 – 29 0/7 weeks) and clinical center. Multiple births were randomized separately in order of birth. The protocol was approved by the Institutional Review Board (IRB) at each participating institution.

Post-discharge visits:

Following discharge, mothers for each neonate were contacted monthly to obtain real-time information and visits were scheduled at 6 and 12 months CGA for clinical evaluation. Parents filled out comprehensive diaries daily for 4 consecutive weeks from 5–6 months and 11–12 months CGA (5). Diaries documented wheezing, coughing, and the use of respiratory medications (steroids, bronchodilators, leukotriene inhibitors) secondary to significant respiratory illness. Positive findings were reported as coughing, wheezing, or respiratory medication use at least 2 days per week for at least 3 consecutive weeks over 4 consecutive weeks. Pulmonary questionnaires previously developed for the SUPPORT trial were also used (12).

Interim Safety Evaluations:

The DSMB performed safety assessments, evaluating all Adverse Events (AE) and Serious Adverse Events (SAE) when 25%, 50%, and 75% of infants were enrolled. A preplanned pause in enrollment occurred at the end of the first cohort. An unplanned pause in enrollment occurred at the request of the DSMB when 75% of infants were enrolled for the purpose of conducting a comprehensive review of all deaths in the study. Upon completion of the review, enrollment was continued and enrollment was completed.

Clinical Trial Sites:

The first low-dose cohort of 44 neonates was enrolled at two centers in Boston, MA (Tufts Medical Center, Brigham and Women’s Hospital). Due to slow enrollment, multiple US sites were contacted during and invited to participate in the second cohort of the study. One site (BayState Medical Center, Springfield, MA) did not have any ongoing studies that would compete with this trial and agreed to participate. All other US sites declined. In addition, orphan drug designation was awarded in Europe, so three new European sites with experience in neonatal clinical trials were added for the second high-dose cohort of 44 neonates, including Ginekologiczno-Położniczy Szpital Kliniczny UM (Poznan, Poland), Instytut Centrum Zdrowia Matki Polski (Lodz, Poland), and Samodzielny Publiczny Zakład Opieki Zdrowotnej Szpital Uniwersytecki w Krakowie (Krakow, Poland).

Primary Outcome Measure:

The primary combined endpoint was survival without evidence of CPIP at 12 months CGA. CPIP was defined a priori as the presence of one or more parent-reported outcomes at 12 months CGA including: 1) evidence of respiratory symptoms (e.g. coughing and wheezing) or use of respiratory medications by parental diaries or pulmonary questionnaires (CPIP-SS), 2) one or more re-hospitalizations for respiratory causes (CPIP-RH), 3) administration of respiratory medications (including oxygen) (CPIP-RM), and 4) at least one non-routine medical visit for respiratory causes (CPIP-DV). Neonatal outcomes were graded as being positive for 1, 2, 3, or 4 of these components. Other exploratory efficacy endpoints of interest were: a) alive without evidence of CPIP at 6 months CGA and b) evidence of CPIP in survivors at 6 and 12 months CGA.

Safety Endpoints:

Safety evaluations included all SAEs before and after NICU discharge and routine laboratory monitoring (CBC, electrolytes, liver function studies, urinalysis). Less serious adverse events (AEs) were also monitored and recorded prior to NICU discharge.

Anti-Drug Antibody (ADA) Testing:

Western blots were performed of serum samples collected at baseline (pre-dosing), 28 or 35 days of life, and/or at 12 months CGA. Briefly, rhCC10 was run on SDS-PAGE (55 mcg/well), reduced and non-reduced, and incubated in 10 µL serum diluted 1:1000 in 5% Blotting Grade Blocker and incubated overnight at 4°C. The samples were washed, then incubated with rabbit anti-human IgG antibody alkaline phosphatase conjugate diluted 1:20,000 in 0.1% Blotting Grade Blocker and incubated 1hr at room temperature. The blot was then washed and developed. Each blot of 4 lanes had a pre-stained size standard, reduced and non-reduced rhCC10, and purified E. coli malate dehydrogenase was used as a positive control. A total of 43/88 subjects had all 3 sequential samples available for analysis.

Growth and Neurologic Outcomes:

Serial physical and neurological examinations at 6 and 12 months CGA were performed. Growth measurements (weight, length, head circumference) were used to examine the relationship between CPIP, treatment, and growth.

Statistical Analysis:

Table 2:

Growth:

There were no significant differences between groups with respect to the mean weight, length, or head circumference of neonates at birth, 6, and 12 months CGA.

Mortality and safety:

The overall survival rate in the study was 90% (79/88 infants). Two of the deaths were in the 1.5 mg/kg rhCC10 group, 5 were in the 5 mg/kg rhCC10 group, and 2 were in the placebo group (both in the second cohort). There were no significant differences in mortality between groups. None of the deaths were thought to be related to study medication. The causes of death were varied and typical of this population.

There were 912 AEs and 198 SAEs in the 88 neonates enrolled in the study.Table 3 shows that there were no significant differences in the number of AEs and SAEs between placebo and rhCC10 treatment groups in each cohort. One hundred forty-five SAEs occurred before NICU discharge and 53 after discharge. Of these 53 post-discharge SAEs, 51 were respiratory infections. There were also no differences between the low and high dose rhCC10 groups with respect to AEs and SAEs. Serial serum samples for ADA testing demonstrated no evidence of anti-drug antibodies in any sample.

Efficacy:

Using our a priori definition of CPIP, only 5% of neonates in the first cohort (a single neonate in each arm) and 14% of neonates in the second cohort had none of the CPIP components at 12 months CGA (Table 4). Overall, administration of a single intratracheal dose of rhCC10 did not appear to significantly improve respiratory outcome in either treatment group when compared to placebo. In addition, results did not change when the two treatment groups were combined and compared to the two placebo groups. Sub-group analyses (not part of the a priori definition) demonstrated that the proportion of neonates with 3 or fewer components of CPIP at 12 months CGA was significantly higher in the placebo group than the low dose rhCC10 group. These analyses also indicated that a greater proportion of neonates in the low dose rhCC10 met criteria for all 4 CPIP components compared to those who received placebo. This difference between arms was not observed in the high dose cohort.

To understand the relative contributions of the four types of exploratory CPIP components, the number of neonates scoring positive for each individual component was evaluated. The distribution at 12 months CGA is shown inTable 5. The number of neonates without any non-routine (unscheduled) respiratory medical visits and respiratory re-hospitalizations was significantly higher in the placebo than the low dose rhCC10 regardless of imputation for death and missing data. However, no significant differences were seen in any CPIP component in the second cohort when placebo was compared to high dose rhCC10.

The most widely used definition of BPD is the use of oxygen at 36 weeks PMA which was an important secondary outcome. There were no significant differences between treatment groups in either cohort, or between cohorts, in the number or percent of neonates that were alive without BPD at 36 weeks PMA, or in the number of survivors that were on oxygen at 36 weeks PMA. In addition, neonates who were alive without BPD at 36 weeks PMA had fewer CPIP components at 12 months CGA (Table 6).

Environmental and genetic risk factors for CPIP:

Table 7 shows the frequencies of neonates by treatment groups (placebo vs rhCC10) with various risk factors for CPIP. Each neonate exposed to cigarette smoke experienced one or more components of CPIP.

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