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

Smith, Johan (2002-12)

In title 12, 22, 11 are in subscript.

Thesis (PhD)--Stellenbosch University, 2002.

Thesis

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 (Glaxo Wellcome), but containing in addition, a sugar, trehalose (TRE). Towards this end, a study was designed to firstly test the hypothesis that the true difference in acute physiological effects between a mixture of oppe, tyloxapol, hexadecanol and trehalose (LPM-l), and Exosurf, (Oppe, tyloxapol and hexadecanol) is zero, in a surfactantdeficient animal 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 of oppe, hexadecanol and tyloxapol, and (2) to assess the effect of the LPM-l surfactant replacement on the epithelial lining fluid composition by means of analysing bronchoalveolar lavage fluid. Thirdly, the effects of TRE and / or calcium were studied on the surface properties of oppe suspensions, by in vitro analysis using the ring detachment method of Du Nouy The in vivo research comprised of two studies, performed in randomised controlled fashion. In the first study, 24 New Zealand White adult rabbits were randomised into 4 groups, while in the second study, 15 animals were randomised into 3 groups. In the first in vivo study, three synthetic surfactants, LPM-l, Exosurf and LPM-2, and a saline group 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 that of commercially available Exosurf, prepared on site. In both studies animals were subjected to repeated lavage with large volumes of warm saline (25 ml/kg) in order to establish surfactant deficiency and acute lung injury. Five minutes after the last lavage, vehicle, i.e. surfactants LPM-l, Exosurf, or LPM-2, or saline, 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. Ventilator settings were standardized before and after lavage. The effects of surfactant treatment on gas 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 vehicle instillation. 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 and LPM-l, rapidly improved the gas exchange and reduced the intrapulmonary shunt, but did not restore the lung to its pre-lavage condition. From the 2nd in vivo study it was evident that trehalose-only, was inefficient as a lung surfactant, failing to improve oxygenation indices or the calculated percentage shunt, or influencing respiratory compliance. 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 of commercial Exosurf, but failed to raise the activity above that of Exosurf. These physiological improvements were sustained for up to 3 hours. Saline-treated animals had no improvement in gas exchange despite management with variable PIP (to maintain a tidal 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 ordinate surface tension values for the OPPC-only and DPPC + TRE mixtures, and the slopes of their respective graphs smaller in magnitude than those of the other formulations, suggesting that these formulations had less surface tension-lowering capability than the other surfactants. At 20°C (20 mg / ml DPPC-surfactants) the mean ordinate values of OPPC and OPPC + TRE, 70.13 and 69.47 dyne / cm, respectively, were not significantly different from each other. The mean ordinate values of LPM-l and the formulation containing OPPC + TRE + tyloxapol + CaCh were lower, but similar, as were the values of LPM-2 (on-site Exosurf) and LPM-2 + CaCho Thus, three internally homogeneous subgroups 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 the mean 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. A similar analysis of mean slope values was performed. Here too a significant difference between substances was found, OPPC alone or in combination with TRE, again being significantly different from the other surfactants. The most prominent light microscopy findings of the lungs of animals included general lymphatic dilatation, congestion and lung polymorphonuclear infiltration, with no difference between study groups. Hyaline membranes were present in all surfactant groups, 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 finding in all of the study groups towards the end of the study protocol. A significant increase in the BAL-fluid neutrophil count occurred in all animals, concurrent with a significant decrease in the BAL macrophage count. No significant change occurred in the peripheral neutrophil count during the 3-hour study, suggesting recruitment of neutrophils from storage pools. Treatment with synthetic surfactant (LPM -1) did not have a significant effect on modifying the inflammatory response, since there was no significant difference in the BAL-derived cell counts between the LPM-1 and -saline groups. Epithelial damage was a consistent finding in all groups. The damage was more evident by electron microscopy examination and included hydropic changes, most readily observed in the mitochondria. The airspaces of study subjects showed the presence of oedema fluid. This luminal 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 microscopical appearance of the epithelial-lined substance ("hyaline membranes") in the present study showed a marked variability within groups as well as within the same case. The majority of cases showed a mix of membrane types with both granular and fibrillar materials present within the same membrane. In some cases there were layering of the membranes into distinct bands. The instillation of LPM-l resulted in the formation of a slightly different type of epithelial lining fluid after lavage, when compared to the prelavage composition. The most pronounced changes occurred within the fatty acids, whilst the phosphatidylcholine values remained unchanged. Palmitic acid concentrations (C16:0) increased significantly, suggesting enrichment of the epithelial lining fluid after instillation of LPM-l. This increase in C16:0 was concurrent with significant decreases in the percentage C16:1, C18:0, and C18:2. In contrast to previous studies, we describe higher levels for phosphatidyldimethylethanolarnine (PEA). An explanation may be that the lipid identified as PEA, was in fact partly phosphatidylglycerol (PG)-a lipid whose accurate 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, but remained statistically unchanged in the animals treated with LPM-l or saline. The change in ratio was mainly accounted for by a decrease in BAL-fluid PC content together with a rise in SM content. A poor correlation existed between the BAL-derived PC/SM ratio and indices reflecting oxygenation status (a/A ratio, Ol), as well as the CRSdynat the time of the final lavage. In conclusion, the primary hypothesis was accepted, LPM-l performed similarly to Exosurf in vivo, improving oxygenation, but not CRSdyn.None was clearly superior to the other. Some questions remain. The reason why LPM-l (LPM-2 + TRE) did not behave in a superior manner, in vivo, to Exosurf, is partly unclear. This finding was somewhat surprising since the chemical composition of Exosurf and LPM-2 did not differ, and the addition of TRE to LPM-2 (on-site Exosurf), did improve the in vivo activity of the resultant LPM-l, above that of LPM-2. A possible explanation for observed differences in 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 in which true commercial Exosurf is prepared.

AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n sintetiese proteïn vrye surfaktant te ontwikkel en die produk te vergelyk met 'n kunsmatige surfaktant reeds in kliniese gebruik. Die bekende uit die literatuur en die onbekende van die produk wat evalueer sou word, lei op tot die samestelling van die nul hipotese van die PhD naamlik dat geen verskil in longfunksie sou gewys word tussen die toetsproduk en reeds gebruikte kommersiële surfaktant nie. Die hipotese was dat 'n suiker (trehalose), in kombinasie met Dipalmitoiel fosfatidielcholine (DPPC), gaswisseling en longfunksies sal verbeter vir 'n long met 'n lae surfaktant konsentrasie. Vir die studie is jong volwasse wit New Zealand konyne gebruik en is hulle met 'n gestandaardiseerde en menslike manier gebruik in eksperimentele werk. Die diere is onder intraveneuse narkose geplaas en verskillende kardiovaskulêre en pulmonologiese aspekte is gemeet. Die long surfaktant is uitgewas deur middel van fisiologiese soutoplossing wat tot liggaam temperatuur verhit is en daarna is die diere prospektief gerandomiseer tot eksperimentele groepe. Met vooraf bepaalde tydsintervalle is die fisiologiese metings herhaal en was die metings toegespits daarop om longmeganiese funksie en gasoordrag vermoë te evalueer. Lig mikroskopiese en elektron mikroskopiese studies is ook op die longe gedoen en verder is brongoalveolêre vloeistof ook ontleed. Die groepe met ondersoek was: I. oppe, heksadekanol, tyloxapol en trehalose (LPM-I). 2. oppe, heksadekanol, tyloxapol (LPM-2 :. LPM-I sonder trehalose). Hierdie is 'n proteïnvrye surfaktant plaaslik berei ( dieselfde samestelling as Exosurf). 3. Exosurf®. (Kommersiële preperaat reeds in gebruik). Hierdie is 'n proteïnvrye sintetiese surfaktant. 4. Trehalose, 'n non-reduserende disakklaried van glukose. Addisioneel is daar ook in vitro studies gedoen waann die oppervlakte spanmngs aktiwiteite van die verskillende surfaktant oplossings vergelyk is. Die statistiese analise is gedoen in samewerking met Prof. J. Maritz wat 'n unieke metode ontwikkel en gepubliseer het om herhalende veranderlikes op 'n statisties verantwoordbare manier te ontleed. In die eerste van die studies, is LPM-I, Exosurf®, fisiologiese soutoplossing en 'n plaaslik bereide "Exosurf" (LPM-2), met 'n chemiese samestelling identies aan dié van kommersiële Exosurf®, evalueer. In 'n tweede studie is die fisologiese effekte van LPM-I vergelyk met trehalose of fisiologiese soutoplossing om die volgende te ondersoek: 1) Die bydrae van trehalose tot 'n mengsel van oppe, heksadekanol en tyloxapol (LPM-2). 2) Die gevolg van LPM-l surfaktant toediening op die konyn se brongo-alveolêre vloeistof samestelling. 'n Derde, in vitro studie, het die oppervlaktespannings-effekte van trehalose en of kalsiumbyvoegings tot DPPC-oplossings gemeet deur middel van die ring metode van Du Nouy, In die eerste in vivo studie verbeter oksigenasie en persentasie longaftakking tot dieselfde mate na LPM-l en Exosurf® toediening en word die hipotese van die proefskrif bevestig. In die breë gesien, is die tydsprofiele van LPM-l en Exosurf® ten opsigte van oksigenasie en persentasie longaftakking statisties betekenisvol beter en van 'n sneller aard, as die tydsprofiele van dieselfde indekse na die toediening van fisiologiese soutoplossing of LPM-2. Die tydsprofiel van dinamiese longvervormbaarheid, na die toediening van LPM-I of Exosurf®, is dieselfde, maar betekenisvol beter as die vervormbaarheid na toediening van LPM-2 of fisiologiese soutoplossing. Alhoewel die oksigenasie indekse in die geval van LPM-l en Exosurf® betekenisvol verbeter oor die studietydperk, vind volkome herstel tot die basislynwaardes (voor spoeling) nie plaas nie. Bykomend, geen van die surfaktante het na toediening enige noemenswaardige verbetering in longvervormbaarheid tot gevolg gehad nie. Die rede vir die swakker vertoning van LPM-2 en Exosurf is onbekend en sal in opvolg studie ondersoek word. In die tweede in vivo studie is dit duidelik dat trehalose op sy eie, 'n oneffektiewe surfaktant is aangesien die preperaat na toediening geen verbetering teweegbring ten opsigte van oksigenasie indekse, persentasie longaftakking, of long-dinamiese vervormbaarheid nie. Die toevoeging van trehalose tot LPM-2, om LPM-l te lewer, neem wel die aktiwiteit van LPM-l tot dieselfde in vivo vlak as dié van kommersiële Exosurf®, maar slaag nie daarim om 'n hoër fisiologiese in vivo aktiwiteit as dié produk te bereik nie. Die diere wat met fisiologiese soutoplossing behandel is toon geen verbetering in enige fisiologiese parameter nie. Die in vitro resultate wat verkry is deur die Du Nouy tensiometer toon hoër gemiddelde ordinaat oppervlaktespannings waardes vir 'n formule wat slegs uit DPPC bestaan, asook vir 'n mengsel van DPPC + trehalose. Die helling van die grafieke van hierdie oplossings is ook kleiner as die van die ander formulas wat daarop dui dat DPPC op sigself, en DPPC + trehalose, weinig vermoë het om oppervlaktespanning te verminder. Daarteenoor verlaag die volgende oplossings die oppervlaktespanning ten opsigte van gedistilleerde water betekenisvol en wel in In konsentrasie afhanklike manier by beide 21°C en 3rc: LMP-I-, LPM-2-, DPPC + trehalose + tyloxapol + CaCf2-, en LPM-2 + CaCf2. Die prominentste ligmikroskopiese bevindinge van die longe van die diere sluit in: Algemene limfvat dilatasie, stuwing, en long neutrofiel infiltrasie. Betreffende hierdie histologiese bevindinge is daar geen verskille aangetoon tussen die groepe nie. Hialienmembrane was teenwoordig in al die groepe, maar betekenisvol meer in die groep wat fisiologiese soutoplossing ontvang as vervangingsterapie. In die tweede in vivo studie is daar 'n betekenisvolle styging in die neutrofiel- en daling in makrofaagtelling, van die brongoalveolêre vloeistof spoeling in al drie die groep aangetoon. Terselfdertyd vind geen noemenswaardige daling in die perifêre (sistematiese) neutrofieltelling plaas nie. Hierdie bevindinge dui daarop dat die brongoalveolêre selveranderinge toegeskryf kan word aan verwerwing van neutrofiele vanuit 'n longstoringspoel eerder as rekrutering vanuit die sistemiese sirkulatoriese poel. Surfaktant (LPM-l), behandeling het geen betekenisvolle vermindering in long inflammasie teweeggebring nie. Epiteelskade was 'n algemene ligmikroskopiese bevinding in al die groepe. Die samestelling van die brongoalveolêre vloeistof verander na installering van LPM-I. Die prominentste verandering word waargeneem in die vetsuur samestelling terwyl die DPPC waardes onveranderd bly. Die vetsuur, palmitiensuur (palmitic acid), (CI6:0), verhoog betekenisvol na toediening van LPM-l. Daarteenoor verminder die konsentrasie van C16:1, C18:0 en C18:2. In kontras met vorige studies, beskryf die huidige studie hoër konsentrasies van fosfatidieletanolamien, moontlik as gevolg van tegniese verskille in die metingsmetodes. 'n Betekenisvolle verlaging in die fosfatidielcholine:sfingomiëlien (PC/SM) verhouding word waargeneem tussen die eerste en die finale longspoeling van die trehalose-groep, terwyl dit onveranderd bly in die diere wat LPM-1 of fisiologiese soutoplossing ontvang.

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