Patent Application
20130029937
The invention relates to a method for enhancing a surfactant, in particular a pulmonary surfactant (also called lung surfactant), as well as a method for producing a surfactant enhancement agent, in particular a pulmonary surfactant enhancement agent. The invention also relates to a surfactant enhancement agent and a use of an enhancement agent for enhancing a surfactant, in particular, a pulmonary surfactant. Further, the invention relates to a method of mitigating oxidative damage to pulmonary surfactant by adding a cholesterol-sequestrating agent such as cyclodextrin. Finally, a method is provided for treating a patient having surfactant dysfunction due to oxidative damage to pulmonary surfactant by administering a surfactant-protective amount of a cholesterol-sequestrating agent to protect the surfactant from the negative effects of oxidative degradation.
It is known that the pulmonary surfactant is a surface-active substance lining the respiratory system of the lung. Type II pneumocytes produce the surfactant, store it as lamellar bodies and finally express it in the alveolar liquid phase. From there it spreads as a molecular thin film on the air/water boundary surface. It displays its effect by dynamically adapting the surface tension of the boundary surface to the current extension of the boundary surface during the breathing cycle.
Pulmonary surfactant prevents alveolar collapse and flooding at end-expiration, affords patency of small airways and reduces the work required for breathing. Pulmonary surfactant consists of 80-90% (by weight) phospholipids, with a large proportion (−30-45%) being disaturated dipalmitoylphosphatidylcholine (DPPC). The surfactant also contains 50-60% unsaturated phospholipids with reported levels of polyunsaturated species >10% of total phospholipids. Surfactant also contains four surfactant-associated proteins—SP-A, B, C, and D. A highly surface-active form of pulmonary surfactant, referred to as the large aggregate fraction, is enriched in the hydrophobic proteins SP-B and SP-C. Neutral lipids, primarily cholesterol, make up the remaining 2-10% of surfactant.
In 1959 it was shown that the lack of pulmonary surfactant in the lungs of premature infants leads to respiratory distress syndrome (RDS), which at that time was the most frequent cause of death for premature infants. For about the past 15 years, extracts from the lungs of cattle or pigs have been used as a medication to treat RDS. Animal preparations are generally expensive and carry the risk of transmitting infectious diseases.
The surfactant (short for surface active agent) consists of glycero-phospholipids, specific proteins, neutral fats and cholesterol. The surfactant covers the alveolar surface and reduces the surface tension, so that after birth the alveoli do not collapse in the human body during exhalation.
Insufficient function of the surfactant can be the cause of respiratory insufficiency, known as Infant Respiratory Distress Syndrome (IRDS) in premature infants and newborns or in adults as Adult Respiratory Distress Syndrome (ARDS). These lung illnesses are the result of a surfactant deficiency, which lead to an inadequate expansion of the lungs (atelectasis) after a collapse of the pulmonary alveoli.
The lung surfactant consists of 90% lipids and 10% proteins. Although cooperation between the surfactant-specific proteins and the lipids is necessary for a completely functional respiratory process, the lipids are essential for the vitally important reduction of surface tension.
The function of pulmonary surfactants and their inhibited function with pulmonary infections and pulmonary diseases have been described in numerous publications. In this regard reference is made to the publications:
Pulmonary surfactants form a complex film, which has or plays a critical role in the reduction of surface tension in the respiratory tract in the hydrated air/lung interface. With inflammatory lung diseases, the molecular profile of pulmonary surfactants in the alveoli and respiratory tract is changed so that the pulmonary surfactant film is quite a lot less effective or ineffective for reducing the surface tension, which is accompanied by a strong decrease in the area available for gas exchange.
Furthermore, it is known that an elevated level of cholesterol in the surfactants is a major cause of surfactant dysfunction. In this regard reference is also made to the following publication:
Furthermore, it has been shown that the release of reactive oxygen species (ROS) from activated leukocytes or following exposure to environmental pollutants may result in a highly oxidizing milieu within the lungs. Increased levels of reactive oxygen species and their byproducts have been detected from bronchoalveolar lavage fluid collected from patients with ARDS, asthma, CF, ventilator induced lung injury (VILI) or chronic obstructive pulmonary disease (COPD), among many other disease states. Several studies have shown that oxidation of various pulmonary surfactants greatly impairs in vitro and in vivo function, which has so far been largely attributed to oxidative alterations of susceptible residues in SP-B and SP-C, whereas peroxidation and hydrolysis of phospholipids were considered less important. In this regard reference is also made to the following publications:
This invention addresses the unaddressed question concerning the contribution of cholesterol to oxidation-induced surfactant dysfunction. In particular, it remained unknown, until this invention; whether oxidative alterations to surfactant interact with normal or increased levels of cholesterol to negatively affect surfactant function.
One aspect of the present invention relates to enhancing surfactants in a simple manner, in particular pulmonary surfactants, which, for example, are inhibited by cholesterol and exhibit dysfunction, where in particular the surface tension of the inhibited surfactants is improved by the treatment.
More particularly, a method for enhancing a surfactant is provided, in particular a pulmonary surfactant, which is further established in that the surfactant is enhanced with a cholesterol sequestrating surfactant enhancement agent, wherein given, in particular cholesterol of the surfactant is selectively sequestrated by means of the surfactant enhancement agent, such that the effect of cholesterol on the surfactant is reduced or reversed and thus also the dysfunction of the surfactant triggered by cholesterol.
In another aspect, a method for treating a patient having surfactant dysfunction due to exposure to reactive oxygen species (ROS) and their derivatives is provided comprising administering to the patient a surfactant-protective amount of a cholesterol sequestrating agent to protect the surfactant from the negative effects of oxidative damage by ROS. Examples of ROS include, but are not limited to, superoxide and hydroxyl radicals. An example of a ROS derivative is peroxynitrite. In another aspect, a method for treating a patient having pulmonary surfactant dysfunction due to oxidative damage to pulmonary surfactant is provided comprising administering to the patient a surfactant-protective amount of a cholesterol-sequestrating agent to protect the surfactant from negative effects of oxidative degradation.
In yet a further aspect, a method of mitigating oxidative damage to pulmonary surfactant by adding a cholesterol-sequestrating agent such as cyclodextrin is provided. In one embodiment, protecting a surfactant from free radical damage due to oxidization of polyunsaturated phospholipids is provided comprising adding to the surfactant a surfactant-protective amount of a cholesterol sequestrating agent.
It was discovered that at the concentrations of cholesterol sequestrating agent used herein, oxidized, cholesterol-depleted surfactant exhibited few defects in surface activity. However, when such surfactants contain physiological levels of cholesterol, severe dysfunction is observed in terms of ability to reach low surface tensions upon compression, efficient film re-spreading upon expansion, and rapid interfacial film formation. Among the many biochemical effects of surfactant oxidation, lipid peroxidation was necessary for dysfunction. ROS are part of most inflammatory lung diseases. For most of these cases, cholesterol levels are not, or not substantially, elevated. Accordingly, a treatment of cholesterol dependent surfactant dysfunction by ROS is applicable to a much broader patient population than the treatment of an elevation of surfactant cholesterol.
For example, but not limiting, the present invention may be useful in treating patients suffering from acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) from causes including shock, bacterial, viral and nosocomial pneumonias, ventilator-induced lung injury (VILI), aspiration, systemic inflammatory response syndrome (SIRS) or inhalation of toxic gases, vapors, fumes and particles. Also, the present invention is useful where the patient is suffering from airway injury including asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or inhalation of toxic gases, vapors, fumes and particles or a patient with infant respiratory distress syndrome (IRDS) that is not responding to treatment or Niemann-Pick disease.
Hence,
Thus,
In the context of the invention, the term “lipid sequestrating” or “cholesterol sequestrating” is understood to mean that lipids or cholesterol of a surfactant are removed from the surfactant in their particularly dysfunctional effect and/or are inhibited in their effect on the surfactant and/or act on the surfactant as a medium which makes lipids soluble in order to reduce or reverse the aforementioned inhibitory effect of the lipids and/or cholesterol. The surfactant dysfunction particularly occurs if there is an increased cholesterol level in the surfactant.
Through the action of the sequestrating surfactant enhancement agent, the dysfunction of the surfactant triggered by the cholesterol as a result of elevated cholesterol content above the corresponding normal level of cholesterol in the surfactants, is at least partially to entirely nullified as well as at least partially to completely reversed.
Lipid sequestrating or cholesterol sequestrating enhancement agents to be considered here are those media which particularly correspond to the action or active substance class of cyclodextrin, in particular of methyl-β-cyclodextrin, on a surfactant and/or a pulmonary surfactant. In this way, for example, the inhibiting effect of cholesterol on a surfactant is weakened or nullified.
Through the inventive enhancement of the surfactant, in particular endogenous surfactant, it is furthermore possible, for example, that a cholesterol-induced or a cholesterol-dependent inhibiting effect on the surfactant can be diagnosed. In addition to that, the properties of the surfactant can also be influenced or modified accordingly by the surfactant enhancement agent, which enables pulmonary diseases, in particular acute pulmonary diseases and chronic pulmonary diseases, to be treated. Examples of acute pulmonary illnesses are acute lung injury (ALI), ARDS, acute respiratory insufficiency, pneumonias, particularly ventilator-induced lung injuries, nosocomial infections or systemic inflammatory response syndrome (SIRS) associated with ALI and bronchiolitis. Chronic lung diseases are for example cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD) and Niemann-Pick disease.
Through the use of a cholesterol sequestrating surfactant enhancement agent, the surfactant is correspondingly influenced in its surface-active properties, so that the surface activity of endogenous, treated pulmonary surfactants can be determined, for instance, using a captive bubble surfactometer, for example.
Moreover, the method is distinguished in that the surfactant is enhanced using a surfactant enhancement agent containing cyclodextrin or using a surfactant enhancement agent corresponding in action to a surfactant enhancement agent containing cyclodextrin or to the activity class of cyclodextrin.
Furthermore, an embodiment is envisaged in which an active substance of the surfactant enhancement agent methyl-β-cyclodextrin (MβCD) or cyclodextrin, especially 2-hydroxypropyl-β-cyclodextrin, or cyclodextrin derivatives which have 2-hydroxypropyl-β-cyclodextrin in particular, is present or dipalmitoylphosphatidylcholine (DPPC) or corresponding derivates of the aforementioned substances or a mixture of the substances mentioned or at least a lipid-selective and lipid-deactivating substance or at least a lipid-sequestrating substance are used.
In particular, the surface tension of the surfactant is reduced by the surfactant enhancement agent compared to an unenhanced surfactant or compared to the surfactant before enhancement.
In addition to that, it is envisaged in an embodiment that cholesterol of the surfactant is dissolved and/or passivated by the surfactant enhancement agent in a solution, particularly an aqueous solution. In this manner the inhibiting effect of cholesterol of the surfactant, for example on its surface activity, is treated accordingly. Consequently the surface activity and/or the surface tension of surfactants can be determined and diagnosed easily subsequent to enhancement.
Furthermore it is envisaged according to one embodiment that the surfactant enhancement agent sequestrate cholesterol of the surfactant which have an effect, particularly an inhibiting one, on the properties of the surfactant, especially the unenhanced surfactant, or which limit its properties.
In addition to that the object is solved by a method for producing a surfactant enhancement agent, in particular a pulmonary surfactant enhancement agent, which is further developed in that at least a portion of at least a cholesterol-selective and at least a cholesterol-sequestrating substance will be or is mixed with the surfactant enhancement agent.
Preferably at least one substance will be or is mixed with the surfactant enhancement agent through which with the application on a surfactant, preferably an endogenous surfactant, cholesterol which have an effect, particularly an inhibiting one, on the properties of the surfactant, especially the unenhanced surfactant, or which limit its properties, are sequestrated.
Furthermore, in accordance with the invention, the surfactant enhancement agent is mixed with an active substance constituted by methyl-β-cyclodextrin (MβCD) or cyclodextrin, especially 2-hydroxypropyl-β-cyclodextrin, or cyclodextrin derivatives which have 2-hydroxypropyl-β-cyclodextrin in particular, or dipalmitoylphosphatidylcholine (DPPC) or corresponding derivates of the aforementioned substances or a mixture of the substances mentioned or at least a cholesterol-selective and cholesterol-deactivating substance or at least a cholesterol-sequestrating substance.
In addition to that, the surfactant enhancement agent can also contain other materials and/or active substances without loss of the aforementioned action of the sequestrating substance, i.e. the effect of this sequestrating substance is or will be retained. For example, during administration as a medication or pharmaceutical, another surfactant, for example an exogenously acting surfactant, can be added to the surfactant enhancement agent.
Furthermore, the object is solved by a surfactant enhancement agent, which is further developed in that at least a portion of at least a lipid-selective and lipid-deactivating substance or at least a lipid-sequestrating substance or cholesterol-sequestrating substance will be or is mixed with the surfactant enhancement agent.
Furthermore, the surfactant enhancement agent is distinguished in that with the use of the surfactant enhancement agent, i.e. during enhancement of a surfactant, the surfactant will be or is enhanced using the surfactant enhancement agent which has as an active substance methyl-β-cyclodextrin (MβCD) or cyclodextrin, especially 2-hydroxypropyl-β-cyclodextrin, or cyclodextrin derivatives which have 2-hydroxypropyl-β-cyclodextrin in particular, or dipalmitoylphosphatidylcholine (DPPC) or corresponding derivates of the aforementioned substances or a mixture of the substances mentioned or at least a cholesterol-selective and cholesterol-deactivating substance or at least a cholesterol-sequestrating substance.
In particular, the surface tension of the surfactant will be or is reduced with the use of the surfactant enhancement agent compared to an unenhanced surfactant or compared to the surfactant before enhancement.
Preferably cholesterol of the surfactant are dissolved and/or passivated by the surfactant enhancement agent in a solution, in particular an aqueous solution.
In addition to that it is envisaged that with the use of the surfactant enhancement agent, cholesterol of the particularly endogenous surfactant which have an effect, particularly an inhibiting one, on the properties of the surfactant, especially the unenhanced surfactant, or which limit its properties will be or are sequestrated by the surfactant enhancement agent.
Moreover, the invention is solved by the use of an enhancement agent for enhancing a surfactant, in particular an endogenous surfactant, in particular a pulmonary surfactant, in which the enhancement agent is or will be made according to the process described above, or in which the surfactant enhancement agent is made from the constituents described above. For this purpose, to avoid repetition, explicit reference is made to the information above.
Advantageously, the application is distinguished in that the enhancement agent is used as a therapeutic agent or pharmaceutical for treating pulmonary illnesses in living beings, in particular humans. For example, the enhancement agent can be present as a pharmaceutical in powder form for exhalative administration or in liquid form for intra-tracheal or intra-bronchial administration. Beyond that in a further development it is possible that the enhancement agent be used with patients in aerosol form. Administration ensues in a manner known to the specialist, preferably by intra-tracheal instillation of a solution or suspension or in the form of an atomized solution or suspension or by atomization of powder.
Furthermore, the object is solved by the use of methyl-β-cyclodextrin (MβCD) or cyclodextrin, in particular 2-hydroxypropyl-β-cyclodextrin, or cyclodextrin derivatives which have 2-hydroxypropyl-β-cyclodextrin in particular, or dipalmitoylphosphatidylcholine (DPPC) or corresponding derivates of the aforementioned substances or a mixture of the substances mentioned or at least a cholesterol-selective and cholesterol-deactivating substance or at least a cholesterol-sequestrating substance, whereby cholesterol of a surfactant is sequestrated, in particular for the production of pharmaceuticals for the treatment or early treatment of pulmonary illnesses in humans, in particular acute pulmonary illnesses.
Furthermore, the object is solved by the use of methyl-β-cyclodextrin (MβCD) or cyclodextrin, in particular 2-hydroxypropyl-β-cyclodextrin, or cyclodextrin derivatives which have 2-hydroxypropyl-β-cyclodextrin in particular, or dipalmitoylphosphatidylcholine (DPPC) or corresponding derivates of the aforementioned substances or a mixture of the substances mentioned or at least a cholesterol-selective and cholesterol-deactivating substance or at least a cholesterol-sequestrating substance, whereby cholesterol of a surfactant is sequestrated, in particular for the investigation, in particular diagnosis, of surfactants, in particular pulmonary surfactants.
In the context of the invention is furthermore possible that by the sequestrating effect of the surfactant enhancement agent the dysfunction will be reduced with respect to the surface tension of a surfactant, in particular an endogenous surfactant, whereby after corresponding enhancement of the surfactant the dysfunction is diminished or nullified by an increased level of cholesterol or lipids in the surfactant.
The invention is based on the idea that a use of methyl-β-cyclodextrin (MβCD) or other cyclodextrin or other cholesterol-sequestrating substances in the activity class or the active substance class of cyclodextrin, including dipalmitoylphosphatidylcholine (DPPC), and a use of substances corresponding to the activity class of cyclodextrin, which reverse or nullify the effect(s) of cholesterol by others means, including vitamin E, as agents makes it possible not only to diagnose or determine the effected or induced inhibition of the surfactant by cholesterol but also to treat the effected or induced inhibition of the surfactant by cholesterol in patients or people with the aforementioned symptoms or those with effected or induced inhibition of the surfactant by cholesterol.
For the enhancement of the symptoms described, the substance or active substance can be delivered to the lung in aerosol form or instilled in the lung as a substance together with at least one exogenous, i.e. externally acting surfactant or delivered to the lung in another manner.
As a means of diagnosis, the inventive substance can be mixed with a specimen of a surfactant from a patient, the function of which is subsequently checked in a surface balance. Alternatively, the substance can be added to an aqueous solution or an aqueous phase of a surface balance to determine the function of the surfactants.
These results and findings were obtained on the basis of systems of model surfactants and pulmonary surfactants of CF patients and ARDS-patients. They will be explained below based on two examples.
In another aspect, the present application shows that severe inhibition of surfactant by oxidation is cholesterol-dependent. Without being bound to theory, it is believed that the mechanism of surfactant dysfunction demonstrated in vitro herein is relevant to diseases. Studies in which oxidized surfactants have been instilled into various animal models have shown good associations between in vitro performance and in vivo activity (Bailey T C, et al (2006) Physiological effects of oxidized exogenous surfactant in vivo: Effects of high tidal volume and surfactant protein A. American Journal of Physiology—Lung Cellular and Molecular Physiology 291: L703-L709; Gilliard N, et al (1994) Exposure of the hydrophobic components of porcine lung surfactant to oxidant stress alters surface tension properties. J Clin Invest 93: 2608).
Moreover, the conditions for dysfunction found in vitro are met in multiple respiratory pathologies. Alveolar oxidative stress is associated with the pathogenesis of, among other diseases, ARDS, asthma, CF, and COPD (Ciencewicki J, Trivedi S & Kleeberger S R (2008) Oxidants and the pathogenesis of lung diseases. J Allergy Clin Immunol 122: 456-468). ROS likely originate to a substantial degree from activated immune cells and/or environmental sources in these diseases (Ciencewicki J, Trivedi S & Kleeberger S R (2008) Oxidants and the pathogenesis of lung diseases, J Allergy Clin Immunol 122: 456-468; Lamb N J, Gutteridge J, Baker C, Evans T W & Quinlan G J (1999) Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: Evidence for neutrophil-mediated hydroxylation, nitration, and chlorination. Crit. Care Med 27: 1738). As well, there are no reports of a decrease of cholesterol in inflammatory lung diseases. To the contrary, cholesterol in surfactant is increased in ARDS (Panda A. K., et al (2004) Effect of acute lung injury on structure and function of pulmonary surfactant films. Am. J. Respir. Cell Mol. Biol. 30: 641-650; Tammi R, Ripellino J A, Margolis R U & Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Invest Dermatol 90: 412-414) and CF (our unpublished results). For these pathologies, the increased level of cholesterol can even explain the dysfunction on its own (Gunasekara L C, et al (2010) Methyl-[beta]-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol. Biochimica Et Biophysica Acta (BBA)—Biomembranes 1798: 986-994; Vockeroth D, et al (2010) Role of cholesterol in the biophysical dysfunction of surfactant in ventilator-induced lung injury. American Journal of Physiology—Lung Cellular and Molecular Physiology 298: L117-L125).
To ensure relevance to disease states, in the Examples below, attempts were made to match surfactant concentration, rate of compression-expansion, surfactant composition, and levels of oxidant exposure to known physiological or pathological parameters. However, by using lipid-extracted surfactants, we removed water-soluble endogenous anti-oxidant molecules present in natural surfactants that may render these more resistant to oxidation (Haddad I Y, et al (1993) Mechanisms of peroxynitrite-induced injury to pulmonary surfactants. American Journal of Physiology—Lung Cellular and Molecular Physiology 265: L555-L564). For example, hyaluronic acid in the alveolar fluid possesses free-radical scavenging properties (Tammi R, Ripellino J A, Margolis R U & Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Invest Dermatol 90: 412-414). On the other hand, surfactant in the inflamed lung could be subject to dilution by alveolar oedema and conversion of LA material into poorly surface-active small aggregates (reviewed in Zuo Y Y, Veldhuizen R A W, Neumann A W, Petersen N O & Possmayer F (2008) Current perspectives in pulmonary surfactant—inhibition, enhancement and evaluation. Biochimica Et Biophysica Acta (BBA)—Biomembranes 1778: 1947-1977; Tammi R, Ripellino J A, Margolis R U & Tammi M (1988) Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe. J Invest Dermatol 90: 412-414), which may enhance oxidative dysfunction. Reactive oxygen species may also enhance the reactivity of reactive nitrogen species found in vivo, which are capable of further damaging surfactant lipids and proteins (Haddad I Y, et al (1993) Mechanisms of peroxynitrite-induced injury to pulmonary surfactants. American Journal of Physiology—Lung Cellular and Molecular Physiology 265: L555-L564; Haddad I Y, et al (1994) Concurrent generation of nitric oxide and superoxide damages surfactant protein A. American Journal of Physiology—Lung Cellular and Molecular Physiology 267: L242-L249).
In a model study, 0, 5 and 10 weight percent cholesterol was added to a ROS-exposed cattle lipid surfactant extract as an example of how even a normal (physiological) amount of cholesterol renders surfactant exposed to ROS and its derivatives dysfunctional; the function of the surfactant model was determined in a captive bubble surfactometer (CBS).
In the absence of methyl-β-cyclodextrin (MβCD) (
In the presence of methyl-β-cyclodextrin (MβCD) (20 mmol) (
In this study with pediatric CF surfactant samples (n=10,
In this study, a murine model of acute lung injury (ALI), the earliest phase of ARDS, was created by acid injury of the lung according to published procedures (Allen, G. B. et al. Neither fibrin nor plasminogen activator inhibitor-1 deficiency protects lung function in a mouse model of acute lung injury. American Journal of Physiology-Lung Cellular and Molecular Physiology 296, L277-L285 (2009)) and analyzed with respect to lung mechanics, oxygenation, lung histology, lung ultrastructure, surfactant function and composition (
In this Example, the role of oxidized polyunsaturated phospholipids in causing cholesterol-dependent surfactant dysfunction was studied. Oxidation of surfactant by the Fenton-like reaction or in the presence of ROS in the inflamed lung results in the formation of lipid-peroxides with polyunsaturated phospholipids the principal target. Lipid peroxides, while otherwise unaltered, possess a hydroperoxide group in their aliphatic tail(s). This introduction of a hydrophilic group in the formerly hydrophobic region will disrupt lipid packing. Addition of 20% w/w oxPLPC or oxCL to BLES+10% cholesterol caused substantial deterioration in surface activity (
Polyunsaturated phospholipid species in addition to forming peroxides are also highly susceptible to oxidant-initiated hydrolysis. As a control, removal of the phospholipid hydrolysis products (as described above) from BLES+10% cholesterol containing oxPLPC or oxCL did not improve surface activity (not shown), thereby excluding the possibility that inhibition was due to the generation and incorporation of large quantities of lysophospholipids and FFAs. In summary, oxidized polyunsaturated phospholipids in conjunction with cholesterol render surfactant dysfunctional with the respective peroxides of the affected lipids being the most likely deleterious molecular species.
Effects of oxidation on the structure of surfactant films was also studied. Low surface tension results from the surfactant forming an incompressible film at the air-buffer interface. In mechanical analogy, surface tension is equivalent to an external lateral pressure compressing a plate and the molecular film at the interface is the continuum plate. If functional, the film will not yield to the lateral pressure and thus offset the surface tension. If dysfunctional, the film will collapse by flowing off the interface into the buffer for fluid films. Rigid dysfunctional films will respond to the pressure by buckling if ductile or cracking if brittle.
Microscopy of Langmuir-Blodgett films (films formed at the air-buffer interface and then transferred onto a solid support) reveals important aspects of how pulmonary surfactant performs its function and how it fails. In the Atomic Force Microscope (AFM), oxBLES films show a pattern of monolayer areas and lipid bilayer regions (
The bilayer stacks are cross-linked to the monolayer by the surfactant proteins B and C. We proposed earlier that these act as a mechanical re-enforcement preventing buckling or cracking of the film (Leonenko, Z. et al. An elevated level of cholesterol impairs self-assembly of pulmonary surfactant into a functional film. Biophys. J. 93, 674-683 (2007)). OxBLES, in contrast to untreated BLES, shows an additional abundance of globular extrusions of sub-micrometer dimensions, possibly representing micelles of defective lipids that have been squeezed out of the active film without compromising its overall performance.
In summary, oxidized surfactant in the absence of cholesterol maintains the functional structure of a lipid monolayer with an abundance of regions of lipid bilayers. AFM shows cholesterol in oxidized surfactant to affect most prominently the assembly of bilayers, resulting in large globular collapse structures in this region. This finding is consistent with bilayer regions that fail to mechanically re-enforce the film but rather become the foci from where lipids are lost to the aqueous phase.
Materials—
BLES was a kind gift from BLES Biochemicals Inc. (London, ON, Canada). 1,2-dipalmitoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), and bovine heart cardiolipin (CL, predominantly 1′,3′-Bis[1,2-dilinoleoyl-sn-glycero-3-phospho]-sn-glycerol) were purchased from Avanti Polar Lipids Inc. (Alabaster, Ala., USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Human SP-A (hSP-A) was isolated from the whole lung lavage of a patient with alveolar proteinosis using butanol extraction as described by Haagsman et al. (Haagsman H P, et al (1989) Studies of the structure of lung surfactant protein SP-A. American Journal of Physiology—Lung Cellular and Molecular Physiology 257: L421-L429). The purity of this preparation was assessed by polyacrylamide gel electrophoresis followed by Coomassie blue staining. All lipids were stored at −20° C. under nitrogen. Surfactant mixtures were stored at 4° C. under nitrogen and used within three days after preparation. Buffers containing HEPES were stored in the dark at 4° C. to avoid inadvertently generating H2O2 (Haddad I Y, et al (1993) Mechanisms of peroxynitrite-induced injury to pulmonary surfactants, American Journal of Physiology—Lung Cellular and Molecular Physiology 265: L555-L564).
In Vitro Oxidation—
BLES was exposed to hydroxyl radicals generated from Fenton-like chemistry for 24 h to produce oxidized BLES (oxBLES) (as described by Rodriguez-Capote et al., Rodriguez-Capote K, Manzanares D, Haines T & Possmayer F (2006) Reactive oxygen species inactivation of surfactant involves structural and functional alterations to surfactant proteins SP-B and SP-C. Biophys J 90: 2808-2821; and Capote K R, McCormack F X & Possmayer F (2003) Pulmonary surfactant protein-A (SP-A) restores the surface properties of surfactant after oxidation by a mechanism that requires the Cys6 interchain disulfide bond and the phospholipid binding domain. J Biol Chem 278: 20461). DPPC, POPC, PLPC, DPPG, and CL were suspended in saline with 1.5 mM CaCl2 and exposed to identical oxidizing conditions and are indicated as oxDPPC, oxPOPC, oxPLPC, oxDPPG, and oxCL in order to indicate exposure to oxidative conditions only. Oxidation of surfactant phospholipids was confirmed by measuring the formation of the secondary lipoperoxidation products malondialdehyde (MDA) and 4-hydroxyalkenal (4-HAE) in 1.0 mg/mL oxBLES (BIOXYTECH LPO-586, OxisResearch, Burlingame, Calif., USA). oxBLES contained significantly more MDA and 4-HAE compared to control BLES (6.69±1.16 and 2.69±0.63 nmol MDA and 4-HAE/mg phospholipid respectively, n=6, p=0.014), consistent with previous studies using this Fenton reaction-like oxidation.
Surfactant Preparation—
To ensure accurate mixing, additional lipids were added to lipid extracts of BLES or oxBLES as organic solutions. The mixtures were then dried under nitrogen and resuspended in CBS buffer (140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2, pH 6.9) to a phospholipid concentration of 27 mg/mL. Phospholipid concentration was assessed using a phosphate assay (Gentaur Molecular Products, Kampenhout, Belgium) after liberation of free phosphate (Duck-Chong C G (1979) A rapid sensitive method for determining phospholipid phosphorus involving digestion with magnesium nitrate. Lipids 14: 492-497). Total phospholipid concentration was 96.8±6.8% (n=6) after lipid extraction and 98.6±11.1% (n=12) after oxidation. BLES contained 2.60% cholesterol (w/w phospholipids) as determined using an enzymatic assay for cholesterol (Amplex Red Cholesterol Assay, Invitrogen, Eugene, Oreg., USA). hSP-A was added to dried films when indicated to a final concentration of 5% (w/w phospholipids). In a subset of experiments, water soluble products of phospholipid hydrolysis (Gilliard N, et al (1994) Exposure of the hydrophobic components of porcine lung surfactant to oxidant stress alters surface tension properties. J Clin Invest 93: 2608; Arnhold J, Osipov A N, Spalteholz H, Panasenko O M & Schiller J (2002) Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid, Biochimica Et Biophysica Acta (BBA)—General Subjects 1572: 91-100) were removed by high-speed centrifugation of dilute surfactant mixtures (40 000 g×30 min×4° C.). The supernatant fluid was discarded and the pellet was resuspended to the initial volume of concentrated surfactant. After high-speed centrifugation, the concentration of phospholipids in oxBLES was 61.9±9.7% that of likewise treated BLES (n=3, p=0.034).
To obtain surfactant containing physiological levels of cholesterol, five female, 12-week old C57BL6 mice (Jackson Laboratories, Bar Harbour, Me., USA) housed in a pathogen-free animal care facility at the University of Calgary Health Sciences Center were humanely euthanized with an overdose of i.p. sodium pentobarbital. A tracheal cannula was surgically placed and three 0.5 mL aliquots of normal saline were used for bronchoalveolar lavage (BAL). BAL fluid was immediately centrifuged (400 g×10 minutes×4° C.) to remove cellular debris, and the large aggregate fraction was then isolated from the supernatant by high-speed centrifugation as described above. These fractions were pooled and lipid extracted to obtain mouse lipid extract surfactant (MLES). After drying, this pooled fraction was resuspended in saline with 1.5 mM CaCl2 to a final concentration of 10 mg/mL phospholipids and subsequently exposed to oxidation as described above to obtain oxMLES. MLES contained 7.3±1.6% cholesterol (w/w phospholipids).
Surface Activity Assessment—
Surface activity of surfactant was determined with a computer-controlled captive bubble surfactometer (CBS) as described in detail by Gunasekara et al. (Gunasekara L, Schoel W M, Schürch S & Amrein M W (2008) A comparative study of mechanisms of surfactant inhibition. BBA—Biomembranes 1778: 433-444), with two important changes: 1) A transparent capillary was used to deposit a ˜1.0 μL bolus of 27 mg/mL surfactant near the air-buffer interface. The quantity and concentration of surfactant were chosen to minimize effects caused by insufficient surfactant or bulk phase adsorption. This amount of native BLES reliably reproduces minimum and maximum surface tensions as measured in situ at functional reserve capacity and total lung capacity (<1 mN/m and ˜30 mN/m, respectively) (Bachofen H & Schtirch S (2001) Alveolar surface forces and lung architecture. Comparative Biochemistry and Physiology—Part A: Molecular & Integrative Physiology 129: 183-193). In a subset of experiments, a transparent capillary was used to deposit 54.0 μL of 0.5 mg/mL surfactant to assess differences in surfactant function caused by concentration. 2) A conditioning step was performed to ensure consistent surface activity in subsequent compression-expansion cycles. The bubble was first quasi-statically compressed to ˜20% of the maximum volume and subsequently expanded to set minimum and maximum volumes. Afterwards, the bubble volume was dynamically cycled over the same volume range at a rate of 20 cycles/minute to condition the film. Four quasi-static cycles and a second set of dynamic cycles were carried out afterwards. Measurements of surface tension and interfacial area were made on this second series of cycles. The compressibility ratio at 20 mN/m (C20) was calculated using the following formula: C20=(δA/δγ)/A, where A=surface area and γ=surface tension.
To evaluate the effect of MβCD, powdered MβCD (Sigma Aldrich, Catalogue-Nr. C4555) was dissolved in buffer to a final concentration of 30 mM and added to the CBS chamber prior to the addition of surfactant. Calcium-free experiments were conducted using CBS buffer without calcium and with 15 mM EDTA added. Surfactants used in these experiments were incubated overnight in EDTA-containing buffer before use.
Isotherms and Film Deposition—
Suspensions of bovine lipid extract surfactant (BLES) were compared with respect to surface activity on a Langmuir trough and surfactant structure as deduced by atomic force- and fluorescence light microscopy. Samples of a) oxBLES, b) oxBLES with 10% w/w cholesterol, and c) oxBLES with 10% w/w cholesterol and hSP-A were prepared as described above. A fluorescent lipid analogue (Rhodamine-DHPE, Invitrogen, Eugene, Oreg., USA) was added to a concentration of 0.1% mol/mol DPPC prior to aqueous suspension.
Surfactant suspensions were spread into a film at the air-buffer interface (750 cm2) of a Langmuir trough (Nima Technology, Coventry, UK) at 37° C. by placing a drop of approximately 10 μl (at a concentration of 27 mg/ml) at the interface. The film area was reduced at a rate of 100 cm2/min from 600 cm2 to 100 cm2 and re-opened. This cycle was then repeated twice. The surface tension was continuously monitored and area-surface tension isotherm recorded. Next, the area was reduced again, until a surface pressure of 45 mN/m was achieved. At this point, films for microscopy were deposited onto round (25 mm) microscope cover slips by the Langmuir-Blodgett technique. For deposition, the previously submerged cover slip was moved perpendicular across the air-buffer interface at a speed of 25 mm/min while the surface tension was kept constant.
Microscopy—
AFM topographical images were collected in air in intermittent contact mode with a NanoWizard II AFM (JPK Instruments, Berlin, Germany) using noncontact mode silicon cantilevers NCH-20 (NanoWorld, Neuchâtel, Switzerland) with typical spring constants of 21-78 N/m, and resonance frequencies of 260-460 kHz. The AFM was fitted on a Zeiss Axio Observer (Zeiss, Jena, Germany). Optical fluorescence images were acquired of sample regions containing the AFM scan regions using a Zeiss 40× Apoplan oil-immersion objective (numerical aperture: 1.4) and an Andor iXon EMCCD camera (Andor, Belfast, Ireland). The optical images were corrected for optical distortions and the magnification accurately calibrated by taking advantage of the AFM scanner to establish reference points (using the “optical overlay” feature of the AFM software). This allowed for an accurate overlay of the two imaging modes.
Statistical Analysis—
All CBS experiments are reported as mean±SEM (n=4-7). Each unique lipid mixture was prepared independently at least twice. Analysis of variance (SPSS 19.0) between two groups was followed by Tukey's HSD for multiple comparisons with p<0.05 used as the standard for significance. Means with the same letter and case in a figure are not statistically different for that cycle.
In summary, oxidized surfactant in the absence of cholesterol maintains the functional structure of a lipid monolayer with an abundance of regions of lipid bilayers. AFM shows cholesterol in oxidized surfactant to affect most prominently the assembly of bilayers, resulting in large globular collapse structures in this region. This finding is consistent with bilayer regions that fail to mechanically re-enforce the film but rather become the foci from where lipids are lost to the aqueous phase.
Without being bound to theory, it is believed that surfactant dysfunction is caused by the combination of oxidation and cholesterol. We show for the first time that ROS lead to lipid peroxides that interact with cholesterol to induce instability in the bilayer regions of the surfactant films. The collapse structures seen in the Figures and the plateau of the isotherm (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2007 025 898.6 | Jun 2007 | DE | national |
This application is a Continuation-in-Part of the National Phase filing under 35 U.S.C 371 of PCT Appl. No. PCT/EP2008/004274 filed May 29, 2008, which claims the benefit of German Application No. DE 10 2007025898 A1 filed on Jun. 1, 2007, both applications of which application are incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2008/004274 | May 2009 | US |
| Child | 12628544 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12628544 | Dec 2009 | US |
| Child | 13629474 | US |
