NZ239933A - Foam comprising pulmonary surfactant produced in situ in lungs of a mammal and use for administration to mammals - Google Patents

Description

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Publication Data: ....?.?, j??5. P.O. Journal, No: ..... y\ ^ ^ os „ w/>\, 4 *23 OCT 1991 Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION NATURAL PULMONARY SURFACTANTS AND LIPID EXTRACTS THEREOF I, FRED POSSMAYER, a Canadian citizen of 944 Wellington Street North, London, Ontario, CANADA N6A 3S9, hereby declare the invention, for which I pray that a patent may be granted to me, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by Page la) \ - lQ~ Natural Pulmonary Surfactants and Lipid Extracts Thereof Technical Field The invention relates to natural pulmonary surfactants obtained from mammals, and more particularly, to foams comprising a natural surfactant, lipid extracts thereof, and processes of producing the same.

Background Art The major cause of perinatal morbidity and mortality in developed nations is the Neonatal Respiratory Distress Syndrome (NRDS) which arises mainly because infants delivered prematurely do not have sufficient pulmonary surfactant stores to stabilize their lungs. Pulmonary surfactant maintains 15 normal lung function by reducing the surface tension at the air-liquid interface of the alveoli in the terminal air spaces of the lungs, thereby preventing collapse of the alveoli and bronchioles. Pulmonary surfactant effectively lowers the surface tension of the liquid film which bathes the entire 20 cellular covering of the alveolar walls to low values sufficient to maintain alveolar inflation during all phases of the respiratory cycle.

It has long been known that pulmonary surfactant, being by definition a surface active material, can readily 25 produce foam. The observation that exposure of animals to phosgene gas resulted in an acute efflux of foam into their major airways led Pattle to the rediscovery of the surfactant system of the lung (Pattle Nature 175:1125-1126, 1955). Acute oedema foam can also be produced by administering mixtures of 30 oxygen and ammonia or by infusing adrenaline into animals in vivo (Pattle 1955, op cit.} Pattle J Path Bact 72:203-209, 1956). Foam can also be obtained by infusing saline into the lungs of anaesthetized animals (Pattle 1956, op cit.). However, the amounts of surfactant obtained by these methods r are very low. On the basis of the weight of the foam, Pattle <s ^ ^ -^calculated that he was able to recover less than 0.25 mg of 7 - °\ ^23 OCT 199* "1 v (followed by page 2) surfactant from an•anaesthetized adult rabbit by his saline-infusion method.

Other prior art processes for producing foams comprising natural surfactant include agitating 5 bronchoalveolar lavages, and passing air or nitrogen through bronchoalveolar lavages or fetal pulmonary fluid (Pattle 1955, op cit.; Enhorning et al. J Exp Med 84:250-255, 1964). Pulmonary surfactant foam can also be produced by subjecting small pieces of lung to reduced pressure and from isolated 10 lungs by perfusing the pulmonary vasculature with saline while compressing and expanding the lungs in a vacuum flask using positive and negative pressures (Klaus et al. Proc Natl Acad Sci 47:1858-1859, 1961; Bondurant et al. J AppI Physiol 37:911-917, 1962).

Though foaming methods were used in the 1950's and 1960's for obtaining small samples of surfactant to study its biophysical or surface tension reducing activity, such prior art methods did not provide sufficient surfactant to allow extensive chemical characterization of surfactant using 20 available methods or physiological testing of surfactant. Furthermore, the foam produced by such prior art methods was not in a convenient form for handling, though it could be dried to a solid and resuspended. For these reasons, the conventional methods for obtaining surfactant for conventional 25 chemical testing and later, physiological testing, were either (1) bronchoalveolar lavage, according to which method saline was infused in the trachea, recovered and then centrifuged; or, (2) lung mincing, according to which method surfactant was obtained from saline extracts of minced lung by a series of 30 differential centrifugation steps.

In current usage, "natural surfactant" typically refers to pulmonary surfactants recovered from alveolar washes or from amniotic fluid by simple centrifugation. A variety of such natural surfactants have been used in clinical trials Prevent and treat NRDS in infants delivered prematurely.

A' [fa o\\ 1^23 OCT1991 fM ri' ' Natural surfactant is primarily composed of phospholipids, neutral lipids and three major protein components. Natural surfactant comprises from 80 to 95 per cent phospholipid (85-95% [w/v]), from two to ten per cent neutral lipid (2-10% 5 [w/v]), and from five to ten per cent protein (5-10% [w/v]).

The phospholipid and neutral lipid compositions of natural surfactant are relatively constant across mammalian species. The major lipid components of natural surfactant are dipalmitoylphosphatidylcholine (DPPC), unsaturated 10 phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylinositol (PI). The ratio of the acidic phospholipids, phophatidylinositol and phosphatidylglycerol, changes during perinatal development.

The composition of bovine natural surfactant has 15 been reported to be 35.6% dipalmitoylphosphatidylcholine (DPPC), 32.5% unsaturated phosphatidycholine (PC), 10% phosphatidylglycerol (PG), 1.5% phosphatidylinositol (PI), 3.0% phosphatidylethanolamine (PE), 1% lyso-jbis-phosphatidic acid, 2.5% sphingomyelin, 3.0% neutral lipids and 10% protein 20 (Yu et al. Lipids 18:522-529, 1983).

Natural surfactant comprises at least three proteins which have been designated surfactant-associated protein A (SP-A), SP-B, and SP-C (Possmayer Am Rev Resp Pis 138:990-998, 1988). Surfactant-associated protein A is a sialoglycoprotein 25 having a molecular mass of 640 kDa, and is composed of 18 monomers each having a molecular mass of approximately 35 kDa. Surfactant-associated proteins B and C are hydrophobic and lipophilic proteins of low molecular mass, and are essential for the biophysical activity of surfactant. Surfactant-30 associated protein B is a dimer of a molecular mass of approximately 15 kDa which migrates at approximately 5 kDa after reduction. Surfactant-associated protein C has been demonstrated to be present in isolated surfactant as a monomer N r°f 3,5 ^Da, and to a lesser extent, as an apparent dimer of ~ 35c:>\kDa.

'W o!\ '4 V ^23 OCT 1991 2399 One problem intrinsic to natural surfactants recovered by alveolar wash procedures is that they contain nonspecific, predominantly plasma proteins as contaminants, and may contain microbial contaminants such as bacteria or 5 viruses. The fact that natural surfactants contain relatively high concentrations of protein, both surfactant-associated protein and also non-surfactant protein, presents problems when preparations comprising natural surfacants are administered clinically. Such preparations are potentially 10 immunogenic due to the presence of significant amounts of protein therein. Immunogenic surfactant preparations derived from any source could lead to sensitization to these foreign proteins among infants to whom the surfactant had been administered. Natural surfactants obtained from non-human 15 mammals are more likely to stimulate adverse immunological responses in human neonates than are those obtained from humans. Theoretically, even preparations comprising human natural surfactant could cause immunogenic complications due to immune responses to non-self human antigens based on minor 20 genetic variations in protein structure. Though natural human surfactant has been used clinically to treat NRDS, viral or bacterial contamination of preparations comprising human natural surfactant remain a problem because natural surfactant can not be autoclaved or sterilized by other means known to 25 the applicants such as irradiation without also losing its biophysical (i.e. surface-tension reducing) activity.

Modified natural surfactants are a second type of surfactant. Modified natural surfactants generally are prepared by extraction of lipids from natural surfactant 30 obtained from lung minces or alveolar lavage, followed in some cases by selective addition (or removal) of certain compounds, and suspension procedures designed to restore the desired surface properties. Early studies demonstrated that protein-depleted lipid extracts of natural surfactant have the ability to reduce the surface tension of a pulsating bubble to near °^mN/m at minimum bubble size and to promote lung expansion }! /•'V "n f(2 \C23 OCT 1991"/ O T A f\ °i' "V 3 y & 5 j and survival of surfactant-deficient prematurely delivered animals. Modified natural surfactants can prove to be biophysically ineffective (i.e. unable to reduce the surface tension of alveoli) depending upon the nature of the means 5 used to finally resuspend the surfactant lipids for administration clinically. In developing modified natural surfactants, the objectives have been to decrease protein content, to achieve sterility, and to standardize, and improve surface properties. In the development of modified natural 10 surfactants, problems relating to sterilization techniques, suspension techniques, reproducibility of preparation and surface properties have been considerable (Jobe et al. Am Rev Resp Pis 136:1256-1275, 1987). The requirements for multiple skills to not only prepare the surfactants but also to test 15 them both in vivo and in vitro have tested the ingenuity of the investigators (Notter et al. J Appl Phvsiol 57:1613-1624, 1984).

It is well known that natural surfactant can be extracted with organic solvents such as chloroform:methanol to yield protein-depleted lipid extracts which retain those biophysical and physiological properties required for clinical use (Metcalfe et al. J Appl Physiol 49:34-41, 1980; Fujiwara et al. Lancet i:55-59, 1980; Tanaka et al. J Jap Med Soc Biol Interface 13:87-94, 1982). Organic solvent extracts of natural surfactant, also known as lipid extract surfactants, have been used extensively in prophylactic trials to prevent NRDS, and also in so-called "rescue" trials to treat the established disease (Jobe et al. Am Rev Resp Pis 1365:1256- 1275, 1987; Robertson et al. Exp Luna Res 14:279-310, 1988).

Such prior art lipid extract surfactant preparations have been prepared from natural surfactant collected either by bronchoalveolar lavage with salt solutions or by salt solution extraction of minced lung. In such prior art methods, the saline solutions used to collect natural surfactant are .35 subjected to a series of differential and gradient £ N r " - . ... c^Ncentrifugation steps in order to obtain crudely purified l! .'V <*23 0CTI99!^ \ * - '■> * - natural surfactant for lipid extraction. The natural surfactant obtained in this manner is subsequently extracted with organic solvents to yield a lipid extract surfactant.

Both the collection and purification of natural 5 surfactant using such prior art methods are labourious and time-consuming. Hence, possible bacterial or viral contamination of natural surfactants derived from unsterilized natural surfactants is a deficiency intrinsic to modified natural surfactants of the prior art.

Because several hours are required to complete collection and purification of natural surfactant using prior art processes, it is possible for preexisting microbial contaminants of the natural surfactant to contaminate lipid extract surfactants produced therefrom, thereby rendering them 15 unsuitable for clinical use. For example, under suitable conditions using prior art processes, bacterial contaminants may (1) secrete lipid soluble toxins co-extractable with the lipid extract surfactant; (2) secrete phospholipases capable of degrading constituent lipid elements of natural surfactant; 20 and (3) proliferate under suitable conditions, thereby increasing the bioburden of any resulting lipid extract surfactant. In the cases of such examples, the resulting lipid extract surfactants would be rendered unsuitable for clinical use. For instance, administration of preparations 25 containing lipid extract surfactant contaminated by lipid soluble toxins to neonates could lead to toxicological complications posing serious health risks. In addition, preparations containing lipid extract surfactant contaminated with non-surfactant lipids (e.g. bacterial or mammalian cell 30 membrane lipids) or having an abnormal phospholipid composition due to degradation of phospholipids by bacterial phospholipases may be biophysically inactive, and thus therapeutically ineffective. Because all contaminated lots of modified surfactants must be rejected as unsuitable for clinical use, even a low rate of contamination of lipid - \ '1991 23 T & v? f* ° f230CTl99l% •tpeiV.-*• extract surfactants produced using prior art collection and purification processes can prove to be very costly.

Artificial surfactants synthesized from mixtures of synthetic compounds that may or may not be constituents of 5 natural surfactant are a third type of surfactant. See for example United States patent 4,826,821 for a review of the prior art (Clements). Artificial surfactants are attractive commercially because all potential complications relating to immunogenicity and sterility are avoidable theoretically. 10 However, when compared with modified surfactants such as lipid extracts of natural surfactant, the record for artificial surfactants has generally not been encouraging. A deficiency intrinsic to prior art artificial surfactants stems from the fact that the biophysical activity of artificial surfactants 15 varies with the different suspension techniques used in their preparation and their surface properties. The biophysical activity in vivo of artificial surfactants having equivalent or very similar phospholipid compositions is not readily predictable, and must be evaluated individually (Jobe et al. 20 Am Rev Resp Pis 136:1256-1275, 1987). Routine testing in vitro of the surface properties of artificial surfactants may not accurately predict their biophysical activity in vivo.

Synthetic natural surfactants comprise the fourth and final type of surfactant known in the prior art, and are 25 not particularly relevant to the invention. Synthetic natural surfactants are reconstructed in vitro from surfactant-specific proteins synthesized using recombinant DNA and molecular biology techniques and from mixtures of phospholipids and neutral lipids. See for example World 30 Patent Number 8904326 (Benson et al.) and British Patent Number 2,181,138 (Schilling et al.) for summaries of this branch of the prior art.

Presently, there exist large stores of natural pulmonary surfactant which are untapped due to the limitations 35 of prior art methods of collecting and purifying natural urfactant. A multiplicity of mammals are slaughtered y -y • - 8 - routinely to produce meat for human consumption. These animals are inspected and certified to be healthy and fit for human consumption, and their lungs contain natural surfactant suitable for processing to provide modified natural 5 surfactants. The lungs of these mammals are either processed without first recovering the surfactant lining the lungs, or simply discarded. It is desireable to have available a method for collecting natural pulmonary surfactant quickly and easily from such slaughtered mammals on site, and for further 10 purifying natural surfactant collected from these mammals for administration into mammalian alveolar spaces.

Disclosure of Invention The invention provides a means for obtaining natural 15 pulmonary surfactant from mammals in quantity and under conditions which obviate purification steps required for natural surfactants obtained by prior art bronchoalveoloar lavage and differential centrifugation methods. The invention involves instilling a saline solution into the lungs of a 20 freshly slaughtered mammal in situ to suspend the natural pulmonary surfactant therein and recovering the surfactant-saline suspension as butchering of the mammal proceeds. When the surfactant-saline suspension is withdrawn from the lungs, it is sufficiently agitated that the surfactant forms a foam 25 in situ which is collected from the mammal in combination with the expelled suspension.

The invention further comprises a lipid extract surfactant separated from the foam of the invention and a process for producing the same. Lipid extract surfactant of 30 the invention possesses excellent biophysical activity and has proven effective for treating NRDS in clinical trials.

Desireable attributes of the invention include features such as the increased yield of natural surfactant collected (per animal) by the method disclosed herein, a significant reduction in the length of time necessary to /f<t^ °^\complete the collection process enabling rapid extraction of .7 ^23 OCT 1991° J "Crs ^ c natural surfactant into an organic solvent mixture and therefore rapid sterilization of the natural surfactant, and simplification of the process of preparing lipid extracts of natural surfactant as compared to conventional processes of 5 the prior art. Sequestering the natural surfactant in a foam which can be skimmed from the expelled saline obviates conventional purification steps of prior art processes.

That larger amounts of natural surfactant can be obtained from a foam produced in situ by use of the process 10 disclosed herein than can be obtained using the conventional bronchoalveolar lavage and centrifugation processes was a novel and unexpected finding. The collection and extraction processes disclosed herein enable natural surfactant to be collected and extracted into an organic solvent mixture in a 15 time span of minutes rather than many hours. The rapid processes of the invention for collecting and extracting natural surfactant are advantageous, and a significant improvement over prior art processes. The invention provides rapid extraction of natural surfactant into an organic solvent 20 mixture which destroys microbial and pathogenic viral contaminants of the natural surfactant, and thereby limits the potential bioburden of lipid extract surfactant produced therefrom. Hence, the improved efficiency of the collection and extraction processes of the invention provide an important 25 quality control feature in addition to commercial benefit.

The processes described herein for obtaining bovine natural pulmonary surfactant are merely for purposes of illustration and are typical of those that might be used. Clearly, other mammals and other procedures may also be 30 employed, as is understood in the art.

Further features of the invention will be described or will become apparent in the course of the following detailed description. °^\ f ... ^ 23 OCT 1991 *' 23 Brief Description of the Drawings Figure 1 shows a flow chart of the process for making lipid extract surfactant; Figure 2 shows a flow chart of the process for 5 preparing a lipid extract surfactant suspension.

Best Mode For Carrying Out The Invention The processes described herein for sequestering bovine natural surfactant in a foam readily collectable from 10 a mammal via its trachea and of preparing a lipid extract surfactant are merely for purposes of illustration, though typical of those that may be used. Mammals other than cattle and other procedures may also be employed, as is understood in the art. For instance, natural surfactant foam may be 15 similarly obtained from mammals such as horses, sheep, rabbits, pigs, dogs and cats.

Preparation of Bovine Surfactant Foam in situ In the preferred embodiment of the invention, a 20 bovine animal is killed. The animal can be bled. The animal's trachea is then exposed without damaging the lungs. (Damage to the lungs may make the animal unsuitable for use.) To permit collection of natural surfactant from the lungs of the mammal, a length of plastic tubing having an 25 outer diameter sized to fit snugly within the animal's trachea (e.g. of 3.75 cm [1.5 inches]) is inserted into the trachea so that a first end of the tubing lies close to the bifurcation of the animal's bronchi. The tubing is then secured within the trachea, either manually or with a length 30 of butcher's cord or the like. After the tubing has been secured, approximately ten to fifteen litres of an ice-cold saline "working solution" (0.15 M NaCl, 10 mM CaCl2, 8 mM MgCla) are introduced into the trachea manually or by pump means. For example, a vessel such as a jerrycan containing '"*5^ working solution is elevated in communication with the trachea // ^ <<y{^o that the required volume of saline flows into the animal's *23 OCT I99| \ * Q 0 X T O -J 1..-V* lungs. Natural surfactant lining the lungs is thereby suspended in the saline solution in situ. After the saline solution has been instilled into the lungs, the resulting surfactant-saline suspension is withdrawn from the lungs and 5 returned to the stock working solution, either by gravity or pump means. Next, the lungs are again refilled with surfactant-saline suspension from the stock working solution. This final volume can be allowed to remain in the lungs until the animal's posterior end or hind quarters are elevated by 10 a hoist or other means, so that the animal's lungs are one-half to one meter below its posterior end. The surfactant-saline suspension in the lungs is then allowed to flow back into the jerrycan by gravity. Under the force of gravity, the surfactant-saline suspension is expelled from the lungs and 15 trachea with force sufficient to cause the natural surfactant to foam in the lungs in situ, with a copious white foam being expelled in combination with the remaining suspension. Preferably, the jerrycan is lowered to collect as much of the suspension/foam combination as possible. 20 Preferably, the animal's viscera are then removed and the lungs are exposed. Preferably, surfaces of the lungs are massaged until no more foam is observed flowing into the tubing, to collect the last of the foam. Massaging of the lungs is best achieved by applying firm pressure with the palm 25 of a hand onto the lung surface. (Any preparations containing significant amounts of blood must be discarded.) Once the last of the foam has been collected, the surfactant-saline suspension in combination with foam is transferred into a large container and let stand for a few 30 minutes to allow the foam to rise to the surface. The copious white foam is then skimmed off into a fresh container.

Preparation of Lipid Extract Surfactant Referring to Figure 1, the foam is strained after 35 collection of foam from a number of animals is complete.

Approximately five litres of foam are strained at a time^-^.

A* \ •V -4 f 23 OCT 19911 ^ Each five litre aliquot of foam is strained through five layers of cheesecloth using a Buchner funnel and collected in a side-arm flask containing 100 ml chloroform:methanol (1:1 [v/v]). (Suction is applied via the side-arm with a water 5 aspirator.) The foam dissolves into the organic solvents virtually instantaneously. Any microbial contaminants of the foam will be killed by this step.

The final volume of the filtrate is measured and sufficient 10% potassium chloride (10% [w/v] KC1) is added to 10 give a chloroform:methanol:1.0% KC1 ratio of 1.0:1.0:0.9 (Bligh et al. Can J Biochem Physiol 37:911-917, 1959). The combined fluids are mixed by gentle rotation and inversion to extract all lipids into the chloroform. The resulting mixtures are then centrifuged in glass flasks at 800 g at 4°C 15 for 20 minutes to produce two phases; namely, a bottom phase containing chloroform and lipids, and an upper phase containing water and methanol. A "pad" of pelleted protein is collected at the interface between the two phases. Both phases of the clear supernatant are decanted into a fresh 20 vessel, leaving the protein pellet in the centrifuge tube. The mixture is swirled briefly and recentrifuged as above for 10 minutes. It is also possible to store these tubes without centrifugation until the two phases separate.

After a suitable interval, the upper aqueous phase 25 and any remaining precipitated protein are removed with a water aspirator. Alternatively, the upper aqueous and lower organic solvent phases may be isolated using a separatory funnel. The organic solvent layer is then evaporated; preferably using a rotary evaporator under reduced pressure. 30 A 500 ml boiling flask is used for 2.5 g of lipid.

To remove any trace amounts of precipitated protein, the resulting viscous oil is re-extracted with chloroform:methanol (1:1) and left at 4°C for several hours. The solution is then centrifuged at 400 g for 15 minutes. The 35 resulting supernatant is decanted, and then evaporated undg.r. reduced pressure to dryness on a rotary evaporator at V ^ ~~~ \ 23 OCT 1991' ">e.. 2 A T 0 13 twice. The resulting lipid extract is then taken up with a small volume of chloroform (t-o give a lipid concentration of approximately 150 mg/ml) and transferred to a 250 ml centrifuge tube. The lipid extract is dried under nitrogen 5 until an oily residue is formed on the walls of the tube, precipitated with 20 volumes (typically 250 ml) of cold acetone, stored for several hours at -20°C, and then centrifuged at 800 g at 4°C for 20 minutes. The resulting precipitate is resuspended in chloroform and the acetone 10 precipitation step is repeated. in a small volume of chloroform:methanol (1:1) (approximately 250 mg/ml) at -20°C indefinitely, or as shown in Figure 2, suspended in saline solution for tests of biophysical activity 15 or administration into mammalian alveolar spaces. lipid extract surfactant suitable for testing the biophysical activity of the lipid extract surfactant purified from the foam or for clinical application, 500 mg of lipid in 20 chloroform:methanol (1:1) is evaporated under reduced pressure in a small boiling flask. Typically, the required volume to give 500 mg is approximately 2 ml. Next, the lipid is redissolved in chloroform:methanol and centrifuged at 800 g for 20 minutes. The resulting liquid fraction is transferred 25 to a 50 ml round bottom screw-cap centrifuge bottle and the lipid is dried under nitrogen onto the walls of the tube, so that the lipid forms an even coating on the lower surface of the centrifuge tube. for 20 minutes to remove any traces of solvent. The lipid is then resuspended by vortexing and bath sonication at 35 to 40°C in a solution of 0.15 M NaCl, 1.5 mM CaCl2, to give a final lipid concentration of 25 mg/ml. Preparations of resuspended surfactant may be pooled, mixed with a stirring 35 bar, and aliquots thereof may be pipetted into sterile serum The final precipitate may be dissolved and stored Referring to Figure 2, to prepare a suspension of After drying, the lipid-coated tubes are lyophilized vials, stoppered with slotted stoppers, and f\ B--S7 "V \iS ij •Tui? 14 evaporated from the vials evacuated under gentle vacuum. Preferably, the vials should be capped with aluminium caps and crimped. Preferably, the vials are autoclaved at 121 eC for 15 to 20 minutes to sterilize the aliquots, cooled, and stored 5 frozen at -20°C.

Characterization of lipid extract surfactant Biophysical activity 1.5 mM CaCl2 share the ability of natural surfactant to reduce the surface tension of a pulsating bubble to near 0 mN/m at minimum bubble radius. To test the biophysical activity of the lipid extract surfactant a 0.2 ml sample is withdrawn from a vial and diluted to 0.5 ml with saline containing 1.5 liM 15 CaCl2 (i.e. to a concentration of 10.0 mg lipid extract surfactant per ml). The biophysical activity is determined with a pulsating bubble surfactometer (Electronetrics Corporation, Amerst, NY, USA). This assay monitors both the adsorption of the surfactant phospholipids to the air-saline 20 interface to form a surface-active monolayer and the squeeze-out of unsaturated phospholipids, leaving a monolayer enriched in dipalmitoylphosphatidylcholine (DPPC) which can reduce the surface tension to low values during the dynamic compression produced during the reduction of bubble surface area 25 (Enhorning J Appl Physiol 43:198-203, 1977? Yu et al. Lipids 18:522-529, 1983; Weber et al. Biochim Biophvs Acta 796:83-91, 1984). a bubble communicating with ambient air is created in a small 30 chamber. The bubble i$ pulsated between radius of 0.4 to 0.55 mm at 20 cycles per minute at 37°C, and acts as a single artificial alveolus. The pressure across the bubble is monitored with a pressure transducer. Surface tension is calculated according to the Law of Young and Laplace which 35 states that the difference in pressure across the bubble is Dispersions of lipid extract surfactant in saline- With the pulsating bubble surfactometer technique, UJ o Surface tensions at maximum bubble radius and minimum bubble radius are calculated. To have acceptable levels of biophysical activity, lipid extract surfactant preparations must reduce the surface tensions to 30 ± 5.0 mN/m at maximum 5 bubble radius, and 2.5 ±' 2.5 mN/m at minimum bubble radius within 50 pulsations. Any preparations not meeting these criteria should be abandoned.

Biochemical characterization 10 The composition of lipid extract surfactant prepared by the processes disclosed herein is of a high degree of purity, and is very similar to that reported for highly purified bovine pulmonary surfactant obtained by bronchoalveolar lavage (Yu et al., Lipids 18:522-529, 1983; 15 Weber et al. Biochim Biophvs Acta 796:83-91, 1984). Tables 1 and 2 show the lipid compositions of bovine pulmonary surfactant and that of the bovine lipid extract surfactant separated from the foam described herein, respectively.

Table 1 Lipid Composition of Bovine Pulmonary Surfactant (From Yu et al., Lipids 18:522-529, 1983) Phospholipid Phosphatidylcholine Phosphatidylglycerol Phosphatidylinositol 35 Phosphatidylethanolamine Lyso-Jbis-phosphatidic acid Sphingomyelin 40 % Total Phosphorus (n = 4) 79.2 ± 1.6 11.3 ± 0.5 1.8 ± 0.3 3.5 ± 0.5 1.5 ± 0.4 2.6 ± 0.5 ^"23 OCT mi 7 r.Q/> 239933 Table 2 Phospholipid Composition of Bovine surfactant- isolated by foaming in situ Phospholipid % Total Phosphorous (n = 3) Lyso-phosphatidylcholine 0.2 ± 0.03 Sphingomyelin 2.0 ± 0.35 Phosphatidylcholine 79.2 ± 0.65 Phosphatidylinositol 1.0 ± 0.03 Phosphatidylethanolamine 3.0 ± 0.18 Phosphatidylglycerol 14.4 ± 0.51 Lyso-jbis-phosphatidic acid Trace The phospholipid content of the lipid extract surfactant is determined by the assay of Rouser et al. (Lipids 5:769-775, 1970). The method of Duck-Chong (Lipids 14:492-497, 1979) provides identical values. A 200 nl sample of 30 lipid extract surfactant is diluted to 500 pi and 50 /ul aliquots are spotted on Whatman 5D plates. Chloroform/etha-nol/water/triethylamine (30:34:8:35) is used to develop the plates according to the method of Touchstone et al. (Lipids 15:61-62, 1980). After development, each plate is dried and 35 sprayed with a phosphate spray as described in Dittmer and Lester (J Lipid Res 5:126-127, 1964). Lipid extract surfactant of the invention contains 75 to 85% phosphatidylcholine, 8 to 16% acidic phospholipids such as phosphatidylglycerol and phosphatidylinositol, 2.5 to 7.5% 40 phosphatidylethanolamine, 0.1 to 3% lyso-Jbis-phosphatidic acid and < 5.0% sphingomyelin. Lipid extract surfactants not conforming to the above lipid profile should be discarded. Similarly, if a lipid extract surfactant were to contain 3.C>% „j» o \ 23 OCT I99| ^ ^•5 I i y u % • \jj 4ci' w >st? Vi lysophosphatidylcholine or more by weight, that preparation should be discarded.

The composition of the lipid extract surfactant of the invention contains lower levels of lysophosphatidyl-5 choline, cholesterol and cholesterol esters than reported for prior art lipid extract surfactants (U.S. Patent Number 4,338,301, issued July 6, 1982; Notter et al. J Appl Physiol 57:1613-1624, 1984; Shelly et al. Luna 160:195-206. 1982; U.S.

Patent No. 4,397,839, issued August 9, 1983; Berggren et al. 10 Exp Luna Res 8:29-51, 1985; King et al. Handbook of Physiology. The Respiratory System [Fishman AP and Fisher AB, eds.], Washington: American Physiological Society, 1:309-336, 1985). When present in excess, these compounds can inhibit the biophysical activity of lipid extract surfactant. It 15 appears that the higher levels of lysophosphatidylcholine and cholesterol present in some prior art preparations arise from contamination of pulmonary surfactant by cellular membranes (Rooney et al. Biochim Bioohys Acta 431:447-458, 1976; Holm et al. J Appl Physiol. 1987). In addition, the lipid extract 20 surfactant of the invention contains relatively low levels of another membrane lipid, namely, sphingomyelin. (The lecithin/sphingomyelin (L/S) ratio is used as an indicator of the relative amounts of surfactant to non-surfactant lipids in surfactant isolated from amniotic fluid and other sources 25 [Gluck et al. Ped Res 1:237-246, 1971].) Because collection of natural surfactant by sequestering it in a foam in the lungs in situ as opposed to mincing avoids damage to the lungs and trachea of the mammal, the foam production and surfactant collection process of the invention precludes the possibility 30 of contamination of the natural surfactant with cellular membranes.

The protein content of the lipid extract surfactant of the invention is measured using the method of Lowry et al.

(J Biol Chem 132:265-275, 1951) in the presence of sodium 35 dodecylsulphate as shown in Possmayer et al. (Can J Biochem . 55:609-617, 1977) using bovine serum albumin as a stanc^nd* ^ ■ y t\ \*23 OCT 1991^ ^..0 je j The lipid does not interfere with samples having 2.0 rag lipid or less in a final volume of 5.0 ml. By this method, the protein content of the lipid extract surfactant is 12.5 ± 6.0 tig protein per mg phospholipid.

Natural surfactant obtained by the standard lavage/centrifugation prior art processes comprises proteins other than SP-A, SP-B and SP-C. These are presumably serum contaminants; albumin is one of these proteins. Electrophoresis of lipid extract surfactant on polyacrylamide 10 gels with sodium dodecylsulphate shows decreased amounts of serum proteins, no SP-A, only SP-B and SP-C (Possmayer Am Rev Resp Pis 138:990-998, 1988). The depletion of non-surfactant proteins characteristic of the lipid extract surfactant of the invention reduces the antigen load of the lipid extract 15 surfactant, and advantageously reduces its potential immunogenicity. Because the gene sequences encoding SP-B and SP-C are highly conserved, and both proteins are small and hydrophobic, they are not likely to be very immunogenic. Hence, lipid extract surfactants derived from a natural 20 surfactant obtained from mammals as disclosed herein are unlikely to pose problems of immunogenicity in clinical use.

It will be appreciated that the above description relates to the preferred embodiment by way of example only. Many variations on the invention will be obvious to those 25 knowledgeable in the field, and such obvious variations are within the scope of the invention as described and claimed, whether or not expressly described.

For instance, organic solvent extractions of the natural surfactant foam are normally performed using 30 chloroform:methanol, but in principle, any extraction method which removes a plurality of the potentially immunogenic non-surfactant proteins and SP-A, but retains the low molecular weight hydrophobic proteins SP-B and SP-C essential for biophysical activity could be used for this purpose. oh, -i, ir\\v: <r> \ 23 OCT I99i Industrial Applicability Organic solvent extraction of the foam of the invention provides a lipid extract surfactant with biophysical and physiological activities appropriate for administration into mammalian alveolar spaces for clinical use to prevent and treat neonatal respiratory distress syndrome, to prevent or treat adult respiratory distress syndromes (e.g. shock lung, pneumonia), or for clinical use in connection with lung transplants.

V 2 23 OCT 1991 :t> •r • ' 2c n o ? WHAT i/W£ CLAIM IS: ° G^sims 1. A foam comprising a natural pulmonary surfactant obtained from a mammal wherein production of said foam is carried out in said mammal's lungs in situ to sequester said natural surfactant in said foam and thereby facilitate collection of said natural surfactant from said mammal. 2. A lipid extract surfactant separated from the foam of Claim 1, said lipid extract surfactant comprising a plurality of said natural surfactant's lipids, particularly phospholipids, and said natural surfactant's low molecular weight hydrophobic proteins, namely surfactant-associated 15 proteins B and C, said lipid extract surfactant further comprising said natural surfactant depleted of surfactant-associated protein A and of a plurality of potentially immunogenic mammalian proteins apart from surfactant-associated proteins B and C. 3. A lipid extract surfactant separated from the foam of Claim 1, said lipid extract surfactant comprising sufficient biophysical ability to reduce the surface tension of a bubble pulsated between a radius of 0.4 to 0.55 mm at twenty cycles per minute at 37 °C, to act as a single 25 artificial alveolus to 30 ± 5.0 mN/m at maximum bubble radius and to 2.5 ± 2.5 mN/m at minimum bubble radius within fifty pulsations. 4. A lipid extract surfactant as claimed in Claim 2 or

Claims (10)

1. 30 Claim 3, comprising, by weight, from 30 to . 50% dipalmitoylphosphatidylcholine, from 30 to 50% phosphatidylcholine, less than 1.0% lysophosphatidylcholine, less than 4.0% cholesterol, less than 2.0% cholesterol esters, less than 5.0% spingomyelin, and from r-0.5 to 2.0% 35 protein. *3) MAR 1994
2. ...••.I'1'- tray k** ?0> 4 M Q "/ ■t'
3. 3.
4. J - 21 -
5. 5. A lipid extract surfactant as claimed in Claim 2 or Claim 3, comprising, by weight, 40% dipalmitoylphosphatidylcholine, 40% unsaturated phosphatidylcholine, 12% phosphatidylglycerol, 3% phosphatidylethanolamine, 2% sphingomyelin, 5 2% phosphatidylinositol, and 1% protein including surfactant-associated proteins B and C.
6. 6. A process of making the foam of Claim 1, said process comprising: 10 exposing the trachea of a freshly killed mammal; introducing an electrolyte solution into said mammal's lungs via said trachea to provide a suspension of said natural surfactant in said electrolyte solution; withdrawing said suspension from said lungs and from 15 said mammal via said trachea with sufficient force to make said suspension foam in situ, and thereby expel said foam from said trachea in combination with said suspension; collecting said expelled foam and suspension from said trachea; 20 separating said foam from said collected suspension.
7. 7. A process as claimed in Claim 6 of making the foam of Claim 1, further comprising either positioning said mammal to locate a posterior end of said mammal above said mammal's 25 lungs to expel by gravity said suspension from said lungs and from said mammal via said trachea with sufficient force to cause said suspension to foam in situ, or pumping said suspension into said lungs and recovering said suspension from said mammal with sufficient force to cause said suspension to 30 foam in situ via a pump member communicating with said trachea.
8. 8. A process of separating from the foam of Claim 1 a lipid extract surfactant, said process comprising: r %\(a) extracting lipids and depleting protein from said if**' 'V^foam filtrate by extracting said foam filtrate in an organic , mil 23 OCT 1991 V -V ,,< * ♦ 2 KJ - 22 - solvent mixture to produce a first solution comprising a plurality of said natural surfactant's lipids and low molecular weight hydrophobic proteins, including dipalmitoylphosphatidylcholine and surfactant-associated 5 proteins B and C, respectively, and a depleted concentration, relative to natural surfactant, of surfactant-associated protein A and of a plurality of potentially immunogenic non-surfactant proteins; (b) adding sufficient potassium chloride to said first 10 solution to provide a biphasic second solution having a 2:0.9 volume ratio of organic solvent mixture to 1% (w/v) potassium chloride; (c) collecting an organic solvent phase of said second solution; 15 (d) extracting lipids from said organic solvent phase to provide a lipid extract surfactant.
9. 9. A method of making a lipid extract surfactant as recited in Claim 8, wherein said mammal is selected from a 20 group comprising cattle, horses, sheep, rabbits, pigs, dogs, and cats; and wherein said organic solvent mixture comprises one of a group of chloroform:methanol having a 1:1 volume ratio, chloroform:methanol having a 2:1 volume ratio, chloroform:ethanol having a 2:1 volume ratio, 25 chloroform:isopropanol having a 1:1 volume ratio, and ethyl ether:ethanol having a 3:1 volume ratio.
10. 10. Use of the lipid extract surfactant made from the foam of Claim 1 for administration into non-human mammalian 30 alveolar spaces. I / r FRED FOSSMAYER By his attorneys BALDWIN, SON & CAREY