The non-intubated, spontaneously breathing, continuous positive airway pressure (CPAP) ventilated pre-term lamb: A unique animal model

Abstract

Neonatologists prefer non-invasive ventilation methods for pre-term neonates, who often require surfactant treatment. Therefore, a technology for non-invasive surfactant administration would be highly appreciated. We have developed a Continuous Powder Aerosolization (CPA) system for the generation of a humidified recombinant surfactant protein-C (rSP-C) surfactant aerosol for non-invasive administration to pre-term neonates via bi-nasal prongs. Before conducting clinical trials, safety testing in an adequate pre-clinical animal model is necessary. In contrast to existing pre-term lamb models, this model should use non-intubated animals to include upper airways for safety testing. Pre-term animals should have already a sufficient respiratory drive to breathe spontaneously on non-invasive continuous positive airway pressure (CPAP) support, but their lungs should still be pre-mature to be comparable with the clinical situation for the treatment of pre-term infants. The aim of this feasibility study was therefore to establish a CPAP-stable, non-intubated pre-term lamb model for the investigation of safety, efficacy, and pulmonary deposition of a humidified rSP-C surfactant aerosol. For this purpose, 19 pre-term lambs with a gestational age of 135–137 days (term: about 144 days) were delivered via Caesarean section. Four animals died before start of treatment, while the remaining animals were treated via customized bi-nasal prongs with rSP-C surfactant aerosol or humidified air as vehicle control. To determine pulmonary deposition, selected animals received rSP-C surfactant labelled with samarium oxide as non-radioactive tracer. Treatment was started at 30 min of age and was continued for 1 or 2.5 h. Investigations during the in-life phase included observation of clinical signs, haematology, blood gas analysis, and determination of minute volume. At 3 h of age, animals were euthanized and organs removed for histopathology investigation or for determination of pulmonary deposition. Administration of humidified, aerosolized rSP-C surfactant was well tolerated, and histopathology investigation of upper airways and lungs revealed no aerosol-related changes. Mean body weight-corrected pulmonary deposition of rSP-C surfactant ranged from 1.7 to 7.7 mg/kg depending on the duration of treatment and aerosolization parameters used. A trend towards reduced spontaneous minute volumes indicating reduced breathing efforts and towards reduced lung weights indicating less fluid in the lungs of surfactant-treated animals compared to animals of the vehicle control group could be seen. Taken together, a CPAP-stable, non-intubated pre-term lamb model was successfully established and the parameters for the investigation of safety, efficacy, and pulmonary deposition of aerosolized rSP-C surfactant for the subsequent main study were identified.

Introduction

Pre-term delivery is the most important cause of perinatal morbidity and mortality in developed countries [1], and pre-term infants often require ventilatory support. For ventilation of pre-term neonates, neonatologists prefer non-invasive ventilator support such as nasal continuous positive airway pressure (CPAP) over invasive positive pressure ventilation (reviewed by Mahmoud et al. [2]). Unfortunately, CPAP failure rates remain high [3], [4], [5], at least partly due to the high surface tension in the lungs of pre-term neonates lacking endogenous surfactant. The standard treatment is a combination of respiratory support (i.e. mechanical ventilation) and surfactant replacement therapy with a natural or synthetic surfactant suspension [6]. However, the currently available surfactant preparations require endotracheal intubation and mechanical ventilation. Mechanical ventilation as well as low gestational age and increased oxygen levels are known risk factors for the development of bronchopulmonary dysplasia [7], [8], [9], [10], a debilitating condition resulting in long-term impairment in general and respiratory health [11]. Newer approaches like the transient intubation technique INSURE (INtubation – SURfactant treatment – Extubation) try to shorten the period of mechanical ventilation, but still require endotracheal intubation for the administration of the surfactant [12], [13], [14]. Recently, a new method has been successfully tested where pre-term infants received a surfactant suspension during spontaneous breathing via a thin catheter inserted into the trachea by laryngoscopy [15]. This technique avoids mechanical ventilation, but requires highly skilled personnel and is still invasive. Since the intubation procedure as well as the laryngoscopy is an invasive method, a non-invasive procedure, such as inhalation of a surfactant aerosol, is desired.

Nycomed GmbH, the Fraunhofer Institute, and the MTF MediTech Franken GmbH have developed a surfactant aerosolization technology based on Continuous Powder Aerosolization (CPA) of recombinant surfactant protein-C (rSP-C) surfactant. The generated aerosol is introduced via a side port directly into customized bi-nasal prongs that are inserted into the nostrils of the baby and that are connected to the ventilatory circuit of a standard ventilator. The advantage of this setting is that it does not require endotracheal intubation as well as no invasive mechanical ventilation as long as the patients breathe spontaneously. The CPA system is a completely new device that has not yet been tested in humans. Before testing of this new device in humans, safety testing in an appropriate animal model is necessary to fulfil ethical and regulatory requirements. Since CPAP failure occurs mainly in pre-term neonates lacking endogenous surfactant, the animal model should be pre-term as well and should have pre-mature lungs to be comparable with the clinical situation. This safety testing should be performed by using the new surfactant CPA system in combination with the customized bi-nasal prongs.

For non-clinical testing of surfactant suspensions, mainly two primary surfactant deficiency models are described in the literature: the pre-term rabbit [16], [17], [18], [19], [20], [21] and the pre-term lamb [16], [22], [23], [24], [25], [26], [27]. In both animal models, pre-term animals are obtained by Caesarean section (C-section). The pre-term rabbit model is a very suitable animal model if the pre-term animals should be mechanically ventilated via an endotracheal tube (after tracheotomy) and the surfactant suspension is administered directly intratracheally. For the present surfactant aerosol, testing of the clinical route of administration (i.e. inhalation via bi-nasal prongs) is desired. However, due to the small size of the pre-term rabbits, placement of bi-nasal prongs is impossible. Therefore, the pre-term rabbit model seems not suitable for testing of an inhaled surfactant aerosol using bi-nasal prongs.

Likewise, the pre-term lamb model is a very suitable animal model if the pre-term animals should be mechanically ventilated via an endotracheal tube (after tracheotomy or after intubation) and the surfactant suspension is administered directly intratracheally. Internal studies have shown that the animals have a sufficient size so that placement of customized bi-nasal prongs into the nostrils of the lambs is possible. Additionally, the pre-term lambs at an “appropriate” gestational age have a sufficient respiratory drive so that they are able to breathe spontaneously on CPAP support. Therefore, the pre-term lamb model was considered to be the appropriate animal species for testing of a surfactant aerosol in a comparable setting as to be used in the clinic.

The aim of this feasibility study was to establish a CPAP-stable, non-intubated pre-term lamb model where the lungs of the animals are still pre-mature, but where the animals have already a sufficient respiratory drive to breathe spontaneously via customized bi-nasal prongs on CPAP support. Additionally, the technical settings of the used CPA and ventilator system for adequate ventilation support of the lambs as well as for sufficient pulmonary deposition of the inhaled surfactant aerosol should be identified. Furthermore, appropriate parameters for safety evaluation of the surfactant aerosol should be selected for the subsequent main study. Ideally, also efficacy parameters should be identified to be included into the following main study.