Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W092/04907 2 0 919 8 0 PCT/CA91/00337
Natural Pulmonary Surfactants and Li~id Extracts Thereof
Ter-hnical 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.
~ckyr~ rt
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
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
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
produce foam. The observation that e~o~e 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
oxygen and ammonia or by infusing adrenaline into animals in
vivo (Pattle 1955, op cit .; Pattle J pAth ~ct 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
are very low. On the basis of the weight of the foam, Pattle
calculated that he was able to recover less than 0.25 mg of
W092/04907 PCT/CA91/00337
2o9~98o
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surfactant from an anaesthetized adult rabbit by his saline-
infusion method.
Other prior art processes for producing foams
comprising natural surfactant include agitating
S 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
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 Appl 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
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
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
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
to prevent and treat NRDS in infants delivered prematurely.
W092/04907 PCT/CA9l/00337
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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
[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
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
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-bis-phosphatidic
acid, 2.5% sphingomyelin, 3.0~ neutral lipids and 10% protein
(Yu et al. T;p;ds 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 ~m Rev ~esp Dis 138:990-998,
1988). Surfactant-associated protein A is a sialogly~G~oLein
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-
associated protein B is a dimer of a molecular mass ofapproximately 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
of 3.5 kDa, and to a lesser extent, as an apparent dimer of
7 kDa.
W092/04907 PCT/CA91/00337
2ogl980 `~
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
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
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
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
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
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
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
0 mN/m at minimum bubble size and to promote lung expansion
2Q91980
W092/~907 PCT/CA9l/~337
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
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
surfactants, problems relating to sterilization techniques,
suspension techniques, reproducibility of preparation and
surface properties have been considerable (Jobe et al. Am Rev
Resp Dis 136:1256-1275, 1987). The requirements for multiple
skills to not only prepare the surfactants but also to test
them both in vivo and in vitro have tested the ingenuity of
the investigators (Notter et al. J Appl Physiol 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 ~ppl 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 Dis 1365:1256-
1275, 1987; Robertson et al. Exp Lung 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
subjected to a series of differential and gradient
centrifugation steps in order to obtain crudely purified
W092/04907 PCT/CA91/00337
209 ~98 - 6 -
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
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
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;
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
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
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
W092/04907 2 0 9 1 9 8 0
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
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.
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
lS 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.
Am Rev Resp Dis 136:1256-127S, 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
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
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
3S of prior art methods of collecting and purifying natural
surfactant. A multiplicity of mammals are slaughtered
W09~ 9 8 0 ~20 9 I q 8 0 PCT/CA91/00337
-- 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
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
purifying natural surfactant collected from these mammals for
administration into mammalian alveolar spaces.
Disclosure of Invention
The invention provides a means for obtaining natural
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
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
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
complete the collection process enabling rapid extraction of
W092/04907 2 0 919 8 0 PCT/CA91/00337
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
S 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
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
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
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
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
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.
W092/04907 PCT/CA91/00337
2ogl98
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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
preparing a lipid extract surfactant suspension.
Best Mode For Carrying Out The Invent;on
The processes described herein for sequestering
bovine natural surfactant in a foam readily collectable from
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
similarly obtained from mammals such as horses, sheep,
rabbits, pigs, dogs and cats.
PrepArA~-;on of Bovine Stlrfactant F~Am in situ
In the preferred embodiment of the invention, a
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
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
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
MgCl2) are introduced into the trachea manually or by pump
means. For example, a vessel such as a jerrycan contA;n;ng
working solution is elevated in communication with the trachea
so that the required volume of saline flows into the animal's
W092/04907 2 0 919 ~ 0 PCT/CA91/00337
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
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
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
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.
Preferably, the animal's viscera are then removed
and the lungs are exposed. Preferably, surfaces of the ~ungs
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
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
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
collection of foam from a number of animals is complete.
Approximately five litres of foam are strained at a time.
W092/04907 PCT/CA91/00337
209~98~ ~
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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
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] KCl) is added to
give a chloroform:methanol:1.0% KCl 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
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
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
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.
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
resulting supernatant is decanted, and then evaporated under
reduced pressure to dryness on a rotary evaporator at least
W092/04907 2 0 919 8 0 PCT/CA91/00337
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twice. The resulting lipid extract is then taken up with a
small volume of chloroform (to give a lipid concentration of
approximately 150 mg/ml) and transferred to a 250 ml
centrifuge tube. The lipid extract is dried under nitrogen
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
precipitation step is repeated.
The final precipitate may be dissolved and stored
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
or administration into mammalian alveolar spaces.
Referring to Figure 2, to prepare a suspension of
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
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
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.
After drying, the lipid-coated tubes are lyophilized
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
bar, and aliquots thereof may be pipetted into sterile serum
vials, stoppered with slotted stoppers, and the solvent
W092/04907 PCT/CA91/~337
209 l98 - 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 C for
15 to 20 minutes to sterilize the aliquots, cooled, and stored
frozen at -20C.
Characterization of lipid extract surfactant
Biophysical activity
Dispersions of lipid extract surfactant in saline-
1.5 mM CaCl2 share the ability of natural surfactant to reducethe 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 ~M
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
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
(Enhorning J Appl Physiol 43:198-203, 1977; Yu et al. Lipids
18:522-529, 1983; Weber et al. Biochim Biophys Acta 796:83-91,
1984).
With the pulsating bubble surfactometer technique,
a bubble communicating with ambient air is created in a small
chamber. The bubble is 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
states that the difference in pressure across the bubble is
equal to two times the surface tension divided by the radius.
W092/04907 2 0 919 8 0 PCT/CA91/00337
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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
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
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;
Weber et al. Biochim Biophys 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 ~ Total Phosphorus
(n = 4)
Phosphatidylcholine 79.2 + 1.6
Phosphatidylglycerol 11.3 + 0.5
Phosphatidylinositol 1.8 + 0.3
35 Phosphatidylethanolamine 3.5 + 0.5
Lyso-bis-phosphatidic acid 1.5 + 0.4
Sphingomyelin 2.6 + 0.5
W092/04907 PCT/CA91/00337
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Table 2
Phospholipid Composition of
5Bovine 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-bis-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 ~l sample of
lipid extract surfactant is diluted to 500 ~l and 50 ~l
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
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%
phosphatidylethanolamine,0.1 to 3% lyso-bis-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 3.0%
W092/04gO7 2 0 319 8 0 PCT/CA91/00337
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-
choline, cholesterol and cholesterol esters than reported forprior 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. L~ag 160:195-206, 1982; U.S.
Patent No. 4,397,839, issued August 9, 1983; Berggren et al.
Exp Lung 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
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 Biophys Acta 431:447-458, 1976; Holm
et al. J Appl Physiol, 1987). In addition, the lipid extract
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
[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
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
dodecylsulphate as shown in Possmayer et al. (Can J Biochem
55:609-617, 1977) using bovine serum albumin as a standard.
W092/04907 PCT/CA91/00337
209198
- 18 -
The lipid does not interfere with samples having 2.0 mg 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
~g 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
gels with sodium dodecylsulphate shows decreased amounts of
serum proteins, no SP-A, only SP-B and SP-C (Possmayer Am Rev
Resp Dis 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
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
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
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
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.
W092/04907 2 0 919 8 0 PCT/CA91/00337
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Industrial Appli~hility
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.
