Poster Number-59 - The Agnostic Respiratory Stimulant ENA-001 Demonstrates Preliminary Positive Results in an Animal Model of Apnea of Prematurity

The Agnostic Respiratory Stimulant ENA-001 Demonstrates Preliminary Positive Results in an Animal Model of Apnea of Prematurity

Purpose: ENA-001 is a novel selective antagonist of large-conductance BK (big potassium) channels located in glomus cells of the carotid bodies. BK channels act as important components of the chemoreception that primarily senses low arterial oxygen levels and establish a feedback loop to brainstem nuclei responsible for initiating spontaneous breathing and maintaining adequate oxygen to tissues.  In a number of animal and human studies, ENA-001 successfully attenuated the respiratory depression induced by a variety of chemical agents, its effect being essentially 'agnostic' to the precipitating drug (e.g., opioid, benzodiazepine, alcohol, propofol). But it was never tested against respiratory depression resulting from a physiological cause, such as apnea of prematurity (AOP).  This proof-of-principle study using a well described animal model of prematurity (premature lamb) was initiated to test the effectiveness of ENA-001 in the setting of an under-developed respiratory control system, similar to that in human AOP.

Methods:

The protocol described one set of twin lambs to be delivered premature via Caesarian section and stabilized as previously described [1,2].  Based on experience with the model, the investigators targeted 135 ± 2 d gestational age (GA) as it was expected that the lambs would have a spontaneous respiratory drive and could be managed for four hours with continuous positive airway pressure (CPAP); i.e., not require mechanical ventilation. Once the umbilical cord was ligated and severed, the lamb was transferred to a heated surgical bed under a radiant warming lamp. An arterial catheter was connected to a transducer for pressure monitoring and a venous catheter was connected to a pump for continuous infusion of 5% dextrose in water (D5W) at a rate of 6 ml/kg/hr. The airway was suctioned and Surfactant was administered intratracheally over a period of 5 min. Lambs were to receive four mechanical breaths for lung recruitment and then started on CPAP. After a stabilization period of 15 min, the protocol called for the first lamb to be started on continuous infusion of ENA-001, with ascending dose hourly, while the second lamb was to serve as a sham (D5W) control. The ENA-001 treatment was designed to be started without a loading dose such that plasma concentrations would climb over the course of each hour and should contribute to an ascending dose effect. The first three doses have been used in human studies of efficacy, where the lower dose had minimal effect, the second dose is the intended adult therapeutic dose (following a loading dose), the third dose is the intended adult human loading dose, and the last hourly dose rate is given to support toxicology analysis in the immature tissues (10-fold the intended adult therapeutic dose). At least ten representative breaths free of artifact from motion or atypical breaths were recorded using a pulmonary function system designed for neonatal research.  For each period, the rate, tidal volumes and minute ventilation were derived from an average of the ten breaths. To maintain a stable plane of anesthesia, repeat doses of fentanyl (1 µg IM) were given as needed based on blood pressure response to stimulation.

Results:

Two male lambs were delivered.  However, neither lamb exhibited a drive for spontaneous breathing.  Each required manual ventilation, with a complete absence of spontaneous effort. Despite the poor prognosis owing to absence of ventilatory effort, continuous infusion of the first dose of ENA-001 was started 20 min following birth. The test animal continued to require manual ventilation, which was continued for an additional 10 min.  The decision was made to give an intravenous bolus infusion of ENA-001 at a dose that has yielded therapeutic plasma levels in previous animal studies.  Nearly instantaneously following the delivery of the IV bolus, the lamb began breathing spontaneously and never again required manual intervention for the remainder of the study.  For the remainder of the four hours following the initiation of the first infusion dose, the doses were changed hourly as per the protocol.

The sham animal was delivered approximately an hour following the test animal. As with the test animal, the sham animal lacked spontaneous breathing efforts.  A decision was made to manually ventilate for 30 minutes to match the course for the test animal, and if still not breathing, would be determined inviable and sacrificed for the control tissue samples. At the 30-minute timepoint, an intravenous bolus infusion of ENA-001 was delivered.  Nearly instantaneously following the delivery of the IV bolus, the lamb began breathing spontaneously.  After several minutes, the spontaneous breathing efforts abated, and manual ventilation was resumed.  The animal was then sacrificed for tissue harvest.

Conclusion: This proof-of-principle study using a well described animal model of prematurity (premature lamb) was initiated to test the effectiveness of ENA-001 in the setting of an under-developed respiratory control system, similar to that in human AOP.  We found that ENA-001 was effective in this model of prematurity. In fact, ENA-001 induced spontaneous breathing, and the lambs were then able to maintain effective spontaneous breathing without additional support. These results suggest that ENA-001 might be effective therapy alone or as comedication for the management of AOP. They further suggest that ENA-001 might have broader applications in situations of neurological ventilatory insufficiency. ENA-001 currently is undergoing further clinical testing and development for a variety of therapeutic and emergency uses.

References: 1. Wolfson MR, Wu J, Hubert TL, Gregory TJ, Mazela J, Shaffer TH. Lucinactant attenuates pulmonary inflammatory response, preserves lung structure, and improves physiologic outcomes in a preterm lamb model of RDS. Pediatr Res. 2012;72(4):375-383.
2. Shashikant BN, Miller TL, Welch RW, Pilon AL, Shaffer TH, Wolfson MR. Dose response to rhCC10-augmented surfactant therapy in a lamb model of infant respiratory distress syndrome: physiological, inflammatory, and kinetic profiles. J Appl Physiol (1985). 2005;99(6):2204-2211.