New Surfactant with SP-B and C Analogs Gives Survival Benefit after Inactivation in Preterm Lambs
et al. (2012) New Surfactant with SP-B and C Analogs Gives Survival Benefit after
Inactivation in Preterm Lambs. PLoS ONE 7(10): e47631. doi:10.1371/journal.pone.0047631
New Surfactant with SP-B and C Analogs Gives Survival Benefit after Inactivation in Preterm Lambs
Matthias Seehase 0 1
Jennifer J. P. Collins 0 1
Elke Kuypers 0 1
Reint K. Jellema 0 1
Daan R. M. G. Ophelders 0 1
Olga L. Ospina 0 1
J. Perez-Gil 0 1
Federico Bianco 0 1
Raffaella Garzia 0 1
Roberta Razzetti 0 1
Boris W. Kramer 0 1
Lynette Kay Rogers, The Ohio State Unversity, United States of America
0 Current address: Department of Pediatric Cardiology, University of Bonn , Bonn , Germany
1 1 Department of Pediatrics, Maastricht University Medical Center , Maastricht , The Netherlands , 2 Research and Development Department, Chiesi Farmaceutici SpA , Parma , Italy , 3 Department of Biochemistry, Faculty of Biology, Complutense University , Madrid , Spain , 4 Department of Physics, Pontificia Universidad Javeriana , Bogota , Colombia
Background: Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo. Methodology/Principal Findings: Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if PaO2 dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals. Conclusions: For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.
Funding: BWK is supported by a VENI grant (No. BWK 016.096.141) from the Dutch Research Council (NWO). OLO and JPG are supported by grants from the
Spanish Ministry of Economy and Competitivity (BIO200909694, CSD200700010) and Community of Madrid (S2009MAT-1507). This study was funded by Chiesi
Pharmaceutici SpA (Parma, Italy). Preparations of CHF 5633 synthetic surfactant and CurosurfH were also supplied by Chiesi Pharmaceutici SpA. FB, RR and RG
participated in the interpretation of the data and review of the manuscript, but otherwise the funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have the following interests. This study was funded by Chiesi Pharmaceutici SpA (Parma, Italy), which is the employer of
authors Raffaella Garzia and Roberta Razzetti. Preparations of CHF 5633 synthetic surfactant and CurosurfH were also supplied by Chiesi Pharmaceutici SpA. As
control treatment, the animal derived surfactant Poractant alfa (CurosurfH, 80 mg/ml, Chiesi Farmaceutici SpA, Parma, Italy) was used, which is frequently used in
clinical practice. All preparations were supplied by Chiesi Farmaceutici SpA (Parma, Italy). There are no further patents, products in development or marketed
products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for
. These authors contributed equally to this work.
Respiratory distress syndrome (RDS) is a significant cause of
morbidity and mortality in preterm infants . RDS is caused
by a deficiency, dysfunction, or inactivation of pulmonary
surfactant [5,6]. Surfactant lowers surface tension and improves
pulmonary dynamic compliance. Numerous surfactants of either
animal extract or synthetic design have been developed and tested
. Although both synthetic and animal derived surfactant
preparations have been shown to be beneficial, studies comparing
animal derived surfactant preparations to synthetic preparations
have demonstrated improvement in immediate ventilator support,
decreased risk of pneumothorax, and decreased risk of mortality in
infants receiving the animal derived products . Furthermore,
there is a marginal decrease in chronic lung disease among
preterm newborns treated with animal derived surfactant
preparations when compared to the synthetic preparations .
Therefore, surfactants from animal derivation including porcine
lung extracts such as Poractant alfa (Chiesi Farmaceutici SpA,
Parma, Italy) are currently the most often used ones in preterm
However the respiratory failure in preterm infants is not only
due to a primary surfactant deficiency but is also caused by
surfactant inactivation as a result of plasma proteins leaking into
the airways from areas of epithelial disruption and injury .
Various conditions which often affect preterm infants, such as
exposure to chorioamnionitis, pneumonia, sepsis, meconium
aspiration and asphyxia, may lead to surfactant inactivation .
Surfactant inactivation of preparations such as Poractant alfa has
already been studied in several animal models of RDS. Calkovska
and colleagues reported the physiological parameters of ventilated
preterm rabbits after surfactant inactivation and treatment with
Poractant alfa .
Mechanisms of inactivation include impairment of surfactant
interfacial adsorption due to steric barriers imposed by serum
and/or inflammatory proteins [11,12] and impairment of
compressibility properties of surfactant films due to incorporation of
spurious components like cholesterol, lysophospholipids or bile
Both surfactant proteins SP-B and SP-C have been proposed to
participate in optimizing the surface behavior of surfactant under
the demanding conditions imposed by the respiratory physiology
 and in particular both SP-B [15,16] and SP-C [17,18] have
been reported to increase the resistance of surfactant to
inactivation by various agents. CHF 5633 is a fully synthetic
surfactant containing two phospholipids and two peptides
analogues of human surfactant proteins B and C, designed to be
resistant to oxidative injury. The phospholipids in CHF 5633
consist of a mixture of DPPC, the most important phospholipid in
terms of physiologic function and POPG sodium salt (POPG, Na)
which has been reported to inhibit lung inflammation [19,20].
Furthermore, Sato et al. demonstrated a superior oxygenation and
lung compliance in ventilated preterm lambs treated with CHF
5633 compared to other, animal-derived surfactant preparations
. Based on these features we hypothesized that CHF 5633
could better counterbalance surfactant inactivation upon
replacement and would improve oxygenation and lung function in
preterm babies with RDS.
The study was approved by the Animal Ethics Research
Committee, Maastricht University, The Netherlands (animal
ethics protocol 2010-129). Texel ewes were date-mated and the
fetuses were randomized to receive Poractant alfa or CHF 5633
before inactivation with albumin. Due to ethical reasons we did
not include control animals, which were treated with CHF5633 or
Poractant alfa only without surfactant inactivation, as previous
studies already demonstrated the physiological parameters of
ventilated preterm lambs after Poractant alfa or CHF5633
treatment [21,22]. Experiments were conducted in 19 preterm
lambs of both genders at a gestational age of 127 days (term = 150
CHF 5633 is a synthetic surfactant comprising
1-palmitoyl-2-oleoyl-sn-glycero-3phosphoglycerol (POPG), sodium salt, synthetic surfactant protein
C analog (IPSSPVHLKRLKLLLLLLLLILLLILGALLLGL) and
synthetic surfactant protein B analog
(CWLCRALIKRIQALIPKGGRLLPQLVCRLVLRCS) as active ingredients. The final
product is a sterile suspension with a total concentration of 80 mg/
ml. As control treatment, the animal derived surfactant Poractant
alfa (CurosurfH, 80 mg/ml, Chiesi Farmaceutici SpA, Parma,
Italy) was used, which is frequently used in clinical practice. All
preparations were supplied by Chiesi Farmaceutici SpA (Parma,
The pregnant ewes underwent cesarean section under epidural
and local subcutaneous analgesia with 2% lidocaine. In addition,
they were sedated with 1 mg midazolam intravenously which was
repeated if necessary. After a lower midline incision, the fetus was
carefully extracted through a small incision of the uterus. An
endotracheal tube (4.5 mm) was inserted and catheters were
placed in the umbilical artery and in the jugular vein and used for
baseline blood sampling (Abbott i-STAT 1 Blood Gas Analyzer,
Abbott Laboratories, Illinois, USA) and for continuous monitoring
of fetal mean arterial blood pressure (MABP) and heart rate (HR).
After the umbilical cord was cut, the fetus was brought to an
open, heated incubator (IW930 Series CosyCotTM Infant Warmer,
Fisher & Paykel Healthcare, Auckland, New Zealand) maintaining
a body temperature of 38uC. The lambs were connected to
intermittent positive pressure ventilation (IPPV) using a ventilator
Babylog 8000 (Drager, L ubeck, Germany) with initial settings as
follows: FiO2 = 1, PEEP 8 cmH2O, PIP 25 cmH2O, frequency
60/min, I:E 1:2. Thereafter, inspiratory pressure was increased by
2 cmH2O if PaCO2 was higher than 80 mmHg or by 4 cmH2O if
PaCO2 was higher than 110 mmHg. When a maximum PIP of
40 cmH2O was reached, no further adjustments were made.
These ventilation settings, which are rather aggressive compared
to clinical ventilation settings, were chosen to test the performance
of the surfactant preparations under unfavorable conditions and to
minimize the variations in ventilation settings between the
individual animals. The lambs were ventilated for 48 hours.
Sedation was maintained with ketamine (4 mg/kg/h) and
midazolam (50 mg/kg/h). For nutrition a solution of 20% glucose
and Ringers lactate was used in a concentration allowing for an
infusion rate of 13 mL/kg/h. After 2 hours of life a urinary
catheter was placed to monitor urine production and kidney
Surfactant inactivation protocol
Before being connected to IPPV, preterm lambs received either
CHF 5633 or Poractant alfa intra-tracheally in a dosage of
200 mg/kg BW (Figure 1). Executors of the experiments were
completely blinded for the type of surfactant given: vials were
wrapped in aluminium foil and marked as A or B by
DRMGO (blinding was only lifted after all experiments and
analyses had been performed). Ninety minutes after birth a
mixture consisting of 9.4 mg/kg human serum albumin (optimal
dose was determined in prior dose-effect experiments) and either
100 mg/kg CHF 5633 or 100 mg/kg Poractant alpha was given
intra-tracheally. The surfactant was used to provide an even
spread of the inactivator in the preterm lung. Blood gases were
taken every hour. If PaO2 dropped below 100 mmHg, the lambs
received every two hours an additional dose of either 200 mg/kg
CHF 5633or 200 mg/kg Poractant alfa until the PaO2 was again
higher than 100 mmHg.
The lambs were euthanized by an i.v. injection of 2 ml T61H
(Veterinaria AG, Z urich, Switzerland) after 48 hours or earlier if
reaching a human endpoint. Humane endpoints were defined as
cardiovascular failure (arterial pH,6.6, heart rate,100 beats per
minute and/or mean arterial blood pressure,30 mmHg) despite
Figure 1. Study design. Preterm lambs received a 200 mg/kg bodyweight intra-tracheal dose of either Poractant alfa or CHF 5633. Ninety minutes
after birth a mixture consisting of 9.4 mg/kg human serum albumin and either 100 mg/kg Poractant alfa or 100 mg/kg CHF 5633 was given
intratracheally to simulate surfactant inactivation. If PaO2 dropped below 100 mmHg, the lambs received an additional dose of either 200 mg/kg
Poractant alfa or 200 mg/kg CHF 5633 every two hours until the PaO2 increased above the 100 mmHg threshold. 48 hours after birth the lambs were
maximum dobutamine administration (20 mg/kg/min), or if either
of these situations occurred in the presence of edema combined
with no response to furosemide treatment (1 mg/kg) and kidney
The thorax was opened to perform a post-mortem pressure
volume curve. The endotracheal tube, which was used for
ventilation during life, was connected to a manometer and glass
syringe. The lungs were inflated until a maximum pressure of
40 cm H2O was reached and the corresponding gas volume was
recorded. Subsequently, the pressure was reduced to 20, 15, 10, 5
and 0 cm H2O and corresponding gas volumes were recorded.
The lungs were removed from the chest and weighed. The left
lung was lavaged three times with 20 mL 0.9% NaCl. The volume
of the resulting bronchoalveolar lavage fluid (BALF) was recorded
and aliquots were used for differential cell counts or snap frozen
for surfactant analysis. The right upper lobe (RUL) was
inflationfixed at 30 cmH2O in 10% formalin for 2 hours for histological
Hematoxylin and eosin staining was performed on paraffin
embedded RUL lung sections (4 mm, transverse). The sections
were deparaffinized in an ethanol series and incubated in Mayers
hematoxylin for 5 minutes. After rinsing in running water for 10
minutes, the sections were incubated in an eosin solution for 1
minute. Subsequently the sections were dehydrated in an ethanol
series and coverslipped. Evaluation was performed by light
microscopy (Leica DM2000) with LeicaQWin Pro v.3.7.0 software
(Leica Microsystems, Wetzlar, Germany).
Disaturated phospholipid analysis in BALF
The disaturated phospholipids content was measured in BALF
of the left lung. Briefly, 1.2 mL of BALF was thawed and
centrifuged for 10 minutes at 300 times g-force/relative centrifugal
force (rcf). One mL of the resultant liquid phase was transferred to
a clean glass tube and evaporated overnight at 60uC. The
remaining BALF condensate was dissolved in 1 mL of
carbontetrachloride and osmiumtetraoxide (1:10) solution, aided by the
addition of a glass bead and intermittent vortexing, and
evaporated for 1.5 hours at 60uC. The condensate was dissolved
in 1 mL chloroform:methanol (20:1) and transferred to a column
filled with glass wool impregnated with 0.8 g activated aluminium
oxide. The columns were subsequently flushed with 8 mL
chloroform:methanol (20:1), followed by 5 mL
chloroform:methanol:ammonia (35:15:1). After passage through the column, the
liquids were collected and evaporated at 60uC until only BALF
condensate remained. After dissolving the condensate in 1 mL
chloroform, 500 mL FeSCN was added. After shortly vortexing to
ensure a homogenous mixture, the samples were centrifuged for
10 minutes at 600 g. The 150 ml of the resulting chloroform phase
was transferred to a 96 well plate and scanned at 488 nm using a
Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific,
Waltham, USA) and Scanit RE for MSS 2.2 software. The
disaturated phospholipid concentration could then be calculated
by using a standard dilution series.
To quantify the hemorrhagic aspect of BALF, 200 ml of each
sample was scanned at 410 nm wavelength using a Multiskan
Spectrum spectrophotometer and Scanit RE for MSS 2.2
software. The resulting optical density (OD) was then corrected
Differential cell counts in BALF (x 106/kg bw):
BALF hemoglobin (OD at 410 nm/kg bw)
CHF 5633 (n = 7)
Poractant alfa (n = 8)
Neutrophils were decreased in the lungs of preterm lambs treated with CHF 5633 compared to Poractant alfa treated lambs after surfactant inactivation by albumin.
BALF bronchoalveolar lavage fluid; bw bodyweight; OD optical density. Data expressed as mean6SEM. * p,0.05, unpaired t-test.
Ventilation efficiency index (VEI) and P/F ratio
The ventilation efficiency index (VEI) was calculated as
VEI = 3,800/(respiratory rate6[PImax2PEEP]6PaCO2], where
3,800 is a CO2 production constant ([ml6mmHg]/[kg6min])
. As a measure for oxygenation, ratios were calculated by
dividing the PaO2 by FiO2 (FiO2 = 1).
Biophysical analysis by captive bubble surfactometry
To analyze the functional behaviour of surfactant samples from
CHF 5633 and Poractant alfa treated animals, material obtained
from the bronchoalveolar lavage was tested in a custom-built
captive bubble surfactometer (CBS), as described elsewhere
[13,24]. Lavages were first centrifuged at 40000 g to obtain the
large aggregates of pulmonary surfactant complexes and
phospholipids were quantitated from pellets by phosphorus analysis
. Samples were then diluted to 25 mg/mL phospholipid
concentration and 200 nL were injected onto the surface of an air
bubble of 50 mL formed in the chamber of the CBS, using a
subphase Tris 5 mM pH 7 containing NaCl 150 mM and 10%
sucrose, thermostated at 37uC and subjected to continuous
stirring. Continuous monitoring of bubble shape with a video
camera allowed determination of surface tension. Once the sample
adsorbed to equilibrium surface tension (initial adsorption, IA), the
bubble was expanded to a volume of 150 mL to allow for
surfactant re-adsorption (post-expansion adsorption, PEA) during
5 min. Then, the bubble was subjected to quasi-static
compression-expansion cycling, in which the bubble size was first reduced
and then enlarged in a stepwise fashion during four consecutive
quasi-static cycles. Finally, after 1 min delay,
compressionexpansion dynamic cycling started, in which the bubble size was
continuously varied at 20 cycles/min, which is a speed
comparable with breathing rates. Illustrative surface tension-relative area
isotherms are presented after repeating at least three experiments
with each of the samples, and averaged relevant parameters such
as minimal surface tension at the end of compression, the percent
of area reduction required to reach minimal tension, and the
maximal tension upon expansion, were compared for each group.
Results are given as means6standard error of mean (SEM). The
groups were compared using a Mann-Whitney u-test or a two-way
repeated-measures analysis of variance where appropriate.
Survival analysis was performed with a Gehan-Breslow-Wilcoxon test.
Statistical analysis was performed by GraphPad Prism v5.0.
Significance was accepted at p,0.05.
At birth fetal lambs from the two different treatment groups
were similar in birth weight and umbilical artery pH (Table 1).
The female to male ratio was also similar at 5:2 for the CHF 5633
group compared to 5:3 in the Poractant alfa group. 4 preterm
lambs were not deemed physically healthy and were therefore not
included into the experiment shortly after birth.
Lambs that were treated with CHF 5633 were more likely to
survive up to 48 hours after birth (46.5 hours after albumin
instillation) than lambs treated with Poractant alfa (5 out of 7
lambs treated with CHF 5633, compared to 1 out of 8 Poractant
alfa treated lambs) (Figure 2). The total amount of administered
surfactant and frequency of redosing did not differ significantly
between treatment groups. However, when the frequency of
redosing was corrected for post-inactivation survival time (in hours),
to correct for the poor survival of Poractant alfa treated lambs,
surfactant requirement was significantly lower among the CHF
5633 treated lambs (Table 2). Minute volumes and tidal volumes
were not statistically different between groups (Figure S1).
Differential cell counts of the bronchoalveolar lavage fluid
(BALF) indicated that there were significantly fewer neutrophils
present in the alveolar spaces of the lungs of CHF 5633 treated
lambs (Table 1). The amount of hemoglobin in the BALF did not
differ between the two treatment groups.
Total surfactant (mg/kg bw)
Frequency of surfactant redosing
Frequency of surfactant re dosing corrected for
hours of survival after inactivation
CHF 5633 (n = 7)
Bw bodyweight. Data expressed as mean6SEM. * p,0.05, unpaired t-test.
Morphologically there was a striking difference between the
lung structures of lambs treated with Poractant alfa or CHF 5633
(Figure 3A and 3B). The lung structure of lambs treated with CHF
5633 was more open, with larger alveolar spaces and thinner
septa, whereas the lungs of Poractant alfa treated lambs did not
inflate well, had thicker alveolar walls and contained more
erythrocytes and lymphocytes. This difference in structure was
reflected in the pressure volume curves (Figure 3C). The lungs of
CHF 5633 treated lambs distended to a larger volume under
increasing airway pressure compared to the lungs of Poractant alfa
treated lambs, although this observation did not reach statistical
significance. Disaturated phospholipids content in the BALF,
which is an indicator for total surfactant content of the lung (both
inherent and administered) did not differ between the two
treatment groups (Figure 3D). However, these results may be
Poractant alfa (n = 8)
confounded by the difference in survival rate of CHF 5633 treated
lambs compared to Poractant alpha treated lambs, or differences
in the amount of surfactant administered. In addition to these
physiological parameters, the efficiency of surfactant treatment
was determined by functional in vivo readouts. The ventilation
efficiency index (VEI) and partial arterial oxygen pressure (PaO2),
which could be calculated by combining ventilation parameters
and blood gas measurements, revealed an increased VEI and PaO2
for animals treated with CHF 5633 during all time points
(Figure 4A and 4B). Statistical significance was however only
reached at 90 minutes of life for VEI and 16.5 hours after
surfactant inactivation for PaO2. The pH of arterial blood showed
a similar trend, and only reached statistical significance at
22.5 hours after surfactant inactivation. Because only one
Poractant alfa treated lamb survived longer than 24 hours,
statistical analysis of VEI, PaO2 and arterial pH was not possible
for these parameters at 34.5 or 46.5 hours after surfactant
To further detail the biophysical capabilities of surfactant from
CHF 5633 treated or Poractant alpha treated animals, we
compared the performance of surfactant complexes obtained from
BALF of the different animals in the CBS. This technique has
been widely used to evaluate the surface behavior of pulmonary
surfactant films under conditions mimicking interfacial breathing
mechanics [15,24]. We compared illustrative
compression-expansion isotherms of original CHF 5633 or Poractant alfa films and of
films formed by surfactant pelleted from BALF of CHF 5633
treated or Poractant treated lungs (Figure 5). Both surfactant
preparations were originally capable to form films able to reach
very low tensions (below 5 mN/m) when subjected to
compression-expansion cycling, either under quasi-static (slow) or dynamic
(rapid, physiological-like) cycling regimes. Material from BALF of
the surfactant treated animals was impaired with respect to their
original capabilities. Surfactant from CHF 5633 treated animals
was in most cases still able to produce very low tensions when films
were subjected to either slow or fast compression-expansion
cycling, although these films required slightly larger area
compression than the original material ones (around 40% instead
of 30%) to produce the minimal tensions. In contrast, films formed
by lavage of Poractant-alfa treated lungs did not produce in most
cases tensions below 20 mN/m when compressed at slow speed
(Figure 5). However, when compressed at fast,
physiologicallycomparable rates, films from Poractant alfa treated animals
underwent reorganization during the first compression cycle to
produce films that were then able to produce minimal tensions
with little compression in the subsequent cycles.
Relevant parameters obtained from compression-expansion
isotherms of films formed by the different samples in the CBS
were summarized in Figure 6. Surfactant from both CHF 5633
treated and Poractant-alfa treated animals adsorbed well to the
air-water interface, producing equilibrium surface tensions
#30 mN/m in less than a minute. Surfactant from Poractant alfa
treated animals exhibited slightly faster adsorption than surfactant
from CHF 5633 animals, a difference that was statistically
significant. The larger difference in the surface performance of
the two groups was observed upon cycling of films at slow speed.
Slow quasi-static compression-expansion cycling usually reveals
intrinsic differences in the compressibility properties of films made
of different materials or having different organization [14,24].
Films from CHF 5633 treated animals produced under quasi-static
cycling in most cases much lower surface tensions, with less area
reduction, compared to films of surfactant from Poractant alfa
treated lungs. Differences in surface behavior practically vanished
when the films were subjected to fast cycling at rates comparable
to those potentially occurring in the lung in vivo.
Figure 5. Surface behavior. Compression-expansion cycling isotherms in the CBS of films formed by original CHF 5633 or Poractant alfa
preparations (left panels) and of films formed by material obtained from the bronchoalveolar lavage of CHF 5633-treated or Poractant alfa-treated
animal lungs (right panels). Isotherms were obtained either under slow quasi-static (upper panels) or fast dynamic (lower panels) cycling regimes.
Quasi-static isotherms include those from the 1st (closed circles), 2nd (open circles), 3rd (closed triangles) and 4th (open triangles)
compressionexpansion cycles, while dynamic isotherms are plotted from the 1st (closed circles), 10th (open circles) and 20th (triangles) cycle.
Surfactant replacement therapy has been the most significant
advance in perinatal care to decrease neonatal mortality since the
late 1980s, equaled only by antenatal corticosteroids . The
chances of survival after preterm birth and low morbidity
increased dramatically in the past decades. Surfactant replacement
therapy is established and well-studied. However, the inherent
costs of surfactant preparations and the risk of inactivation urge
the search for new surfactant preparations. Been et al. for example
found that the exposure to antenatal inflammation
(chorioamnionitis) resulted in a poor response to exogenous surfactant
replacement therapy . This is a clinical relevant example that
the success of surfactant replacement therapy can be reduced by
inactivation due to inflammatory changes in the lung. The dose of
surfactant and/or the preparation of the surfactant might present
clinical alternatives in the best clinical care after preterm birth.
We developed a model to study surfactant inactivation in
premature, virtually surfactant deficient lambs and showed that
CHF 5633 has a survival benefit and a trend for improved
ventilation and oxygenation after in vivo inactivation. An additional
benefit of CHF 5633 is that it had a more durable effect, as
redosing was not required as frequently as in the Poractant alfa
treated lambs. To our information this is the first study that shows
superiority of a surfactant preparation for inactivation in vivo.
Albumin was chosen as inhibitor since it is well studied as inducer
of surfactant inactivation in vitro and in vivo [28,29]. In addition,
albumin is a plasma protein and part of the inflammatory cascade
induced by various stimuli such as mechanical ventilation or
infection [30,31]. The inhibitory effect of albumin was clearly seen
during our experiments as most lambs, particularly the ones
treated with the conventional Poractant alfa surfactant, showed a
decrease in arterial oxygenation levels and ventilation efficiency
index shortly after albumin instillation.
The study has several limitations. First of all, the sudden onset of
inactivation after birth (besides the injury caused by mechanical
ventilation) is not very common in clinical practice as are the
ventilation strategies used in this model. However, we chose the
setup of sudden inactivation and aggressive ventilation strategies to
reduce variability between individual animals and study the effects
of the two surfactant preparations in the most unfavorable
conditions. Secondly, we have limited the follow-up to 48 hours,
which is rather long for an experimental study but still far away
from clinical practice. Furthermore, we did not include control
animals treated with CHF 5633 or Poractant alfa only nor test
different doses of CHF 5633 or inhibitors other than albumin.
CHF 5633 was developed with properly designed synthetic
analogs of the two natural SP-B and SP-C proteins. Natural
surfactant as produced by the lungs consists of a complex mixture
of several phospholipids and the proteins SP-A, SP-B, SP-C and
SP-D, of which SP-B and SP-C play a key role in decreasing
surface tension in the lung [14,32]. Experimental research showed
that the inclusion of both SP-B and SP-C in exogenous surfactant
preparations improved the biophysical effects in preterm rabbits
Figure 6. Parameters defining interfacial performance. Comparison of parameters defining the interfacial performance in the CBS of films
formed by material obtained from the bronchoalveolar lavage of CHF 5633 treated and Poractant alfa treated animals. Data plotted are the minimal
surface tension reached after 1 min of initial or post-expansion adsorption (upper panel), and the minimal surface tension, the percent of area
reduction required to reach minimal tension and the maximal tension during quasi-static (middle panels) or dynamic (lower panels)
compressionexpansion cycling. Data expressed as mean6SEM. *p,0.05, two-way repeated-measures analysis of variance.
. The addition of SP-B to natural surfactant preparations that
already contained SP-B further improved the function in vivo .
The experience with the importance of both SP-B and SP-C in
natural surfactants has been repeated with synthetic surfactants.
Most previously reported synthetic surfactants either contain SP-B
or SP-C , but for an optimal functional effect both proteins
are required. Almlen and colleagues [38,39], have demonstrated
that a synthetic preparation containing SP-B and SP-C was
superior to single-peptide surfactants in premature ventilated
rabbits. Our study can now confirm that a similar synthetic
preparation has proven to have superior resistance to inactivation
Our biophysical measurements suggest that the higher
resistance of CHF 5633 surfactant to inactivation in vivo could be
related to better compressibility properties of CHF 5633 films after
exposure to inflamed and injured lungs. In vitro studies established
that the main effect of albumin as a surfactant-inactivating agent
could be related to impairment of the ability of surfactant to form
surface active films at the air-liquid interface, as a consequence of
the competition of albumin and surfactant to reach the interface.
However, the impairment of surface activity observed in the
surfactant from the BALF from our lambs, particularly in the case
of Poractant alfa, resembles the impairment of surfactant due to
incorporation of inhibitory substances such as cholesterol or bile
acids . Interfacial adsorption was not impaired in surfactant
from Poractant alfa treated animals, but was even slightly better
than that from CHF 5633 treated lambs, probably because the
fluidization of Poractant alfa that impairs compressibility and
favors adsorption. This suggests that the introduction of albumin
into the lungs causes surfactant inactivation not so much due to the
primary impairment of interfacial adsorption but also indirectly
through the release of some surfactant inhibitors as a consequence
of lung inflammation. In these conditions surfactant films become
highly deformable and are not able to reach the lowest tensions
(very high surface pressure) when subjected to compression. Our
data suggest that either Poractant alfa is more susceptible than
CHF 5633 to incorporate spurious components leaked to the
airspaces, or that the particular lipid and protein composition of
CHF 5633 can accept higher amounts of inhibitors before losing
the compressibility properties required to reach and sustain the
low surface tensions required to stabilize the lungs. Further studies
are required to understand the origin of the different inhibitory
susceptibility, but the apparently higher resistance to inhibition of
CHF 5633 may provide improved treatment opportunities not
only for preterm infants who have decreased oxygenation due to
surfactant inactivation by inflammation, but also for patients
suffering of Acute Respiratory Distress Syndrome (ARDS).
In conclusion, this is the first study which shows that a synthetic
surfactant with properly designed analogs of SP-B and SP-C
results in a similar oxygenation in preterm lambs but also conveys
a survival benefit to preterm lambs over the gold standard
treatment Poractant alfa with the same doses. The promising
results reported here support the introduction of CHF 5633 as a
possible new therapy for surfactant deficiency or dysfunction
conditions. The superiority in this model will have to be confirmed
in a clinical trial.
Figure S1 Minute and tidal volume. Recordings of the
minute (A) and tidal volume (B) did not show any significantly
difference between CHF 5633 treated animals and Poractant alfa
treated animals for the duration of the experiment. Grey
spheres = CHF 5633; Black cubes = Poractant alfa. Data expressed
as mean6SEM. *p,0.05, two-way repeated-measures analysis of
We would like to thank Lilian Kessels, Pim Boden and Anne-Claire
Bosmans for their excellent technical assistance.
Conceived and designed the experiments: MS JJPC BWK OLO JPG.
Performed the experiments: MS JJPC EK RKJ DRMGO BWK OLO
JPG. Analyzed the data: MS JJPC EK BWK OLO JPG. Contributed
reagents/materials/analysis tools: RG RR FB. Wrote the paper: MS JJPC
EK BWK JPG. Data interpretation: MS JJPC EK JPG FB RG RR BWK.
Manuscript revision: MS JJPC EK RKJ DO JPG FB RG RR BWK.
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