A comparison of synthetic surfactants : evaluation of a novel surfactant (1,2-dipalmitoyl-sn-phosphatidycholine and trehalose [C12H22O11]) and comparison with other synthetic formulations

[摘要] ENGLISH ABSTRACT:The aim of this study was to test a synthetic protein-free surfactant preparation, LPM-l,with the same chemical composition as commercially available Exosurf (GlaxoWellcome), but containing in addition, a sugar, trehalose (TRE). Towards this end, astudy was designed to firstly test the hypothesis that the true difference in acutephysiological effects between a mixture of oppe, tyloxapol, hexadecanol and trehalose(LPM-l), and Exosurf, (Oppe, tyloxapol and hexadecanol) is zero, in a surfactantdeficientanimal model. A second study addressed the physiological effects of oppe,hexadecanol, tyloxapol and trehalose (LPM-l) compared to treatment with trehalose(TRE) or saline, in order to determine (1) the contribution of TRE to the mixture ofoppe, hexadecanol and tyloxapol, and (2) to assess the effect of the LPM-l surfactantreplacement on the epithelial lining fluid composition by means of analysing bronchoalveolarlavage fluid. Thirdly, the effects of TRE and / or calcium were studied on thesurface properties of oppe suspensions, by in vitro analysis using the ring detachmentmethod of Du NouyThe in vivo research comprised of two studies, performed in randomised controlledfashion. In the first study, 24 New Zealand White adult rabbits were randomised into4 groups, while in the second study, 15 animals were randomised into 3 groups. In thefirst in vivo study, three synthetic surfactants, LPM-l, Exosurf and LPM-2, and a salinegroup were tested. LPM-l is a new formulation that consists ofa mixture of Df'PC, TRE,hexadecanol and tyloxapol. LPM-2 is a formulation with a composition equivalent to thatof commercially available Exosurf, prepared on site. In both studies animals were subjected to repeated lavage with large volumes of warmsaline (25 ml/kg) in order to establish surfactant deficiency and acute lung injury. Fiveminutes after the last lavage, vehicle, i.e. surfactants LPM-l, Exosurf, or LPM-2, orsaline, in the first in vivo study, and LPM-l, TRE or saline in the second in vivo study,was instilled, and the course of the animals followed over the next 3 hours. Ventilatorsettings were standardized before and after lavage. The effects of surfactant treatment ongas exchange (arterial Pa02, oxygenation index (Ol), arterial-alveolar oxygen (a/A)ratio), percentage calculated shunt, and total dynamic respiratory compliance (CRSdyn),and histopathological changes were compared with changes in saline treated controls.Arterial blood gases in 100% oxygen and CRSdynwere measured before and after lavage,at 15 minute intervals for the first 30 min, then at 60, 90, 120, and 180 min after vehicleinstillation.Oxygenation improved to a similar extent after LPM-l and Exosurf instillation,surpassing that of LPM-2 or saline. Overall, intratracheal instillation of both Exosurf andLPM-l, rapidly improved the gas exchange and reduced the intrapulmonary shunt, butdid not restore the lung to its pre-lavage condition. From the 2nd in vivo study it wasevident that trehalose-only, was inefficient as a lung surfactant, failing to improveoxygenation indices or the calculated percentage shunt, or influencing respiratorycompliance. The addition of the sugar, trehalose (TRE), to the on-site 'Exosurf mixture(LPM-2) brought the activity of the resultant LPM-l to the same level as that ofcommercial Exosurf, but failed to raise the activity above that of Exosurf. Thesephysiological improvements were sustained for up to 3 hours. Saline-treated animals hadno improvement in gas exchange despite management with variable PIP (to maintain atidal volume of -1 0 ml / kg) and constant PEEP of 5 cm H20.In-vitro results, obtained by the Ou Nouy tensiometer, showed higher mean ordinatesurface tension values for the OPPC-only and DPPC + TRE mixtures, and the slopes oftheir respective graphs smaller in magnitude than those of the other formulations,suggesting that these formulations had less surface tension-lowering capability than theother surfactants. At 20°C (20 mg / ml DPPC-surfactants) the mean ordinate values ofOPPC and OPPC + TRE, 70.13 and 69.47 dyne / cm, respectively, were not significantlydifferent from each other. The mean ordinate values of LPM-l and the formulationcontaining OPPC + TRE + tyloxapol + CaCh were lower, but similar, as were the valuesof LPM-2 (on-site Exosurf) and LPM-2 + CaCho Thus, three internally homogeneoussubgroups could be identified which differed significantly, namely: DPPC and DPPC +TRE, LPM-2 and LPM-2 + CaCh, and DPPC + TRE + tyloxapol + CaCh and LPM-l.Similar conclusions apply to the ordinate values of the surfactants at 37°C, and to themean slope values at 20°C, with the exception that the subgroups, LPM-2 and LPM-2 +CaCh, and LPM-l and OPPC + TRE + tyloxapol + CaCh are not so clearly separated. Asimilar analysis of mean slope values was performed. Here too a significant differencebetween substances was found, OPPC alone or in combination with TRE, again beingsignificantly different from the other surfactants.The most prominent light microscopy findings of the lungs of animals included generallymphatic dilatation, congestion and lung polymorphonuclear infiltration, with nodifference between study groups. Hyaline membranes were present in all surfactantgroups, but significantly more so in the saline treated group. In the first in vivo study, the presence of neutrophils in the lung interstitiwn as well as alveoli, was a common findingin all of the study groups towards the end of the study protocol. A significant increase inthe BAL-fluid neutrophil count occurred in all animals, concurrent with a significantdecrease in the BAL macrophage count. No significant change occurred in the peripheralneutrophil count during the 3-hour study, suggesting recruitment of neutrophils fromstorage pools. Treatment with synthetic surfactant (LPM -1) did not have a significanteffect on modifying the inflammatory response, since there was no significant differencein the BAL-derived cell counts between the LPM-1 and -saline groups. Epithelial damagewas a consistent finding in all groups. The damage was more evident by electronmicroscopy examination and included hydropic changes, most readily observed in themitochondria. The airspaces of study subjects showed the presence of oedema fluid. Thisluminal oedema appeared to be more prominent in the control group and LPM-2 (on site'Exosurf') group. Organellar debris, probably originating from lysis of epithelial cells,was present, despite treatment with synthetic surfactant. The electron microscopicalappearance of the epithelial-lined substance (hyaline membranes) in the present studyshowed a marked variability within groups as well as within the same case. The majorityof cases showed a mix of membrane types with both granular and fibrillar materialspresent within the same membrane. In some cases there were layering of the membranesinto distinct bands. The instillation of LPM-l resulted in the formation of a slightlydifferent type of epithelial lining fluid after lavage, when compared to the prelavagecomposition. The most pronounced changes occurred within the fatty acids, whilst thephosphatidylcholine values remained unchanged. Palmitic acid concentrations (C16:0)increased significantly, suggesting enrichment of the epithelial lining fluid afterinstillation of LPM-l. This increase in C16:0 was concurrent with significant decreasesin the percentage C16:1, C18:0, and C18:2. In contrast to previous studies, we describehigher levels for phosphatidyldimethylethanolarnine (PEA). An explanation may be thatthe lipid identified as PEA, was in fact partly phosphatidylglycerol (PG)-a lipid whoseaccurate identification was precluded for technical reasons.After surfactant instillation, the PC/SM ratio, a reflection of the lecithin / sphingomyelin(LIS), decreased significantly in the TRE-group between the first and final lavage, butremained statistically unchanged in the animals treated with LPM-l or saline. The changein ratio was mainly accounted for by a decrease in BAL-fluid PC content together with arise in SM content. A poor correlation existed between the BAL-derived PC/SM ratio andindices reflecting oxygenation status (a/A ratio, Ol), as well as the CRSdynat the time ofthe final lavage.In conclusion, the primary hypothesis was accepted, LPM-l performed similarly toExosurf in vivo, improving oxygenation, but not CRSdyn.None was clearly superior to theother.Some questions remain. The reason why LPM-l (LPM-2 + TRE) did not behave in asuperior manner, in vivo, to Exosurf, is partly unclear. This finding was somewhatsurprising since the chemical composition of Exosurf and LPM-2 did not differ, and theaddition of TRE to LPM-2 (on-site Exosurf), did improve the in vivo activity of theresultant LPM-l, above that of LPM-2. A possible explanation for observed differencesin performance include methodological issues, i.e. the preparation of the on-site formulations, especially that of LPM-2 (on-site Exosurf), may differ from the way inwhich true commercial Exosurf is prepared.

[发布机构] Stellenbosch University