In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits.

Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation.

Introduction

Neonatal respiratory distress syndrome (RDS) is a condition of pulmonary insufficiency affecting preterm infants. With an incidence as high as 50% in very and extremely preterm infants (born below 33 and 28 weeks of gestational age, respectively), the development of RDS is associated with a high risk of perinatal morbidity and mortality.[1,2] At low gestational age, alveolar epithelial cells are structurally and functionally immature and the ability of surfactant synthesis and secretion is therefore compromised.[3] Consequently, surfactant pools in both alveolar and parenchymal compartments are noticeably low at birth,[4] leading to high intra-pulmonary pressure and alveolar collapse.[5] RDS develops shortly after birth and evolves rapidly towards a life-threatening hypoxemic condition unless vigorous rescue treatment is provided to facilitate adequate alveolar expansion and intrapulmonary perfusion,[6] of which surfactant replacement therapy is the most efficient pharmacological treatment.

Animal-derived surfactants contain several classes of phospholipids, including relatively high amounts of disaturated phospholipids, namely dipalmitoyl- phosphatidylcholine (DPPC) as one of the main active components, and the hydrophobic surfactant proteins (SP)-B and SP-C.[6,7] As an emerging country, neonatal-perinatal care in China has been advanced in the past decade. With 16–17 million annual births and a preterm birth rate of 4–5%, 100,000 cases are presumed to be affected by RDS and require surfactant therapy every year in China.[8,9] Poractant alfa and Calsurf are two animal-derived surfactant preparations widely used in China for the treatment of RDS. Poractant alfa is extracted from porcine minced lungs and is licensed to be administered at phospholipid doses of 100 or 200 mg/kg, with salient pharmacodynamics and pharmacokinetic effects in the treatment of RDS.[10-13] Moreover, Poractant alfa has undergone large randomized clinical trials comparing its efficacy to other surfactant preparations.[13-16] On the other hand, little is known about Calsurf, which is extracted from lung washes of calves and is indicated for the treatment of RDS at an initial phospholipid dose of 40–100 (average 70) mg/kg (manufacturer information). To our knowledge, very few clinical studies addressing the pulmonary efficacy of Calsurf have been published to date in international, peer-reviewed journals.[17] Neither surface properties nor clinical effects compared to porcine or other bovine surfactants were provided. Only the information that Calsurf has a similar formulation in terms of phospholipid concentration (roughly about 30 mg/ml) as other bovine-derived surfactants has been reviewed.[18] As a comparison between the clinical efficacy of Poractant alfa and Calsurf is not available yet, we performed a direct comparison of both surfactant preparations in vitro and in vivo. Our aim was to perform a preliminary preclinical characterization of Calsurf, providing insights into both biophysical and physiological properties as well as the pharmacodynamics of this surfactant preparation in comparison with Poractant alfa.

Materials and methods

Surfactant preparations

Poractant alfa (Curosurf®, Chiesi Framaceutici, Parma, Italy) is a natural surfactant suspension, prepared from minced porcine lungs, containing almost exclusively polar lipids, in particular, phosphatidylcholine (PC, about 70% of the total phospholipid content), and about 1% of specific low molecular weight hydrophobic proteins SP-B and SP-C, at a phospholipid concentration of 80 mg/mL (product information). Calsurf (Kelisu®, Shuanghe Pharmaceuticals, Huarun Group, Beijing, China) is a natural surfactant, prepared from calf lung washes, containing approximately 80% phospholipids, at least 55% phosphatidylcholine, and about 1–2% of SP-B and SP-C at a phospholipid concentration of 35 mg/mL (product information). Calsurf is presented as a lyophilized product and must be re-suspended with distilled water before use.

Liquid-chromatography mass-spectrometry for phospholipids analysis

A detailed description of the method is provided in Supporting information (S1 Method). Liquid chromatography solvents, acetic acid and ammonia solution were purchased from Sigma Aldrich Italy (Milan, Italy). The chemical standards of phospholipids and the internal standards used for quantitative purposes were obtained from Avanti Polar Lipids (Alabaster, AL, USA).

Normal phase liquid chromatography mass spectrometry (NPLC-MS) was performed on a Thermo Scientific (Bremen, Germany) system composed by a high performance liquid chromatography (HPLC) surveyor, with column oven and auto-sampler, coupled to an LTQ ion trap mass spectrometer via electrosprayinterface (ESI) (Thermo). Surfactant samples were separated on a Varian Polaris Si A, 250 x 2.1 mm, 5 μm, 200 Å (Agilent Technologies, Santa Clara, CA, USA) with a ternary gradient.

Acquisition was performed by polarity switching, recording both negative and positive ions during the entire elution program. Data were acquired in full scan mode, from 200 to 1600 m/z, excluding cholesterol that was acquired in positive ion MS/MS mode, recording the two product ions at m/z 161.1 and 243.2 deriving from the precursor ion at m/z 369.3, [M+H-H2O]+ (isolation window 2.6 m/z, normalized collision energy 20). Data were acquired and elaborated by Xcalibur software version 2.0.7. Semi-quantitative data were accessed by manual peak integration of the analytes of interest.

Atomic force microscopy measurements

A 70 mg mass of commercial lyophilized Calsurf powder was re-suspended in a vial using 2 mL of sterile water for injection and gently mixed by vial rotation for 20 min at room temperature. Poractant alfa was analyzed by AFM at a phospholipid concentration of 80 mg/mL. Further, it was diluted 2.2 times in saline solution to a final concentration of 35 mg/mL so that the results of both preparations could be directly compared at the same phospholipid concentration. Volumes of 30 μl of Poractant alfa and Calsurf were deposited onto freshly cleaved mica substrates for 2 min. Thereafter, the surface was rinsed with MilliQ water (≥18.2 MΩ-cm) and dried with a nitrogen gas flow. AFM imaging was carried out “in air” with a Nanoscope IIIA microscope (Digital Instruments, Santa Barbara, CA, USA) equipped with the J scanner and commercial silicon cantilevers (MikroMasch, Tallinn, Estonia) operating in tapping mode. Images of 512 × 512 pixels were collected with a maximum scan size of 10 μm, at a scan rate of 1 or 2 lines per second. While imaging, height, amplitude and phase signals were recorded. A surface roughness analysis was carried out using the dedicated tool of the Gwyddion v2.53 software. The Rtm parameter represents the mean peak-to-valley roughness and it was determined by the difference between the highest peak ant the lowest valley within multiple samples in the evaluation length. Roughness data were collected on three independents images (10 μm x 10 μm) from independent experiments of Poractant alfa and Calsurf. For each image, 11 local roughness observations were performed, by means of an evaluation line of 10 μm in length x 100 nm in thickness, shifted along the entire image by a step of 1 μm.

Langmuir Blodgett measurements

Both Poractant alfa and Calsurf were subjected to lipid extraction according to the method described by Bligh and Dyer.[19] The chloroform phase containing surfactant components was diluted to a final phospholipid concentration of 1 mg/mL and used for the Langmuir Blodgett trials. Before each measurement, the ultra-pure water subphase (Milli-Q ≥18.2 MΩ-cm) was quickly compressed to verify the absence of possible organic contaminations. Surfactant was spread onto the subphase of a ribbon barrier trough (KSV NIMA, Finland) by means of a 50 μl glass microsyringe (Hamilton Company, Bonaduz, Switzerland). The subphase operational area was 156 cm2 and the temperature was maintained constant at 25 ± 0.5°C using an external circulating water bath.

The chloroform phase containing surfactant components was spread onto the water subphase to reach a surface pressure of approximately 10 mN/m. Surface pressure is the amount by which surface tension is reduced. For instance, a surface pressure of zero mN/m indicates no reduction at all of surface tension, whereas a surface pressure value of 70mN/m in a water (surface tension of water at 37° C is 70 mN/m) indicates that surface tension has been fully counteracted by surfactant.[20] After spreading, the film was left undisturbed for 15 min to allow solvent evaporation. The film was then symmetrically compressed at a rate of 40 cm2/min. A surface pressure/area (π/A) isotherm was recorded in real time with the built-in software. The value of compressed area at a surface pressure of 68 mN/m was used to compare both formulations. For each surfactant, at least three independent experiments were performed. Two-dimensional compressibility of surfactant films was obtained from the compression isotherms. Film compressibility (Cm) is defined as the inverse of the compression modulus and is given by: where A is the surface area and π is the surface pressure. Cm provides information concerning phase transitions and fluidity/elasticity of the monolayer. Large Cm values are indicative of the state in which the film displays high compressibility and fluidity, whereas small Cm values reflect a high packing of the phospholipid molecules. For each measurement, Cm was plotted as a function of the surface pressure π.

Standardized positive inspiratory pressure (PIP) ventilation loop experiments in preterm rabbits

Seven pregnant, date-mated New Zealand White rabbits were obtained from Shanghai Songlian Experimental Animal Center and housed until the twenty-sixth day of gestation under standard conditions according to the current procedures for animal housing and handling of the center. The does were transported to the experimental site one day prior to the experiments with full shelter, food and water ad libitum. The study protocol was approved by the ethics committee of the Children’s Hospital of Fudan University (No. 2016240), and all efforts were made to minimize animal suffering.

One the experimental days, does were sedated with 2 mL i.m. 0.5% Diazepam (Shanghai Xudong Haipu Pharmaceutical Co. Ltd., Shanghai, China) and paralyzed with 10 mL i.m. 20% Urethane (Ethyl carbamate, BBI Life Sciences, Shanghai, China), followed by intravenous catheterization for additional 10–15 mL Urethane infusion. A maintenance dosage of Urethane was given at 1 mL/kg per hour to keep does unconsciousness until the end of the cesarean section. The does were then sacrificed by an overdose of potassium chloride.

Preterm rabbit fetuses were obtained by cesarean section after 27 days of gestation (27+0h-27+6h days of gestation, term 31 days). After delivery, rabbit pups were weighed and immediately anaesthetized with 0.1 mL i.p. 1% Lidocaine hydrochloride (Shandong Hualu Pharmaceutical Co. Ltd., Shandong, China). Pups were then tracheostomized, intratracheally intubated with a thin, short metal cannula (18G needle, 1.2 mm outer diameter, 10–12 mm in length), and connected through silica tubing to a multi-plethysmograph-ventilator system.[21] The pressure delivered to each rabbit was measured by a pressure transducer (Shanghai Yangfan Electronic Co. Ltd., Shanghai, China) and tidal volume (VT) was recorded by a pneumo-tachometer (RSS100-HR, Hans Rudulph, inc. Kansas City, USA). Both were connected to an automated physiologic monitoring system (PowerLab, ADInstruments Pty Ltd, Bella Vista, Australia).

In total 60 rabbit pups were allocated to one of the six experimental groups (n = 10 per group) in natural order by litter and delivery, in sequence and consecutive manner. To compare the in vivo effects both in licensed doses and head-to-head way, two groups of animals received intratracheal Poractant alfa at doses of 200 mg/kg and 100 mg/kg (P200 and P100 groups), which were 2.50 and 1.25 ml/kg as for fluid volume, and three further groups received Calsurf at doses of 200 mg/kg, 100 mg/kg and 70 mg/kg (C200, C100, and C70 groups), which were 5.71, 2.86 and 2.00 ml/kg as for fluid volume. A control (Ctrl) group received only sham volume of air. The rabbit pups in six groups were paralleled submitted to mechanical ventilation (Siemens 900C ventilator, Siemens-Elema, Solna, Sweden) with 100% oxygen, for 30 min according to a fixed ventilation protocol, as previously described.[22,23] Briefly, the pups were initially ventilated for 15 min with a positive inspiratory pressure (PIP) of 25 cmH2O, followed by 5 min at a PIP of 20 cmH2O, another 5 min at a PIP of 15 cmH2O, and a final step of 5 min in which the PIP was restored to the initial level of 25 cmH2O. 0.5 mL 2% lidocaine was intracranially injected at the end of ventilation.

Since positive end-expiratory pressure (PEEP) modifies the response to surfactants in ventilated immature rabbits,[24, 25] two different experimental sessions were carried out. In the first set of experiments, PEEP was not applied. In the second session, a PEEP of 2–3 cmH2O was included in the ventilation protocol to the same animals in groups to see if different responses existed between the two surfactants. VT, PIP and PEEP were measured at 5 min intervals for each animal. All pups were anaesthetized throughout the study. The dynamic compliance of the respiratory system (Cdyn) was derived from: where VT was in mL/kg birth weight, PIP and PEEP in cmH2O.

Prophylactic surfactant treatment in standardized VT ventilation experiment in preterm rabbits

Rabbit pups were delivered and prepared for mechanical ventilation in the same way as described in the PIP ventilation loop experiments to assess survival after surfactant administration (P200, P100, C200, C100, C70, and Ctrl groups; N = 25 pups per group from another 22 litters). For this set of experiments, the ventilator was set at 40 breaths/min, with an inspiration-to-expiration ratio (I:E) of 1:2, and a fraction of inspired oxygen of 1.0. A PIP ranging between 10–25 cmH2O was applied to generate a standardized VT of 4–6 mL/kg body weight. PEEP was provided at 2–3 cmH2O. PIP was titrated every 3–5 minute interval during the first 30 min of ventilation, and subsequently 10–15 minute interval afterwards. The anesthesia was the same as in the PIP loop experiments, and it was maintained for preterm rabbit pups by i.p. 0.1 mL mixed solution (0.01 mL of 2% Lidocaine, 0.03 mL 5% NaHCO3 and 0.06 mL 10% Glucose) at 60–90 min intervals.

Survival time was assessed at 3 h, or at an early death. Death occurring during ventilation was determined by body skin color change and confirmed by chest wall pulsation sign of heartbeats. The determination was made continuously during ventilation. At 180 min, euthanasia was provided by intracranial injection of 0.5 mL 2% lidocaine.

Biochemical analysis of broncho-alveolar lavage and lung tissue samples

Lung samples from 8–10 animals per group from the prophylactic surfactant treatment experiments were preserved immediately after exitus by broncho-alveolar lavage (BAL) with 30 ml/kg body weight of normal saline (0.9% NaCl). BAL fluid was immediately centrifuged at 2000 rpm for 15 min at 4 oC to remove cell debris. Further, BALs underwent organic extraction with methanol/chloroform (1:2) to extract the total amount of phospholipids. Similarly, lung tissue homogenates were also extracted with methanol/chloroform (1:2). Total phospholipids (TPL), disaturated phosphatidylcholine (DSPC) and total proteins (TP) were determined by the Bartlett assay,[26] the Mason’s osmium digestion and aluminum tetroxide column chromatography,[27] and the Lowry’s method, respectively, as previously described.[28] For these experiments, a group of delivered but non-ventilated pups (n = 9) was included as an additional control group (C0).

Lung examination

Post mortem, the lungs of another 13 animals per group (other than those for biochemical analysis, both were allocated randomly) from the prophylactic surfactant treatment experiments were examined for pneumothorax followed by intrapulmonary arterial perfusion fixation with 4% formalin at 10 cmH2O for 30 min. The perfusion-fixed lungs were removed en bloc and fixed continuously in 4% formaldehyde for 72 h. The lung blocks were embedded in paraffin, cut into thin sections (5–6 μm) and stained with hematoxylin and eosin. Alveolar expansion (VV) was determined based on the image-analysis of 50 microscopic fields per lung sample at 200x magnification, using a semi-quantitative theorem of lung morphometry by point-counting method. VV reflects the magnitude of average alveolar expansion using total lung parenchyma as denominator on aerated alveolar spaces of each individual animal bilateral lung.[29] CV (Vv) denotes the standard deviation / mean of VV for individual lungs, reflecting the homogeneity of alveolar expansion.[30] The appendix lobe of the right lung was ligated and removed for wet-to-dry lung weight (W/D) measurement before perfusion to estimate lung tissue fluid content.[31]

Lung sections were inspected by an expert pathologist in a blind manner and assessed according to lung injury scores (LIS), which was based on four items of pathological impairment in lung tissue: small airway epithelial damage (desquamation), edema, hemorrhage, and neutrophil infiltration. Severity for each item was estimated by scores of 0 for none, 1 for mild-to-moderate (item present in < 25% of the field), 2 for moderate-to-severe (item present between 25%-50% of the field), 3 for moderate-to-severe (item present between 50%-75% of the field), and 4 for severe (item predominates in > 75% of the field).[30] The sum of all items provides an overall injury severity on an individual lung basis.

Statistical analysis

Continuous data are presented as mean and standard deviation (SD) and subjected to analysis of variance (ANOVA) followed by post hoc comparison of between-group difference with Student-Newmann-Keuls test. Categorical data are presented as ratio or percent (%) and analyzed with Chi square test, or by non-parametric ANOVA with Kruskal-Wallis test followed by Wilcoxon-Mann-Whitney test for between-group differences. The AFM and Langmuir Blodgett datasets were subjected to ANOVA and Student’s t-test. Survival analysis was subjected to Log-rank (Mantel-Cox) test. A P value <0.05 is regarded as a statistically significant difference.

Results

LC-MS analysis of the lipid composition of Poractant alfa and Calsurf

The relative amount of lipid species in Poractant alfa and Calsurf are summarized in Table 1. The LC-MS analysis revealed qualitative differences between surfactant preparations. Indeed, determined amounts of PC, DPPC, phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were respectively 3-, 2-, 10-, and 3-fold as high in Poractant alfa as Calsurf. Moreover, the two preparations differed in phospholipid chemical properties; in Calsurf 65% of the phospholipid fraction was saturated, whereas Poractant alfa was composed of 50% of unsaturated acyl chains phospholipid with higher molecular weight. Plasmalogens were not detected in Calsurf, whereas they account for 4% of the total lipid composition of Poractant alfa. On the contrary, the presence of cholesterol was only detected in Calsurf, which occupied about 3% of the total lipid.

Table 1

Lipid analysis of Poractant alfa and Calsurf.

Lipid class	Calsurf (n = 4)		Poractant alfa (n = 8)
	mg/mL	%/PL	mg/mL	%/PL
PC**	15.0±0.2	43.1	43.3±1.2	49.4
DPPC	15.9±0.2	45.7	28.2±1.1	32.2
PG	0.7±0.1	2.0	0.8±0.2	0.9
BMP	0.6±0.1	1.7	0.1±0.03	0.1
PE	0.5±0.1	1.4	5.9±0.9	6.7
PI	0.9±0.2	2.6	3.0±0.8	3.4
SM	0.1±0.03	0.3	2.7±0.8	3.1
PLPE	ND	ND	3.7±0.6	4.2
Cholesterol	1.1±0.2	3.2	ND	ND
	%/PL		%/PL
Saturated PL	66.3		45.6
Mono-unsaturated PL	27.1		24.9
Poly-unsaturated PL	6.6		29.5

Data are expressed as mg of each lipid class (mean±SD) and weight % with respect to 1 mL of surfactant (mean values of the indicated numbers “n” of independent observations). Notice that the lipid concentration of Calsurf and Poractant alfa is 35 mg/mL and 80 mg/mL, respectively. The phospholipid species listed in the top panel were quantified using internal phospholipid standards. Abbreviations: PC, phosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; PG, phosphatidylglycerol; BMP, bis (monoacylglycero) phosphate; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipids; SM, sphingomyeline; PLPE, 1-palmitoy l,2-linoleoylphosphatidyl-ethanolamine

**, total content of PC with the exception of DPPC, expressed individually; ND, not detectable.

At any phospholipid concentration, the ultrastructure of Poractant alfa was characterized by overlapping phospholipid domains, which showed complex multilamellar organizations, composed of 12–15 overlapped lipid bilayers (Fig 1). Single bilayers of Poractant alfa showed a height ranging between 5.4–6 nm. Conversely, the aforementioned multilamellar structures were not detected in Calsurf, which showed a maximum of 2–3 overlapping bilayers of a lower height, in the range of 3.5–4.5 nm.

Fig 1

Representative atomic force microscopy amplitude images of Poractant alfa and Calsurf.

Comparison of representative atomic force microscopy amplitude images of Poractant alfa at a phospholipid concentration of 80 mg/mL (A) and 35 mg/mL (B), and Calsurf at a concentration of 35 mg/mL (C). Selected regions of interest in A-C (squares delineated by white dashed lines) were color-coded in order to emphasize the height of overlapping phospholipid domains in D-F. The color switch was set every 5.5 nm for Poractant alfa (D-E) and 4.5 nm for Calsurf (F), based on the bilayer thickness measured for each surfactant preparation. In Calsurf, three overlapped homogeneous bilayers composed the bottom of the deposition and poor tridimensional complexity was detected. At a comparable phospholipid concentration, Poractant alfa showed peculiar multilamellar structures widespread above a multilayered ultrastructure. Dimensions of multilamellar structures increased with Poractant alfa concentration (maximum height greater than 100 nm). Detail of a lamellar body-like structure of Poractant alfa composed of 10 overlapped bilayers (G) and Calsurf ultrastructure (H). Dashed lines in (I) and (J) are height profiles marking each bilayer-bilayer (BL) transition in overlapped surfactant structure in Poractant alfa and Calsurf.

Differences in three-dimensional complexity between two surfactants were quantified by means of a surface roughness analysis. A mean peak-to-valley roughness Rtm of 28.8 ± 6.7 nm and 16.4 ± 2.5 nm was measured in Poractant alfa and Calsurf, respectively, demonstrating that surfactant preparations differed significantly in terms of microstructure (P<0.05).

Langmuir Blodgett analysis

Isotherms for Poractant alfa and Calsurf were generated at 25°C, compressing symmetrically the film spread on a pure water subphase. At the starting surface pressure of 10 mN/m, the molecular areas of Poractant alfa and Calsurf were 57 Å2/molecule and 48 Å2/molecule, respectively, suggesting for a more expanded monolayer in the case of Poractant alfa. The slope of the curves in the range 10–45 mN/m, in which phospholipid chains with a low (liquid-expanded phase) and high packing (liquid-condensed phase) coexist were equivalent for both preparations (Fig 2A). The coexistence of two phases makes surfactant films poorly compressible; indeed, the minimum value of compressibility of 0.02 m/mN was reached by both surfactants in this pressure range (Fig 2B).

Fig 2

Poractant alfa and Calsurf in compression isotherms and film compressibility.

(A) shows the compression isotherms of Poractant alfa and Calsurf, recorded at 25°C using MilliQ ultra-pure water as subphase. Surfactants were spread to an initial surface pressure of ≈ 10 mN/m and subjected to symmetric compression at a rate of 40 cm2/min until film collapse. (B) shows film compressibility (Cm) of Poractant alfa and Calsurf as a function of surface pressure. Each Cm data set was calculated using compression isotherms shown in (A). Cm = -1/A*(δA/δπ), where A is the surface area and π is the surface pressure.

At surface pressure of 45 mN/m Poractant alfa and Calsurf generated a plateau, a region of the isotherm representing the transition from monolayer to multi-layer with selective exclusion (squeeze-out) of the more fluid-like unsaturated lipids, cholesterol and proteins from a monolayer progressively enriched in DPPC. The two surfactants differed in the length of the plateau, more extended in Calsurf. In this region of the isotherm, surfactant films are highly compressible, due to the squeeze out of fluid molecules from the interface. In the case of Poractant alfa a maximum value of compressibility of 0.11 m/mN was measured, slightly higher than 0.08 m/mN in Calsurf, although the latter maintained a status of high compressibility for a more extended surface pressure range (from 45 to 60 mN/m). The comparison of the compressed area at a surface pressure of 68mN/m did not reach statistical significance, being 14.6 ± 0.35 Å2/molecule for Poractant alfa and 13.8 ± 0.76 Å2/molecule for Calsurf.

Standardized PIP ventilation loop experiments

There were no statistical differences in body weight between the experimental groups (see S1 Table). Untreated animals (Ctrl) showed extremely low Cdyn values (less than 0.1 mL/kg/cmH2O), indicative of severe lung structural immaturity with surfactant deficiency mimicking human RDS (Fig 3). Compared to untreated animals, all surfactant-treated groups had a significantly higher Cdyn during the first 15 min and the last 5 min of the experimental period, when PIP was 25 cmH2O (P<0.01). Lowering PIP from 25 to 20 cmH2O and then to 15 cmH2O was associated with a decrease of Cdyn in all surfactant-treated groups. At PIP levels of 20 and 15 cmH2O, there were no significant differences between surfactant-treated groups and the control group, except for P200, which registered significantly higher mean Cdyn values compared to untreated control animals (p<0.01 at PIP 20 cmH2O and p<0.05 at PIP 15 cmH2O). Irrespective of the surfactant preparation or the administered dose, no significant differences were found if surfactant-treated groups were compared. The response to surfactant administration in terms of Cdyn was similar at a PEEP level of 2–3 cmH2O or in the absence of PEEP.

Fig 3

Poractant alfa and Calsurf in preterm rabbits with primary surfactant deficiency in PIP loop experiment.

The dynamic compliance of the respiratory system (Cdyn) was determined during mechanical ventilation with a standardized PIP loop (25-25-25-20-15-25 cmH2O, each PIP for 5 min). Two independent experimental sessions, with the PEEP set at 2–3 cmH2O (A) and without PEEP (B) were conducted. Symbols and ticks represent mean±SD. Group definitions for symbols: P200, 200 mg/kg of Poractant alfa, black triangles; P100, 100 mg/kg of Poractant alfa, grey triangles; C200, 200 mg/kg of Calsurf, black circles; C100, 100 mg/kg of Calsurf, grey circles; C70, 70 mg/kg of Calsurf, white circles; Ctrl, untreated animals served as controls, white squares; (n = 10 animals in each group). One-way ANOVA with S-N-K post-hoc test was used for between-group comparisons. # P<0.01 vs. all surfactant-treated groups; ** P<0.01, * P<0.05 vs. Ctrl group.

Comparison of survival-time after prophylactic surfactant treatment in standardized VT ventilation experiment

A second experimental session with the same experimental groups as above, but with another 25 animals per group, was designed to investigate survival following surfactant administration. The delivery of a 200 mg/kg dose of Poractant alfa was associated with a significant reduction of mortality compared to untreated but identically ventilated control animals (Fig 4, p<0.05). After 180 min, the survival rate across groups followed the order: P200 (76%) >P100 (60%)>C200 (56%)>C100 (48%) >C70 and Ctrl (40%). However, no significant differences were detected after comparing surfactant-treated groups. The 50% survival time for the Ctrl group was around 100 min, which was extended to 130 min for the C70 group, 180 min for the C100 group and extended to over 180 min for P100, P200 and C200 groups. In our additional experiment with the same standardized VT ventilation protocol of 360 min, the 50% survival time for P200 could reach 280 min while Ctrl and C70 groups confirmed a 50% survival time around 100 and 130 min (see S1 Fig, n = 13 in each group, P<0.05 in log-rank (Mantel-Cox) test).

Fig 4

Survival rate of preterm rabbits after surfactant treatment in standardized VT ventilation for 180 min.

Lines for each group are defined as survival rate over time. At 180 min, the survival rate across groups followed the order: P200 (76%) >P100 (60%)>C200 (56%)>C100 (48%) >C70 and Ctrl (40%). The 50% survival time was 100 min in Ctrl group, 130 min in C70 group, 180 min in C100 group and more than 180 min in C200, P100 and P200 groups. P200 and P100, 200 and 100 mg/kg of Poractant alfa, C200, C100, and C70, 200, 100 and 70 mg/kg of Calsurf. Ctrl, control as untreated but ventilated. For group symbols: P200, thick black dash line; P100, thick dark grey dash line; C200, thin black dotted line; C100, thin dark grey dotted line; C70, thin light grey dotted line; Ctrl, black solid line. * P<0.05 vs. Ctrl in log rank (Mantel-Cox) test, n = 25 in each group.

Statistically significant differences in survival rate between P200 and control groups could be already detected 45 min after the initiation of mechanical ventilation (P<0.01) and were maintained until the end of the experimental period (see S1 Table). Over the 180 min ventilation with standardized VT, there was 50–150% increment of values of Cdyn in all surfactant-treated groups compared to the control group (all P<0.05). However, no significant difference was detected between surfactant groups with different doses. At 180 min, the values of Cdyn across groups followed the order: P200, P100>C200, C100, C70>Ctrl. (see S2 Table).

Biochemical analysis of broncho-alveolar lavage fluid and lung tissue samples

The measurements of phospholipids and proteins in the lungs after 180-min of mechanical ventilation revealed that, there were approximately 50–60 mg/kg more of TPL, 30–35 mg/kg more of DSPC, and 80–100% relative increment of DSPC/TPL, in the lung tissue homogenates of both P200 and C200 groups, compared with the corresponding baseline values of the control group (Fig 5A–5C). Similar trends were found for TPL and DSPC in the BAL fluid although, in this case, the DSPC/TPL ratio increased only modestly in both P200 and C200 groups (Fig 5D–5F). In contrast, values of TPL, DSPC and DSPC/TPL in P100, C100 and C70 had mild to moderate increment in both lung homogenates and BAL fluid. The amounts of TP (Fig 5G) were 30–40% higher in all mechanically ventilated animals compared to non-ventilated controls (C0). The values for DSPC/TP (Fig 5H) were 2-4-fold higher in all surfactant-treated groups compared to Ctrl and C0 groups. These results indicate that a higher initial phospholipid dose (e.g. 200 mg/kg) may provide a significant benefit in improving the intracellular surfactant pool.

Fig 5

Total phospholipids and proteins in lung tissue homogenates and bronchoalveolar lavage.

Comparison of total phospholipids and proteins in lung tissue homogenates (A-C) and in bronchoalveolar lavage (BAL) fluid (D-F) of preterm rabbits after treatment with Poractant alfa or Calsurf followed by 180 min standardized VT of mechanical ventilation. G-H show total proteins in BAL fluid. P200 and P100, 200 and 100 mg/kg of Poractant alfa, C200, C100, and C70, 200, 100 and 70 mg/kg of Calsurf. Ctrl, control as untreated but ventilated, and C0, untreated and non-ventilated. Abbreviations: TPL, total phospholipids; DSPC, disaturated phosphatidylcholine; TP, total proteins. Values are mean±SD or ratio in percentage (%). * P<0.05 vs. Ctrl and C0; n = 8–10 in each group.

After completion of the experimental follow up, the W/D ratio was determined as an estimate of the total lung fluid capacity as well as postnatal lung fluid clearance, versus maturation. The P200 group showed the lowest W/D and was the only surfactant-treated group with significantly improved W/D values compared to untreated controls (Table 2), suggesting that lung fluid clearance was accelerated along the mechanical ventilation period in the P200 group.

Table 2

Lung conditions in preterm rabbits after 180 min of standardized VT mechanical ventilation.

Groups	PTX	W/D	Vv	CV [Vv]
P200	3 (12)	6.17±1.48*	0.50±0.06*	0.23±0.06*
P100	6 (24)	7.50±1.84	0.42±0.07	0.25±0.04
C200	6 (24)	7.78±1.57	0.48±0.05*	0.27±0.04
C100	4 (16)	7.69±1.25	0.42±0.06	0.26±0.05
C70	4 (16)	8.08±1.52	0.38±0.06	0.28±0.04
Ctrl	5 (20)	8.58±1.43	0.39±0.08	0.31±0.05

Group definitions: P200, Poractant alfa 200 mg/kg; P100, Poractant alfa 100 mg/kg; C200, Calsurf 200 mg/kg; C100, Calsurf 100 mg/kg; C70, Calsurf 70 mg/kg; Ctrl, control. Abbreviations: PTX, pneumothorax; W/D, wet-to-dry lung weight ratio; Vv, morphometry of alveolar expansion, as aerated vs. total lung parenchyma by point counting; CV [Vv], coefficient of Vv, as homogeneity of alveolar expansion. Values are number (%) for PTX, n = 25, and ratio as means±SD for the rest of variables, n = 13.

* P<0.05 vs. Ctrl group (one-way ANOVA with S-N-K post-hoc test).

VV accounts for improved alveolar expansion at the end of expiration [32] and is a good estimation of residual volume reflecting the maintenance of functional residual capacity (FRC) under various treatment. VV was the lowest in the control group, most probably due to the high incidence of atelectasis in this group. All surfactant-treated groups showed higher mean VV than controls, although only P200 and C200 reached statistical significance. CV (VV) is a parameter that reflects the homogeneity of alveolar expansion. The mean CV (VV) values showed a similar trend as VV. Nevertheless, for CV (VV), the only significant difference was observed for P200, suggesting a more homogeneous alveolar expansion after delivery of 200 mg/kg dose of Poractant alfa.

Lung sections were evaluated by an expert pathologist for edema, hemorrhage, neutrophil infiltration, and epithelial desquamation. All surfactant-treated groups were scored with significantly less epithelial desquamation compared to untreated control animals, which highlights the lung protective function of surfactant therapy in the setting of lung injury (Table 3). Poractant alfa-treated groups showed a significantly lower incidence of hemorrhage compared to controls. On the other hand, neutrophilic infiltration in both P200 and C200 was significantly lower compared to Ctrl. For comparison between two surfactants, epithelial desquamation was significantly higher in C70 than P200 group (P<0.05), and P200 was significantly lower than all other four surfactant-treated groups in sum of LIS, including C200 group (P<0.05 vs. C200, C100 and P100 groups; P<0.01 vs. C70 group).

Table 3

Lung injury scores of preterm rabbits after 180 min of standardized VT mechanical ventilation.

Groups	Lung injury scores
	Edema	Hemorrhage	Neutrophil infiltration	Epithelial desquamation	Total
P200	1.62±0.34	0.58±0.25*	1.11±0.46*	0.10±0.19**	3.41±0.97**
P100	1.90±0.59	0.64±0.34*	1.80±0.50	0.64±0.31**	4.98±1.12**#
C200	1.60±0.54	0.98±0.34	1.40±0.48*	0.50±0.33**	4.48±1.07**#
C100	1.89±0.46	0.80±0.36	1.90±0.57	0.54±0.33**	5.13±1.23**#
C70	2.10±0.56	1.10±0.45	2.10±0.36	0.98±0.44*#	6.28±1.43*##
Ctrl	2.40±0.46	1.40±0.48	2.50±0.48	2.30±0.53	8.60±1.96

Values are means±SD.

* P<0.05 and

** P<0.01 vs. Ctrl group

## P<0.01 and

# P<0.05 vs. P200 group (Kruskal-Wallis test followed by Wilcoxon-Mann-Whitney test, n = 13 in each group). P200 and P100, 200 and 100 mg/kg of Poractant alfa, C200, C100, and C70, 200, 100 and 70 mg/kg of Calsurf. Ctrl, control as untreated but ventilated.

Discussion

In the present work, we have compared the in vitro and in vivo performance of Poractant alfa and Calsurf. In vitro, we found differences in the lipid composition of both preparations, which might partly explain evident ultrastructural differences as determined by AFM. In vivo, irrespective of the surfactant preparation and dose, we found that surfactant treatment significantly improved Cdyn, survival time, W/D and CV [Vv] compared to untreated control animals, with only P200 group reached the statistical significance. If the highest licensed clinical dose of Poractant alfa (200 mg/kg) and the routine clinical dose of Calsurf (70 mg/kg) were compared directly, treatment with Poractant alfa was associated with a significantly lower LIS but not in other aspects. To carry out a sound head-to-head scientific comparison between surfactants, we included additional experimental groups of Calsurf-treated rabbits with dosages of 100 and 200 mg/kg, which would match the licensed doses of Poractant alfa. Considering that Poractant alfa is not licensed to be administered at doses below 100 mg/kg, we did not include a group of animals treated with Poractant alfa at a dose of 70 mg/kg. The intratracheal delivery of either preparation at a 200 mg/kg dose was associated with a marked increase of alveolar expansion and higher intracellular DSPC pools. Nevertheless, a significant reduction of RDS-associated mortality compared to untreated but ventilated controls was only achieved after treatment with 200 mg/kg of Poractant alfa.

Surfactant replacement reduces RDS-associated mortality and morbidity and is widely used for the treatment of preterm infants with moderate-to-severe RDS. In clinical practice, animal-derived surfactant preparations, which contain relatively high amounts of DPPC as well as SP-B and SP-C, are still recommended over synthetic preparations.[6,7] The performance of Poractant alfa has been systematically compared, in vitro and in vivo, to that of other animal-derived as well as synthetic surfactants,[10,11,22,33,34] and has been confronted to other preparations in several clinical trials.[13-16] Significant differences in clinical outcomes were found between Poractant alfa and other bovine preparations, both from minced lung and lung lavaged fluid. However, differences were only limited to studies with a higher initial dose of Poractant alfa. Thus it could not be clarified whether it was related to animal source or initial dose.[35] The phospholipid concentration at which Poractant alfa is formulated (80 mg/mL), allowing delivering a high phospholipid dose (200 mg/kg) at relatively low fluid volumes (2.5 mL/kg), has been pointed out as an intrinsic advantage of this preparation in terms of reduction of RDS-associated mortality and re-dosing.[36] To date, no clinical studies have been conducted for comparisons with a dose of 200 mg/kg of bovine surfactants because of ethics.[18] The overall efficacy in the present study in regard to survival rate, Cdyn, biochemical measurements of intrapulmonary surfactant pool (including TPL, DSPC, DSPC/TP in BAL fluid and lung homogenate), lung morphometry and LIS was in favour of the high dose (200 mg/kg) of Poractant alfa not only in manufacturer-recommended doses comparison but also in amount-equivalent (i.e. mg for mg of phospholipids) comparison with Calsurf. We speculate that a low dose of surfactant may be still associated with its uneven distribution in the premature lungs, leading to alveolar and small airway damage and pneumothorax, and eventually to early death in the control and low-dose surfactant-treated groups. This may account for the lower LIS observed for the high dose groups compared to the low-dose groups, whereas Cdyn was not a sensitive indicator.

We found differences in the ultrastructure as well as in the lipid composition of Poractant alfa and Calsurf. AFM analysis revealed a marked difference in the height of lipid bilayers, which were of a lower height for Calsurf compared to Poractant alfa. Such differences may be explained by the presence of cholesterol in Calsurf, which increases the mobility of phospholipids and decreases their packing,[37] accounting for more fluid phospholipid organization in contrast to the tightly-packed, pure phospholipid bilayers of Poractant alfa. Moreover, we observed multilamellar structures of up to 15 overlapping lipid bilayers in Poractant alfa that were absent in Calsurf. We speculate that such multilamellar structures in Poractant alfa may occur due to a higher content of DPPC and resemble the lamellar bodies observed in vivo. However, it cannot be ruled out that the presence/absence of such structures may also be related to different phospholipid extraction methods employed for the production of the surfactants. During tidal breathing, the structure of lamellar bodies is crucial to transfer packed lipid structures into the air-liquid interface, supporting a rapid surfactant replenishment of the respiratory interface. Highly packed lamellar bodies are less prone to inactivation by binding serum components than the more exposed single bilayers.[38] Differences in lipid composition between surfactants were also suggested by the compression isotherms. Except for the very last portion of the curve, the isotherm of Poractant alfa was shifted to the right compared to that of Calsurf. These data reflect the more expanded monolayer of Poractant alfa and its higher content in unsaturated phospholipids. Indeed, the presence of double bonds along the alkyl chain, not only introduces more distance between phospholipids tails, accounting for a higher surface occupied by a single phospholipid molecule, but also disturbs the monolayer transition from a liquid expanded phase to the tightly packed liquid condensed phase (a transitional liquid-crystal state when the highest surface pressure is achieved during cyclic breathing).[39] Moreover, the more abundant fraction of unsaturated lipids in Poractant alfa, can support the higher value of film compressibility compared to Calsurf, because, upon compression, unsaturated phospholipid fraction preferentially segregates into more fluid domains easier to be compressed than those composed by saturated lipids.[40] Another difference in the compression isotherm was detectable in plateau extension, more pronounced in Calsurf. The plateau is representative of the removal from the interface of the more fluid surfactant components. Although unsaturated phospholipids take part to this event, the higher extension of the Calsurf plateau could be ascribed to the presence of cholesterol, absent in Poractant alfa. In fact, the plateaus differed of about 1.5-fold in our measurements, well reflecting a peculiar difference already observed upon compression among Poractant alfa and other commercial surfactant preparations containing cholesterol.[41]

In vivo, surfactant treatment was associated with significant improvement of Cdyn and reduced lung injury, irrespective of doses and preparations. The short-term pulmonary response indicates that a phospholipid dose as low as 70 mg/kg, which is the routine dose for Calsurf administration, may suffice to improve lung mechanics. However, we found a significant benefit in terms of lung expansion if either Calsurf or Poractant alfa were administered at a dose of 200 mg/kg. This benefit seems to be related to the development of an intracellular surfactant pool following a high-dose surfactant administration. The amount of DSPC in BAL fluid was significantly increased in all surfactant-treated groups, which may very well explain the short-term pulmonary response observed in surfactant-treated groups compared to untreated control animals. Nevertheless, the amounts of DSPC and DSPC/TPL in lung tissue were only significantly higher in both P200 and C200 groups, suggesting that more phospholipids were taken up by alveolar epithelial cells, presumably by retaining its large aggregate form, and be readily secreted into the alveolar space, or catabolized and reutilized to synthesize and ensemble new, endogenous surfactant (containing SP-A).[42] This intracellular surfactant pool could eventually replace the “spent” alveolar surfactant, enabling a more sustained pulmonary response after a single surfactant dose, a mechanism accounting for biological half-life and re-dosing, or bioavailability, of a surfactant preparation. So far, Calsurf is not licensed to be delivered at a dose of 200 mg/kg, although some domestic clinical study aimed to verify its efficacy.[43] At its present phospholipid concentration (35 mg/ml), delivering 200 mg/kg of Calsurf to preterm infants would involve a relatively high airway fluid load, which has been associated with clinical instability.[44]

With regard to the benefits of a surfactant dose of 200 mg/kg on lung mechanism improvement, Poractant alfa also achieved the lowest W/D, the most uniform alveolar expansion and lowest LIS, even compared to the same dosage of Calsurf. We speculate that such differences between surfactants could be related to their phospholipid concentration, accounting on the volume delivered to get the same dose, and their different biochemical composition. In particular, the more abundant fraction of unsaturated phospholipids and the presence of plasmalogens in Poractant alfa could support surfactant absorption and transition of phospholipids from the multi-layered reservoir to the active monolayer at the air-water interface.[10,45,46] We, therefore, suspect that the significant differences observed between Poractant alfa administered at a dose of 200 mg/kg and untreated control animals in the PIP loop experiments, under the most astringent ventilation conditions (PIP of 15 cmH2O without PEEP support), may be in part explained by the presence of plasmalogens, which may induce a fast transition from bilayer to monolayer structures under the demanding physiological breathing rates.[45] We speculate that such differences in performance between surfactants derive from differences in lipid composition and concentration, which significantly influence the ultrastructure as well as the pulmonary performance. The results of this investigation set the rationale to look into more clinical studies which may facilitate the comparison of the two preparations based on large sample sizes.

Conclusions

We have performed a preclinical characterization of Calsurf comparing its performance to that of Poractant alfa. Both preparations improved lung mechanics and protected lung injury in the ventilated preterm rabbit model of primary surfactant deficiency to resemble human RDS. The overall and specific effects were in a dose-dependent response pattern with maximum benefits achieved at a dose of 200 mg/kg in both preparations. Poractant alfa delivered at 200 mg/kg exerted better effects in terms of alveolar expansion, lung fluid clearance and protection of lung parenchyma, along with a significantly reduced mortality in comparison to untreated animals.

Liquid-chromatography mass-spectrometry expanded method for phospholipids analysis.

(DOCX)

Click here for additional data file.

Birth weight (BW) and survival rate of preterm rabbits assessed for 180 min standardized VT mechanical ventilation.

Values are mean+SD, or n (%). Group definitions: P200, Poractant alfa 200 mg/kg; P100, Poractant alfa 100 mg/kg; C200, Calsurf 200 mg/kg; C100, Calsurf 100 mg/kg; C70, Calsurf 70 mg/kg; Ctrl, control. ** P<0.01 and * P<0.05 vs. Ctrl group by Chi square test. Initial numbers = 25 in each group.

(DOCX)

Click here for additional data file.

Dynamic compliance of respiratory system (Cdyn) over time in preterm rabbits with prophylactic surfactant treatment and standardized VT mechanical ventilation.

Values are mean+SD. a: p values are for one-way analysis of variance (ANOVA) by F test; * p<0.05 for between-group differences between Ctrl and any surfactant-treated groups by Student-Newmann-Keuks post hoc test. For group definitions and sample sizes at each time point see S1 Table in the Supporting information.

(DOCX)

Click here for additional data file.

Survival rate of preterm rabbits after prophylactic treatment in standardized VT ventilation for 360 min.

Lines for each group are defined as survival rate over time. The 50% survival time was 100, 120 and 280 min in Ctrl, C70 and P200 group, respectively. For group definition and symbols see Fig 4. * P<0.05 vs. Ctrl in log rank (Mantel-Cox) test, n = 13 in each group.

(TIF)

Click here for additional data file.

26 Jul 2019

PONE-D-19-15621

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa (Curosurf) and Calsurf (Kelisu) in ventilated preterm rabbits

PLOS ONE

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Reviewer #1: The tables and figures were imbedded in the manuscript instead of at the end (out of order), and the figure legends don't tell a story of what it is they are trying to show. The take home message of each figure is not clear.

The language and labeling is confusing- for example- C200 corresponds to poractant 200 mg/kg and K200 is calsurf. why would calsurf start with the K and not the C?

Also, the numbers in the tables don't always correspond with the results section. Relevance of results is not well explained.

Statistics are lacking- for example table 1.

Reviewer #2: With interest, I read the manuscript by Bo Sun et al about the characterization and comparison of two types of surfactant in preterm rabbits. The authors performed an impressive and complete vivo and vitro investigation. The study is well performed, well described and interpretable by readers with limited background in animal and in vitro studies. The study showed a favourable outcome for poractant. This study, although more detailed and done in animals, is one of many trying to identify a difference between the different surfactant products. Studies done in preterm infants have shown contradictory results, although generably in favour of poractant. Reviewing the literature, it is remarkable how much of the studies, as is this one, have been sponsored by surfactant manufacturers. It is my opinion that the surfactant research needs more independent studies. The study is a head to head experiment, eventually using 60 rabbit pups in several experiments. The study was approved by the medical ethics counsel and suffering for the animals was minimised. Thoughout the manuscript it remains somwhat unclear which rabbit underwent which investigation(s).

Some questions and suggestions remain,

How is it possible that Calsurf is marketed without any preclinical or clinical studies? For bovine or calf derived surfactant there is enough evidence and clinical studies, how does Calsurf relate to these products?

Line 351-352 PEEP or no PEEP, If I look at figure 3, I see, possibly not significant, a positive effect of PEEP on the Cdyn. The positive effect of C200 is amplified by PEEP. Would have liked to see higher PEEP levels, although not investigated in this study.

When looking furhter at figure 3, it strikes me that K100 outperforms C100 and that C100 is so much ‘worse’ than C200. Any thoughts on that?

Line 365 – 377 This seems to be an irrelevant finding and does not support the use of Poractant. The control group was not treated with surfactant. It is well know that they do not survive. Also for Table 2. Of course there is a difference between the control group and the poractant group. But this was not the object of the investigation. Also for 473 – 474 and 554-555, the control group is not under investigation.

In figure 4 it is hard to disciminate between the different lines

Reference 6 has a 2019 update

Reviewer #3: This is a bench research work comparing two surfactant preparations with a multitude of methods.Practant alfa and Calsurf are compared by various in vitro tests and also in vivo in rabbit parts. Methods include the measurement of lipid composition, atomic force microscopy, Langmuir Blodgett analysis, and in vivo effects in premature rapid pups. In the latter, compliance, survival time, and lung histology were measured. By all means, results point in the same direction, with both surfactants having typical effects, but poractant alfa function was superior. In that respect, this reviewer cannot follow the author's conclusion that large clinical study comparing both surfactants might be warranted. Such a study might be unethical in view of the superior results found in all aspects with poractant alfa, because it is likely to expose a large number of infants to an inferior surfactant preparation. Equipoise, the prerequisite of randomization, can no longer be claimed after the described results have been found.

The abstract does not really reflect what was done in the paper, and whant the resutls were. It may be misleading in referring only to Calsurf dosages of 70-100 mg per kilogram, whereas in fact, Calsurf was also tested at 200 mg per kg. Revising the abstract might be warranted.

line 477: practice, not praxis

Reviewer #4: The authors of this manuscript present a very comprehensive in vitro and in vivo analysis of these two surfactant preparations. Upon revision, these data will be an important contribution to the exogenous surfactant literature.

Major Criticisms

1. The authors use matching doses of 100 mg/kg and 200 mg/kg of both surfactants but only 70 mg/kg of Calsurf. The authors need to explain in the manuscript why there is no Poractant alfa 70 mg/kg for comparison.

2. For all experiments, the authors report statistical comparisons between experimental and control animals (which is important), but neglect to report statistical comparison between experimental groups (except for compliance (line 391) and lung injury score C200 vs K70 (line 456)). Despite this omission, they repeatedly state that one surfactant is superior to the other (lines 445, 467, 493, 553, 574, etc)

3. Methods (line 196) and associated Results/Fig 3 – There needs to be both a discussion and references to why the authors used no PEEP and a low PEEP such as 2-3 cm H2O. In part, this could be a reason why administration of surfactant did not statistically improve compliance (except for C200)(line 349, Fig 3) which is surprising. In figure 3, given the small SD for the control, it is surprising that there is no statistical difference. The authors may need to recheck their statistical analysis. For the figure 3 legend, it is not clear what “*”, “**”, and “#” refer to.

4. Methods (line 208) – For the prophylactic surfactant treatment, the authors report “A PIP ranging between 10-25 cmH2O was applied to generate a VT of 4-6 mL/kg body weight” (line 208). It is clear that pulmonary compliance changes quickly after administration of surfactant. During the 3 hour experiment, how often was the PIP titrated to maintain a goal VT of 4-6 mL/kg?

5. Figure 3 and Table S2 – There needs to be some discussion of why dynamic compliance was so different between the PIP ventilation experiment and the survival experiment. For example, at 15 minutes for C200, compliance was ~0.5 in figure 3A but was 0.28 in table S2. The authors seem to pick the data they prefer by stating in the discussion, “…was associated to significant improvement of Cdyn..” (line 531). This may be true for the data in Table S2 but not for the data in Figure 3.

6. Results – For the AFM, Langmuir Blodgett and lipid analysis, why is there no quantitative comparison or statistical analysis? How do we know if the samples are statistically different? For the lipid analysis, why were only 4 samples tested for Calsurf but 8 samples tested for Poractant alfa and why are standard deviations omitted?

7. Results (line 373) – for the survival analysis, the authors mention a 360 minute ventilation experiment but don’t include these data. These data should be included and the protocol needs to be described in the methods section.

8. Discussion – the authors should add speculation for why the lipid species are so different in Table 1, yet when given to rabbits, they are not very different in lung homogenate or BAL fluid (Figure 5).

9. Discussion – the authors should add speculation to reconcile the lack difference in compliance between surfactants yet improved survival. What is the mechanism?

10. Discussion (lines 487, 551) - The authors point out the importance of the fluid volumes of the instilled surfactant, yet don’t include this data in the methods or results. For each of the experiments presented, what volumes of surfactant were instilled?

Minor Criticisms

1. It is noted in the PLOS One competing interests statement that four of the authors are employees of Chiesi including Davide Amidani. For Davide Amidani, this needs to be noted in the manuscript itself for readers to be aware of (line 5).

2. Throughout the manuscript, the use of abbreviations based on the trade names is confusing. Consistent with the generic nomenclature, the abbreviations should be Calsurf (C70, C100, C200) and Poractant alfa (P100, P200). Trade names should be omitted from the manuscript except maybe once in the methods section.

3. For the pathology analysis (line 246), the authors need to specify whether or not the pathologist was blinded or not to the experimental group.

4. Results (line 410) – The authors state “…suggesting DSPC/TP to 410 be a sensitive indicator for lung injury protection.” Surfactants have phospholipids and surfactants can help, in some ways, to prevent lung injury. This does not mean, nor does the data provided, support that this ratio is a sensitive indicator. This statement should be omitted.

5. Results (line 437) – “VV accounts for improved alveolar expansion at the end of expiration and is a good estimate to assess the maintenance of functional residual capacity (FRC)” – a reference should be provided to support this statement.

6. Results (line 283) – DPPC is spelled wrong as “pipalmitoylphosphatidylcholine”

7. Results (line 409) – “folds” should be “fold”

8. Discussion (line 477) – While the term “praxis” may technically be correct, the term “practice” is more understood

**********

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7 Sep 2019

Authors reply to each query (Q) and additional changes.

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Response: We thank the editors for the instructions. We hope that our manuscripts meet the specified requirements.

2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: 1) Please provide details of animal welfare for the does (e.g., shelter, food, water, environmental enrichment); 2) Please confirm that the pups were anaesthetised throughout the study and never gained consciousness; Please also clarify whether the does were anaesthetised when they were euthanized and 3) Please provide the source of the porcine lungs used in this study to isolate Poractant alfa. If the pigs were euthanized specifically for this study, please include the method of euthanasia. If they were obtained from a slaughterhouse, please provide the name, location, and accreditation. Thank you for your attention to these requests.

Response: 1) All does were housed until the twenty-sixth day of gestation under standard conditions of the animal center and were transported to the experiment site one day prior to the experiments with full shelter, food and water. Related contents are added to Method part, page 8, line 176-178, of R1-marked. 2) All does and pups were anaesthetized and never gained consciousness throughout the study. Does were sacrificed by overdose of potassium. Pups were euthanized by intracranial injection of 0.5 mL 2% lidocaine. Related content are add to Method part, page 8, line 187-189 and page 9, line 215-216, of R1-marked. 3) Poractant alfa was purchased by the Research and Development Department of CHIESI Farmaceutici, from the Sales Department of the same company. Therefore, all Poractant alfa vials used in this study were produced in compliance with all European regulations concerning the development of pharmaceutical products. Technically, all Poractant alfa vials are from a commercial source and no pigs were specifically euthanized for this study.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: S1 Fig is added to Supporting Information to show the survival rate of groups over the 360 min observation period (also see response in Q8), and the phrase has been removed in R1.

4. Thank you for stating the following in the Financial Disclosure section:

The study was supported by Chiesi Farmaceutici S.p.A. The company contributed to the study design but had no influence in the performance, analysis, and interpretation of experimental data and in writing the manuscript. YD is a recipient of research grants from the National Natural Science Foundation (No. 81501288) and Shanghai Municipal Commission of Health (Project Young Physician Investigator).

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Response: We have extended the Competing Interests Statement as follows, and is added to cover letter.

The study was supported by Chiesi Farmaceutici S.p.A, which owns the marketing rights for Poractant alfa. The company contributed to the study design but had neither influence on the performance, analysis, and interpretation of experimental data nor in writing the manuscript. BP, SC, and FS are employees of Chiesi Farmaceutici S.p.A. XM served as consultant for Chiesi in this study. YD is a recipient of research grants from the National Natural Science Foundation (No. 81501288) and Shanghai Municipal Commission of Health (Project Young Physician Investigator). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Reviewer reports:

Reviewer #1:Q1 The tables and figures were imbedded in the manuscript instead of at the end (out of order), and the figure legends don't tell a story of what it is they are trying to show. The take home message of each figure is not clear.

Response: We thank for the reviewer’s comments. We submitted the tables and figures according to the PLOS requirements, in which the tables and figure legends were embedded in the manuscript and the figures were submitted separately as TIFF forms. The figure legends have been rephrased for your reading convenience.

Q2 The language and labeling is confusing- for example- C200 corresponds to poractant 200 mg/kg and K200 is calsurf. why would calsurf start with the K and not the C?

Response: We thank for the reviewer’s comments and feel sorry for the confusion. Reviewer 4 also mentioned this problem in Q25. Considering the generic nomenclature and to keep coincidence, the abbreviations of experimental groups are modified into C70, C100, C200 for 70, 100, 200 mg/kg Calsurf and P100, P200 for 100, 200 mg/kg Poractant alfa in R1.

Q3 Also, the numbers in the tables don't always correspond with the results section. Relevance of results is not well explained.

Response: We thank for the comments and rephrased results related to tables and figures in R1.

Q4 Statistics are lacking- for example table 1.

Response: We have analyzed and determined the surface roughness of Poractant alfa and Calsurf, which is a parameter representative of the microstructural complexity of the surfactant. We performed an ANOVA and then a t-test between Poractant alfa and Calsurf and both tests revealed a significant difference between preparations, showing a more complex three-dimensional structure of Poractant alfa.

In Table 1, we have completed the descriptive statistics by adding the standard deviation of the measurements for each class of phospholipids in the new version of the manuscript. For PL classes less represented in the surfactants, our NP HPLC-MS methods showed approximately a 20-30 % of difference in the quantitative estimation. This was in part due to the presence of some single species with intensities near the quantitative detection limit, and in part to the fact that data were acquired in full scan mode, by switching the two polarities, using an ion trap mass spectrometer. This approach, in our opinion, was adequate for the target of this work, a general comparison of the two surfactants.

Reviewer #2: With interest, I read the manuscript by Bo Sun et al about the characterization and comparison of two types of surfactant in preterm rabbits. The authors performed an impressive and complete vivo and vitro investigation. The study is well performed, well described and interpretable by readers with limited background in animal and in vitro studies. The study showed a favourable outcome for poractant. This study, although more detailed and done in animals, is one of many trying to identify a difference between the different surfactant products. Studies done in preterm infants have shown contradictory results, although generally in favour of poractant. Reviewing the literature, it is remarkable how much of the studies, as is this one, have been sponsored by surfactant manufacturers. It is my opinion that the surfactant research needs more independent studies. Q5 The study is a head to head experiment, eventually using 60 rabbit pups in several experiments. The study was approved by the medical ethics counsel and suffering for the animals was minimised. Throughout the manuscript it remains somewhat unclear which rabbit underwent which investigation(s).

Response: We feel sorry for not having clarified enough the in vivo experimental design. Actually, in total, two in vivo sessions of experiments were performed: one was standardized PIP loop ventilation experiment, and the other, standardized VT ventilation experiment.

In the PIP loop session, 60 rabbit pups were randomly allocated into six ventilation groups, namely P200 (Poractant alfa 200 mg/kg), P100 (Poractant alfa 100 mg/kg), C200 (Calsurf 200 mg/kg), C100 (Calsurf 100 mg/kg), C70 (Calsurf 70 mg/kg) and Ctrl (shame air) groups in this revised manuscript (n=10 in each group).

In the standardized VT session, in addition to the former six groups (n=25 in each group in this session), we added one non-ventilated group (C0), which served as an additional control group (n=9) for biochemical analysis of broncho-alveolar lavage and lung tissue samples.

Some questions and suggestions remain,

Q6 How is it possible that Calsurf is marketed without any preclinical or clinical studies? For bovine or calf derived surfactant there is enough evidence and clinical studies, how does Calsurf relate to these products?

Response: We appreciate this comment, which raised a relevant concern. We have conducted an additional search of articles on PubMed with the keywords “Calsurf” or “Kelisu”. As a result, we find only one additional research published: Rong Z, et al. A multicetered randomized study on early versus rescue calsurf administration for the treatment of respiratory distress syndrome in preterm infants. Am J Perinatol. 2019 Feb 4. doi:10.1055/s-0039-1678530. This publication has been added to Introduction part as reference [17]. Neither surface properties nor comparisons to other surfactants have been published yet to the best of our knowledge. We can only know Kelisu has similar formulation in terms of phospholipid concentration (roughly about 30 mg/ml) as other bovine derived surfactants (reviewed in [18] in revised manuscript). Related contents are added or rephrased in page 4, line 82-88, in R1-marked.

Q7 Line 351-352 PEEP or no PEEP, If I look at figure 3, I see, possibly not significant, a positive effect of PEEP on the Cdyn. The positive effect of C200 is amplified by PEEP. Would have liked to see higher PEEP levels, although not investigated in this study.

When looking further at figure 3, it strikes me that K100 outperforms C100 and that C100 is so much ‘worse’ than C200. Any thoughts on that?

Response: We thank for the comment. To clarify this issue, we replaced the acronyms of experimental groups in the revised manuscript (modified according to comments by Reviewer 1 and 4 in Q2 and Q25): P200 (Poractant alfa 200 mg/kg), P100 (Poractant alfa 100 mg/kg), C200 (Calsurf 200 mg/kg), C100 (Calsurf 100 mg/kg), C70 (Calsurf 70 mg/kg) and Ctrl (shame air) groups.

PEEP indeed had a positive effect on Cdyn, not only in P200 group but also in all groups including Ctrl group, with no statistic significance. However, the effect of PEEP was not the aim of this study, so we didn’t highlight this point in the manuscript. Showing both results in Fig 3A and 3B was only to clarify the similar effects of surfactants both in dose and category way. Higher PEEP was not applied because of the higher requirements of ventilation instruments.

Considering deviations, Cdyn in C100 didn’t outperform P100, and the possible reason why P100 was so much “worse” than P200 may be related to the little fluid volume of exogenous surfactant, which was only 1.25 ml/kg in P100 group (about 0.03 ml per pup assuming birth weight was 27 g). The little fluid volume may have a negative effect on surfactant diffusion in premature lung.

Q8 Line 365 – 377 This seems to be an irrelevant finding and does not support the use of Poractant. The control group was not treated with surfactant. It is well known that they do not survive. Also for Table 2. Of course there is a difference between the control group and the poractant group. But this was not the object of the investigation. Also for 473 – 474 and 554-555, the control group is not under investigation.

Response: We thank for the comments and queries. This protocol of mechanical ventilation, along with intermittent intraperitoneal injection of mixed solutions (Lidocaine+NaHCO3+Glucose), aiming at providing extra anaesthesia, energy and counterbalancing acidotic deterioration, ensured consistently the assessment of surfactant effects when the total length of ventilation may be 3-6 hours (Fig. 4 and S1 Fig. in R1). S1 Fig. is added in R1 to illustrate the survival length in the Ctrl, P200 and C70 groups. The survival length for the other three surfactant-treated groups (P100, C200 and C100) may be estimated thereupon. We applied a log-rank test as stated in the Methods (page 13, line 289) of the body text for comparison of the survival status across the groups. With this test statistic, the survival length in the surfactant-treated groups was compared step-wise in time interval with those in the control group which received identically standardized mechanical ventilation. This enabled estimation of either 50% survival (and death as reciprocal) at different time(s) or overall survival rate at a designated time over the whole ventilation period. Thus, it supports the notion that the length of survival is one of the major advantages in assessment of dose-effect of surfactant preparation.

Q9 In figure 4 it is hard to discriminate between the different lines

Response: Thanks for your comments. Lines in figure 4 have been modified as P200, Poractant alfa 200 mg/kg, thick black dash line; P100, Poractant alfa 100 mg/kg, thick dark grey dash line; C200, Calfsurf 200 mg/kg, thin black dotted line; C100, Calsurf 100 mg/kg, thin grey dotted line; C70, Calsurf 70 mg/kg, thin light grey dotted line; Ctrl, shame air, black solid line.

Q10 Reference 6 has a 2019 update

Response: We thank for the comments. Reference 6 has been updated.

Reviewer #3: This is a bench research work comparing two surfactant preparations with a multitude of methods. Practant alfa and Calsurf are compared by various in vitro tests and also in vivo in rabbit parts. Methods include the measurement of lipid composition, atomic force microscopy, Langmuir Blodgett analysis, and in vivo effects in premature rabbit pups. In the latter, compliance, survival time, and lung histology were measured. By all means, results point in the same direction, with both surfactants having typical effects, but poractant alfa function was superior.Q11 In that respect, this reviewer cannot follow the author's conclusion that large clinical study comparing both surfactants might be warranted. Such a study might be unethical in view of the superior results found in all aspects with poractant alfa, because it is likely to expose a large number of infants to an inferior surfactant preparation. Equipoise, the prerequisite of randomization, can no longer be claimed after the described results have been found.

Response: We thank the reviewer for the comments. So far, no clinical trials have been conducted to compare the effects between Poractant alfa and Calsurf in preterm infants. Besides, the effects of Poractant alfa and other bovine or calf surfactants are not clear. Meta analysis in 2017 (reference [34] in R1) concluded Poractant alfa may be better, but could not tell whether it was related to animal source or initial dose (related discussion is added to page 24, line 540-541, of R1-marked). Accordingly, page 28, line 636-639, of R1-marked, the last sentences are modified or deleted.

Q12 The abstract does not really reflect what was done in the paper, and what the results were. It may be misleading in referring only to Calsurf dosages of 70-100 mg per kilogram, whereas in fact, Calsurf was also tested at 200 mg per kg. Revising the abstract might be warranted.

Response: We thank for the comments. The arrangement and results regarding the comparison between the two surfactant preparations in abstract have been modified, as suggested.

Q13 line 477: practice, not praxis

Response: Thanks, amended.

Reviewer #4: The authors of this manuscript present a very comprehensive in vitro and in vivo analysis of these two surfactant preparations. Upon revision, these data will be an important contribution to the exogenous surfactant literature.

Major Criticisms

Q14 1. The authors use matching doses of 100 mg/kg and 200 mg/kg of both surfactants but only 70 mg/kg of Calsurf. The authors need to explain in the manuscript why there is no Poractant alfa 70 mg/kg for comparison.

Response: We thank the reviewer for the comments. From the manufacturer information, Poractant alfa is licensed to 100-200 mg/kg and Calsurf is 40-100 (average 70) mg/kg. Considering the comparisons in both licensed dose and mg-to-mg ways, we set Poractant alfa in 100 and 200 mg/kg doses, Calsurf in 70, 100 and 200 mg/kg doses, so that 100 and 200 mg/kg of two surfactants were for head-to-head comparison and Calsurf 70 mg/kg was purposely added for licensed dose comparison with Poractant alfa 100 and 200 mg/kg. Considering that Poractant alfa will never be administered at doses below 100 mg/kg, we do not see the reason to add a Poractant alfa 70 mg/kg group for comparison.

Q15 2. For all experiments, the authors report statistical comparisons between experimental and control animals (which is important), but neglect to report statistical comparison between experimental groups (except for compliance (line 391) and lung injury score C200 vs K70 (line 456)). Despite this omission, they repeatedly state that one surfactant is superior to the other (lines 445, 467, 493, 553, 574, etc)

Response: We thank for the comments and have rephrased our results in in vivo studies. In Table 3, we report significance in lung injury scores between P200 (Poractant alfa 200 mg/kg) vs. other surfactant-treated groups in the Results, page 22, line 501-507, of R1-marked. We draw the statement that Poractant alfa was superior to Calsurf for two reasons: one was the direct comparison between the two surfactants, in which LIS was significantly lower in P200 group than in all other surfactant-treated groups (Table 3); the other was the indirect comparison for the two surfactants compared to the Ctrl group, in which only P200 group could reach the statistical significance in terms of Cdyn (PIP=20 and 15 cmH2O), survival time, W/D and CV [Vv] but no shown in the other surfactant-treated groups (Fig. 3, 4 and Table 2).

Q16 3. Methods (line 196) and associated Results/Fig 3 – There needs to be both a discussion and references to why the authors used no PEEP and a low PEEP such as 2-3 cm H2O. In part, this could be a reason why administration of surfactant did not statistically improve compliance (except for C200)(line 349, Fig 3) which is surprising. In figure 3, given the small SD for the control, it is surprising that there is no statistical difference. The authors may need to recheck their statistical analysis. For the figure 3 legend, it is not clear what “*”, “**”, and “#” refer to.

Response: We thank for the kind comments and feel sorry for the irrelevant presentation. For the Fig 3 legend, # refers to P<0.01 vs. all surfactant-treated groups; ** to P<0.01 and * to P<0.05 vs. Ctrl group. Accordingly, all surfactant-treated groups had a significantly higher Cdyn during the first 15 min and the last 5 min of the PIP loop experiment (when PIP=25 cmH2O). When lowering PIP to 20 and 15 cmH2O, only P200 group exerted significance compared to Ctrl group (P<0.01 when PIP=20 cmH2O, P<0.05 when PIP=15 cmH2O). The response to surfactant administration in terms of Cdyn was similar with or without PEEP=2-3cmH2O. The reason why the PIP loop was performed in both no- and low-PEEP was to verify whether there exists any differences of the surfactants response to PEEP. Related comments are also provided by Reviewer 1 in Q7. Two references [23, 24] and two sentences have been added in the Method part, page 9, line 217-218, and page 10, line 222, and Result part, page 17, line 379-381, and page 18, line 401-402, of R1-marked.

Q17 4. Methods (line 208) – For the prophylactic surfactant treatment, the authors report “A PIP ranging between 10-25 cmH2O was applied to generate a VT of 4-6 mL/kg body weight” (line 208). It is clear that pulmonary compliance changes quickly after administration of surfactant. During the 3 hour experiment, how often was the PIP titrated to maintain a goal VT of 4-6 mL/kg?

Response: We thank for the comments. During the first 30 min of ventilation, PIP was titrated in 3-5 minute interval, and subsequently, when the Cdyn (dynamic compliance of respiratory system) was relatively stable, PIP was adjusted at 10-15 minute interval. Related content is added to Method part of the body text, page 10, line 235-237, of R1-marked.

Q18 5. Figure 3 and Table S2 – There needs to be some discussion of why dynamic compliance was so different between the PIP ventilation experiment and the survival experiment. For example, at 15 minutes for C200, compliance was ~0.5 in figure 3A but was 0.28 in table S2. The authors seem to pick the data they prefer by stating in the discussion, “…was associated to significant improvement of Cdyn..” (line 531). This may be true for the data in Table S2 but not for the data in Figure 3.

Response: To clarify the differences of Cdyn levels (at 15 min, e.g.) in Fig 3 (as in the response to Q16) and S2 Table, different PIP was used in the PIP loop and the Vt standardized ventilation for survival. In the PIP loop ventilation, PIP=25-25-25-20-15-25 cmH2O, when PIP=25 cmH2O with or without PEEP, tidal volume could reach 8-10 ml/kg in P200 (Poractant alfa 200 mg/kg) group as a very good response while Cdyn reached ~0.5 ml/kg/cmH2O. In the survival experiment with standardized VT ventilation, PIP was adjusted in a range of 10-20 cmH2O, with PEEP at 2-3 cmH2O, to achieve 4-6 ml/kg of VT. Accordingly, Cdyn in the two different ventilation modes were not comparable. One sentence is added to Discussion, page 27, line 595, of R1-marked to clarify the issue.

Q19 6. Results – For the AFM, Langmuir Blodgett and lipid analysis, why is there no quantitative comparison or statistical analysis? How do we know if the samples are statistically different? For the lipid analysis, why were only 4 samples tested for Calsurf but 8 samples tested for Poractant alfa and why are standard deviations omitted?

Response: We have analyzed and determined the surface roughness of Poractant alfa and Calsurf, which is a parameter representative of the microstructural complexity of the surfactant. We performed an ANOVA and then a t-test comparing Poractant alfa and Calsurf. Both tests revealed a significant difference between preparations, showing a more complex three-dimensional structure of Poractant alfa. For this application, we considered the Langmuir Blodgett results as qualitative data and we did not perform any statistical analysis.

In Table 1, we have completed the descriptive statistics by adding the standard deviation of the measurements for each class of phospholipids in the new version of the manuscript. For PL classes less represented in the surfactants, our NP HPLC-MS methods showed approximately a 20-30 % of difference in the quantitative estimation. This was in part due to the presence of some single species with intensities near the quantitative detection limit, and in part to the fact that data were acquired in full scan mode, by switching the two polarities, using an ion trap mass spectrometer. This approach, in our opinion, was adequate for the target of this work, a general comparison of the two surfactants. Unfortunately, due to the limited Calsurf availability (e.g. not in Europe, only in China) we could only perform 4 LCMS runs with this particular preparation.

Q20 7. Results (line 373) – for the survival analysis, the authors mention a 360 minute ventilation experiment but don’t include these data. These data should be included and the protocol needs to be described in the methods section.

Response: We thank for comments. The additional experiment was conducted in the same standardized VT ventilation protocol except for the observation time for 360 min in P200 (Poractant alfa 200 mg/kg), C70 (Calsurf 70 mg/kg) and Ctrl (sham air) groups. We confirmed that the 50% survival time of Ctrl and C70 group was around 100 and 130 min and that of P200 group could reach 280 min. These results have been submitted as S1 Fig in Supporting information. See also on response to Q8. Related content is added to Results part, pages 18-19, lines 415-421, of R1-marked.

Q21 8. Discussion – the authors should add speculation for why the lipid species are so different in Table 1, yet when given to rabbits, they are not very different in lung homogenate or BAL fluid (Figure 5).

Response: We thank for the comments. There may exist two reasons for this speculation. For the first, although lipid species detected in Table 1 were abundant including cholesterol and several kinds of phospholipids, the percentage of saturated PL were both around 50%, which was consistent with Fig 5F though lacking significant differences among groups. For the second, as with the metabolism of exogenous surfactants in premature lungs, the surfactants were taken up by alveolar epithelial cells and reutilized as ingredients. A speculation is already presented in the Discussion, pages 25-26, lines 565-569, of R1-marked, regarding the biophysical behaviour of phospholipid composition of the two preparations. The total phospholipids (TPL) and disaturated phosphatidycholine (DSPC) detected in the experiment were those in the alveolar and intracellular pools, not identical to those from exogenous surfactant itself. In other words, these phospholipids were mixed up with endogenous phospholipids in each compartment pool.

Q22 9. Discussion – the authors should add speculation to reconcile the lack difference in compliance between surfactants yet improved survival. What is the mechanism?

Response: We thank for the comments. The facts that increment of surfactant dosage (by phospholipids) may be responsible for the prolonged survival time with association of stabilized compliance. There were not significant differences in Cdyn among surfactant-treated groups in standardized VT ventilation. We speculate that the relatively low VT should have exerted protective role in the difference of LIS in Table 3. A low dose of surfactant may be associated with its uneven distribution in the premature lungs provocative of alveolar and small airway damage and pneumothorax, leading to early death in the control and low-dose surfactant-treated groups. This is added in the Discussion, page 25, line 552-557, of R1-marked.

Q23 10. Discussion (lines 487, 551) - The authors point out the importance of the fluid volumes of the instilled surfactant, yet don’t include this data in the methods or results. For each of the experiments presented, what volumes of surfactant were instilled?

Response: We thank for the kind comments. The fluid volume for P200 (Poractant alfa 200 mg/kg), P100 (Poractant alfa 100 mg/kg), C200 (Calsurf 200 mg/kg), C100 (Calsurf 100 mg/kg) and C70 (Calsurf 70 mg/kg) groups were 2.50, 1.25, 5.71, 2.86 and 2.00 ml/kg respectively. Related contents have been added in Methods, page 9, line 205-208, of R1-marked.

Minor Criticisms

Q24 1. It is noted in the PLOS One competing interests statement that four of the authors are employees of Chiesi including Davide Amidani. For Davide Amidani, this needs to be noted in the manuscript itself for readers to be aware of (line 5).

Response: We have corrected the authors’ affiliations and we have extended the competing interest statement as:

The study was supported by Chiesi Farmaceutici S.p.A, which owns the marketing rights for Poractant alfa. The company contributed to the study design but had neither influence on the performance, analysis, and interpretation of experimental data nor in writing the manuscript. BP, SC, and FS are employees of Chiesi Farmaceutici S.p.A. XM served as consultant for Chiesi in this study. YD is a recipient of research grants from the National Natural Science Foundation (No. 81501288) and Shanghai Municipal Commission of Health (Project Young Physician Investigator). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Q25 2. Throughout the manuscript, the use of abbreviations based on the trade names is confusing. Consistent with the generic nomenclature, the abbreviations should be Calsurf (C70, C100, C200) and Poractant alfa (P100, P200). Trade names should be omitted from the manuscript except maybe once in the methods section.

Response: Thanks for the comments. The experimental groups and trade names of surfactants have been replaced accordingly in the revised manuscript.

Q26 3. For the pathology analysis (line 246), the authors need to specify whether or not the pathologist was blinded or not to the experimental group.

Response: We thank for the comments. The pathologist was blinded to the experimental design. Related content is added to Methods, page 12, line 274, of R1-marked.

Q27 4. Results (line 410) – The authors state “…suggesting DSPC/TP to be a sensitive indicator for lung injury protection.” Surfactants have phospholipids and surfactants can help, in some ways, to prevent lung injury. This does not mean, nor does the data provided, support that this ratio is a sensitive indicator. This statement should be omitted.

Response: We thank for the comments. This statement has been omitted in the revised manuscript.

Q28 5. Results (line 437) – “VV accounts for improved alveolar expansion at the end of expiration and is a good estimate to assess the maintenance of functional residual capacity (FRC)” – a reference should be provided to support this statement.

Response: Reference [31] has been provided in page 22, line 490, of R1-marked.

Q29 6. Results (line 283) – DPPC is spelled wrong as “pipalmitoylphosphatidylcholine”

Response: It has been changed.

Q30 7. Results (line 409) – “folds” should be “fold”

Response: It has been changed.

Q31 8. Discussion (line 477) – While the term “praxis” may technically be correct, the term “practice” is more understood

Response: It has been changed.

Other changes in R1 not related to reviewers’ and editors’ comments:

Minor changes about language for more readable. Reference [25-26] is added for the original method in phospholipid analysis.

Submitted filename: Response to Reviewers-1.docx

Click here for additional data file.

2 Dec 2019

PONE-D-19-15621R1

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa (Curosurf®) and Calsurf (Kelisu®) in ventilated preterm rabbits

PLOS ONE

Dear Mr Sun,

Thank you for submitting your revised manuscript to PLOS ONE. After consideration, we feel that minor changes or additions are still necessary before it can be published in PLoS One.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #4: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed many of the reviewers comments. I believe there are a few more adjustments that are necessary before publication.

1. In the body of the abstract, the authors need to include in the methods section that the company Chiesi Farmaceutici S.p.A. contributed to the study design.

2. Q14 and Q15 revision response should be added to the discussion of the manuscript.

Reviewer #2: I would like to thank the authors for their answers to my questions and wish them the best with their investigations.

Reviewer #4: The authors have addressed many, but not all, of my previous concerns. Although the original manuscript was written in acceptable English, the revision has many statements that are not intelligible and not written in standard English.

Remaining concerns:

Q15. The authors still did not perform a statistical analysis to determine if the two surfactants are different in Figures 3 and 4 and Table 2. Without this, it is not accurate to repeatedly state that one surfactant is superior to the other. An indirect comparison for the two surfactants compared to the control group is not an acceptable substitute.

Q19. The authors still did not perform a statistical analysis for the Langmuir Blodgett analysis (Figure 2) or for the Lipid analysis (Table 1). Both of these appear to very quantitative experiments. In the current manuscript, there is no way to know if there is a proven difference between surfactants for the experiments in Figure 2 and Table 1. For example, in the Langmuir Blodgett analysis, it is not valid to comment on any difference in plateau surface pressure or peak compressibility without a statistical test.

Q25. The Trade names continue to be in the article title. These should be removed.

Q28. The authors added reference 31 (Ennema JJ et al) to support the statement “VV…is a good estimate to assess the maintenance of functional residual capacity (FRC).” The referenced publication makes no mention of FRC and therefore does not support a previously proven correlation between VV and FRC.

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Reviewer #1: No

Reviewer #2: Yes: Daniel Vijlbrief

Reviewer #4: No

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20 Dec 2019

Authors reply to the remaining query (Q) and additional changes.

Reviewer #1: The authors have addressed many of the reviewers comments. I believe there are a few more adjustments that are necessary before publication.

1. In the body of the abstract, the authors need to include in the methods section that the company Chiesi Farmaceutici S.p.A. contributed to the study design.

Response: We take into consideration the Reviewer’s comment. According to our previous experience, in the final layout of PLOS ONE, the conflict of interest is declared in the front page of the article, just below the abstract. We have already declared our conflict of interest during the submission process, which will be accordingly displayed in the front page, provided that this work is accepted.

That said, if this would not be enough, we are willing to include the sentence suggested by Reviewer#1 in the abstract if the editor finds it more appropriate.

2. Q14 and Q15 revision response should be added to the discussion of the manuscript.

Response: Regarding Q14, we have included the following sentence in the discussion section explaining why we did not include a group of 70 mg/kg of Poractant alfa (in line 530-535, page 25, of R2-marked):

“To carry out a sound head-to-head scientific comparison between surfactants, we included additional experimental groups of Calsurf-treated rabbits with dosages of 100 and 200 mg/kg, which would match the licensed doses of Poractant alfa. Considering that Poractant alfa is not licensed to be administered at doses below 100 mg/kg, we did not include a group of animals treated with Poractant alfa at a dose of 70 mg/kg”.

Regarding Q15, we would like to clarify that, from the beggining, we always compared statistically all groups between them (including surfactant-treated groups) with the statistical methods reported in the “statistical analysis” section (line 287-288, page 13, of R2-marked). However, we only reported significant differences in the figures if P<0.05. For instance, in the ventilation loop and in the mortality curve, there were no significant differences between surfactant-treated groups; only between P200 and untreated but ventilated controls. To make this point clear for the readers, we have included new sentences in the results and discussion sections clarifying in which cases no significant differences were observed between surfactant-treated groups and also sentences indicating when the significant differences were observed between P200 and the control group:

Line 391-392, page 18, of R2-marked: “Irrespective of the surfactant preparation or the administered dose, no significant differences were found if surfactant-treated groups were compared”.

Line 418-419, page 20, of R2-marked: “However, no significant differences were detected after comparing surfactant-treated groups”.

Line 442-443, page 21, of R2-marked: “However, no significant difference was detected between surfactant groups with different doses”.

Line 537-539, page 25, of R2-marked: “Nevertheless, a significant reduction of RDS-associated mortality compared to untreated but ventilated controls was only achieved after treatment with 200 mg/kg of Poractant alfa”.

Line 635-638, page 29, of R2-marked: “We therefore suspect that the significant differences observed between Poractant alfa administered at a dose of 200 mg/kg and untreated control animals in the PIP loop experiments…”.

Reviewer #2: I would like to thank the authors for their answers to my questions and wish them the best with their investigations.

Response: We thank the reviewer for the criticism, which has improved the quality of the manuscript.

Reviewer #4: The authors have addressed many, but not all, of my previous concerns. Although the original manuscript was written in acceptable English, the revision has many statements that are not intelligible and not written in standard English.

Response: We thank the reviewer for the advice. We have revised the manuscript and shortened and English-proofed some sentences to make the statements clearer and more comprehensible for the readers.

Remaining concerns:

Q15. The authors still did not perform a statistical analysis to determine if the two surfactants are different in Figures 3 and 4 and Table 2. Without this, it is not accurate to repeatedly state that one surfactant is superior to the other. An indirect comparison for the two surfactants compared to the control group is not an acceptable substitute.

Response: Regarding Q15, we refer to the response given to the 2nd concern of Reviewer #1. In the present version of the manuscript, we clearly state that in figures 3 and 4 and table 2 statistical analysis between all groups has been performed, and there are not significant differences between surfactant-treated groups and that the significant differences were only detected between P200 and untreated control animals.

This has also been amended in the discussion section.

Q19. The authors still did not perform a statistical analysis for the Langmuir Blodgett analysis (Figure 2) or for the Lipid analysis (Table 1). Both of these appear to very quantitative experiments. In the current manuscript, there is no way to know if there is a proven difference between surfactants for the experiments in Figure 2 and Table 1. For example, in the Langmuir Blodgett analysis, it is not valid to comment on any difference in plateau surface pressure or peak compressibility without a statistical test.

Response: We agree with Reviewer#4 that these techniques yield quantitative data. However, we believe that the most interesting observations from Table 1 and Figure 2, and also Figure 1 (AFM) are typically qualitative (as applied already many times in the available literatures. See below additional references).

The LC-MS data revealed qualitative differences between both formulations and in particular the absence of plasmalogens in Calsurf and the absence of cholesterol in Poractant alfa. It must be noted that the analysis was performed using 1 mL of native surfactant preparations, which are formulated at concentrations of 35 mg/mL (Calsurf) and 80 mg/mL (Poractant alfa), which accounts for a 3-, 2-, 10-, and 3-fold higher PC, DPPC, PE and PI content in Poractant alfa compared to Calsurf. We believe that these data already show evident differences between both surfactants.

Regarding the Langmuir Blodgett analysis, we have measured the value of the compressed area reached by Poractant alfa and Calsurf at a surface pressure of 68 mN/m. We have chosen 68 mN/m because both surfactants reached this very high value of pressure in these trials.

Under these analytical conditions, the comparison between surfactants did not reach statistical significance (P>0.05), being 14.6 ± 0.35 Å2/molecule for Poractant alfa and 13.8 ± 0.76 Å2/molecule for Calsurf. We have included this comparison in the present version of the manuscript (line 376-378, page 18, of R2-marked).

That said, we believe that the shape of the isotherms is qualitative a much more relevant information and it indicates some differences between both preparations which are discussed in the manuscript. This is the reason why we included just qualitative observations in previous versions of the manuscript.

We include below some references in which LB experiments have been used to qualitatively characterize composition and activity of pulmonary surfactants:

1. H Zhang, Q Fan, YE Wang, CR Neal, YY Zuo. Comparative study of clinical pulmonary surfactants using atomic force microscopy. Biochimica et Biophysica Acta. 2011; 1808: 1832–1842.

2. D Lukovic, A Cruz, A Gonzalez-Horta, A Almlen, T Curstedt, I Mingarro, J Pérez-Gil. Interfacial behavior of recombinant forms of human pulmonary surfactant protein SP-C Langmuir. 2012; 28: 7811−7825.

3. DM Schenck, J Fiegel. Tensiometric and phase domain behavior of lung surfactant on mucus-like viscoelastic hydrogels ACS. Appl. Mater. Interfaces. 2016; 8: 5917−5928.

4. RP Valle, CL Huang, JSC Loo, YY Zuo. Increasing hydrophobicity of nanoparticles intensifies lung surfactant film inhibition and particle retention ACS. Sustainable Chem. Eng., 2014; 2: 1574−1580.

Q25. The Trade names continue to be in the article title. These should be removed.

Response: We understand the concern of the reviewer. Following the Reviewer’s advice, we have removed the trade names of the products from the title.

Q28. The authors added reference 31 (Ennema JJ et al) to support the statement “Vv…is a good estimate to assess the maintenance of functional residual capacity (FRC).” The referenced publication makes no mention of FRC and therefore does not support a previously proven correlation between Vv and FRC.

Response: What we meant in this sentence is that Vv accounts for improved alveolar expansion at the end of expiration, as well FRC is essential to prevent alveoli collapse at the end of expiration. The method of Vv in this animal model was developed in late 1970’s and 1980’s by Dr. Bengt Robertson and his colleagues adopting Weibel’s theorem of stereometric measurement of lung volume to estimate expansion magnitude of alveoli (a gas volume approximate to FRC, which was further verified by nitrogen wash-out methods in clinical settings for human neonates in 1990’s). Different from the previous experience, the animals in our study were succeeded to be ventilated with 2-3 cmH2O PEEP in contrast to 5-6 cmH2O in clinical settings for RDS, which should have achieved a better, though still lower, level of FRC. Furthermore, the method of perfusion fixation of the animal lungs in this study may not ensure the expanded alveoli to be commensurable to FRC. Therefore, Vv should at best approximate to residual volume (RV), a part of FRC, in these premature lungs, in the presence or absence of exogenous surfactants.

Considering these, we additionally think that this parameter can be a good estimate to assess the manteinance of FRC. To clarify this concern, we have modified the position of reference 31 as follows: (Line 492-494, page 23, of R2-marked)

“VV accounts for improved alveolar expansion at the end of expiration[31] and is a good estimation of residual volume reflecting the maintenance of functional residual capacity (FRC) under various treatment”.

Additional changes in R2:

We included two short sentences and reference [20] in the methods section of the Langmuir Blodgett measurements of the main body text (Line 159-163, page 8, of R2-marked) to better understand the relationship of surface tension and surface pressure.

Submitted filename: Response to Reviewers-R2.docx

Click here for additional data file.

26 Feb 2020

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits

PONE-D-19-15621R2

Dear Dr. Sun,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Umberto Simeoni

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

2 Mar 2020

PONE-D-19-15621R2

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits

Dear Dr. Sun:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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