11 Taeush HW et al / Acta Pharmacol Sin 2002 Oct; 23 Supplement: 11-15

E-mail btaeusch@sfghpeds.ucsf.edu
2002, Acta Pharmacologica Sinica
ISSN 1671-4083
Shanghai Institute of Materia Medica
Chinese Academy of Sciences
http://www.ChinaPhar.com
Improving pulmonary surfactants
H William TAEUSCH, Karen LU, Daniela RAMIEREZ-SCHREMPP
Department of Pediatrics, University of California-San Francisco, San Francisco, CA 94110, USA
Since its introduction to clinical medicine in 1980
by Fujiwara and coworkers, surfactant therapy of res-
piratory distress syndrome (hyaline membrane disease)
has revolutionized the care of newborn infants in neo-
natal intensive care units
[1]
. Their work was based on
the work of many others stemming from the seminal
discovery of pulmonary surfactant by Clements in 1957.
Neonatologists (the older ones) now refer to the pre
and post surfactant eras in neonatology. Tab 1 lists
some of the major investigators and research milestones
in the forty plus years since that discovery.
Despite the overall success of surfactant treat-
ment, a small percentage of infants either respond tran-
siently or fail to respond. In addition, other pediatric
and adult lung diseases characterized by acute lung in-
jury are less responsive to surfactant treatment than
infants with RDS. A common denominator of non-
responsiveness is plasma leakage into airspaces and in-
activation of surfactant. Much work is now aimed at
finding surfactants that are resistant to inactivation, the
hypothesis being that more lung diseases will respond
to surfactant therapy.
There are four surfactants, (Exosurf, Survanta,
Curosurf, and Infasurf) now available and approved by
the Federal Drug Administration (FDA) for use in the
United States, and several others either available or un-
der development in the United States and Europe. Tab
2, 3 compare the compositions of the lipids and the
proteins of the currently available (in USA) therapeutic
surfactants with those of natural surfactant. To the
best of our knowledge, all surfactants are delivered in
0.9 % saline.
Alveofact (SF-RI 1, Thomae GmbH, Biberach/
Riss, Germany) is a chloroform-methanol extract of
surfactant obtained by broncho-alveolar cow lung
lavage. This surfactant is widely used in Germany.
Curosurf (Poractant, Chiesi Farmaceutici,
Parma, Italy) is a modified natural surfactant prepared
from minced porcine lungs by washing, centrifugation,
Tab 1. Highlights of surfactant research.
Pattle and Gruenwald Inferred the presence and importance
of surface active substance in the lung.
Clements Discovered and described mammalian surfactant.
Avery and Mead Found that infants dying of RDS have
impaired surface activity in lungs
Tierney First described surfactant inactivation
King and Clements Characterized biochemistry and surface
behavior of surfactant
Enhorning Along with Fujiwara, Robertson, and Adams
showed that surfactant replacement improved lung function
of immature animals
Gluck Amniotic fluid constituents reflects risk of RDS
Gregory, Tooley CPAP splints the surfactant deficient lung
and improves survival
Liggins Induction of lung maturity with glucocorticoids
Jobe and Ikegami Extensive studies on surfactant turnover
Fujiwara First successful use of surfactant treatment of in
fants
Hallman Characterized surfactant in adult RDS
Shapiro and Notter Developed antecedent of bLES and
Infasurf
Clements Invented Exosurf
Bangham and Morley Developed synthetic surfactant
Curstedt Along with Johannsen and Robertson developed
Curosurf
Whitsett Molecular biology of surfactant proteins
Hawgood Mechanisms of surfactant function
Taeusch HW et al / Acta Pharmacol Sin 2002 Oct; 23 Supplement: 11-15 12
extraction with 2:1 chloroform:methanol, and liquid-gel
affinity chromatography. Curosurf was approved by
the FDA on November, 1999
[2]
.
Exosurf: Glaxo-Wellcome, Burgwedel, Germany.
This surfactant is a synthetic protein-free surfactant
that was invented by John Clements. It was the first
surfactant approved for clinical use in the United States.
It is composed of dipalmitoyl phosphatidylcholine
(DPPC) mixed with cetyl alcohol and tyloxapol. Our
understanding is that marketing of this product is now
limited to countries other than the United States.
Infasurf (Calfactant) is manufactured for For-
est Pharmaceuticals, Inc, St Louis, MO, by ONY, Inc,
Amherst NY. It is an chloroform-methanol extract of
surfactant obtained from calf lung lavage.
KL-4 is a synthetic surfactant composed of
DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palm-
itic acid combined with a 21 amino acid synthetic pep-
tide that mimics structural characteristics of SP-B.
Repeat subunits of one lysine and four leucines form an
amphipathic helix.
Pumactant: (ALEC) Made in England by
Britannia, this is a synthetic surfactant composed of
DPPC and PG. Marketing is currently suspended be-
cause of results summarized below.
Survanta: (Beractant, Abbott Ltd, Ross Laborato-
ries, Columbus, OH). This is a modified natural sur-
factant prepared by lipid extraction of minced bovine
lung then addition of DPPC, palmitic acid, and tripalm-
itin are added to specified concentrations.
Venticute: rSP-C (Byk Gulden, Konstanz,
Germany) surfactant contains DPPC, PG, and palmitic
acid and 2 % rSP-C.
Surfactant TA: Similar to Survanta, it was the
first surfactant used clinically. It is made and marketed
only in Japan. It is a cow lung minced extract made by
Tokyo Tanabe Co (Tokyo, Japan).
A number of studies have been carried out com-
paring in vitro effects of some of these surfactants,
and composite results are shown in Tab 4. A more
stringent way of testing surfactants in vitro is to con-
sider their resistance to various inactivating agents. In
these experiments, surfactants are tested for surface
activity in a pulsating bubble surfactometer before and
after mixing them with various inactivating substances
(lipids, meconium, serum) that may coexist with sur-
factant in alveolar spaces after acute lung injury. Re-
sults from comparisons with inactivation are shown in
Tab 4. Note that all of the modified natural surfactants
are readily inactivated (surface tension >10 mN/m), but
that natural surfactant is the most resistant to inactiva-
tion.
In vitro studies are limited because of the unclear
relevance to in vivo activity and because of the speci-
ficity of the characteristics that are tested. For ex-
ample a group of investigators have measured suscep-
tibility to oxidation of surfactant surmising that oxida-
tion may not only denature surfactant but also may pro-
duce agonists that are proinflammatory
[3]
. Other in vitro
tests evaluate antiinfective capabilities, large aggregate
conversion rates, interactions with calcium, infrared
spectroscopy, thermal characteristics, electron micro-
Tab 2. Lipid composition (%, g/g of total lipid) of surfac-
tants available in the United States.
Native Curosurf Infasurf Survanta Exosurf
Surfactant (23) (25) (23) (29)
(21) (2) (26) (28) (5)
(5)(22) (24)(22) (27) (22)
PC 70-85 67-74 70-74 79-87 90
-DPPC 36-54 50-56 41-61 45-74 100
(%PC)
LPC 0.2 6.9 ? 2.2 0
SPH 2 8.1 2 4.8 0
CHOL 5 0 5 0 0
PI 4-7 3.3 ca. 2 0.5 0
PS 5 ? ca. 2 ? 0
PE 3 4.5 3 2.2 0
PG 7-10 1.2 6 3.2 0
Unknown - - 4.3 - -
Hexadecanol 0 0 0 0 10
Tyloxapol 0 0 0 0 7
Tab 3. Estimated average protein composition of surfac-
tants (g protein/mol L
-1
phospholipid) (24) (30) (2) (26)
(31).
Native Curosurf Infasurf Survanta Exosurf
Surfactant
SP-A 300 0 0 0 0
SP-D 42 0 0 0 0
SP-C 22-34 5-11.6 8.1 1-20 0
SP-B 10-11 2-3.7 5.4 0-1.3 0
13 Taeush HW et al / Acta Pharmacol Sin 2002 Oct; 23 Supplement: 11-15
scopic appearance, fluorescence and dark field micros-
copy
[4]
. Scanning probe microscopy and other tests of
surface activity such as adsorption rates in captive
bubble surfactometers and the respreading characteris-
tics of surface films
[5]
.
TESTING IN ANIMAL MODELS
Animal models can be viewed as belonging to one
of two categories. 1.Relatively pure surfactant
insufficiency, such as premature rabbits or lambs with
respiratory failure that mimics respiratory distress of
premature infants, or lung lavage models as popular-
ized by Lachmann wherein alveolar surfactant is re-
moved by multiple intratracheal saline lavages
[6]
. 2.
Acute lung injuries with the injurious agent causing in-
flammation and capillary leak of serum into the alveolar
spaces. These models are associated with surfactant
inactivation rather than surfactant deficiency. Such
experiments use oxygen, barotrauma, meconium, acid,
endotoxin or other agents, either singly or in combination.
The injuries resemble human acute lung injury (ALI)
and adult (acute) respiratory distress syndrome (ARDS).
A variety of generalizations can be drawn from
the many studies that fall within the categories named
above.
1. Modified natural surfactants give better re-
sponses than protein-free surfactants.
2. Animals with surfactant insufficiency respond
better to surfactant treatment than those with surfac-
tant inactivation.
3. Response to surfactant treatment is less, the
greater the degree of lung injury.
4. Native surfactant generally gives better re-
sponses than modified natural surfactants.
5. Surfactant preparations that contain higher
concentrations of SP-B and SP-C give better responses
than surfactants with lower concentrations.
For example, Cummings et al
[7]
compared natural
sheep surfactant, Exosurf, Infasurf, and Survanta with
no treatment in 29 newborn lambs at 126 d gestation.
All control lambs died within 8 h after delivery. Natural
surfactant, Survanta, and Infasurf prolonged survival
and improved arterial oxygenation (5-6greater than un-
treated controls) and ventilatory compliance. Limita-
tions of this study include large variability and small
sample sizes. Rider et al
[8]
tested surfactants contain-
ing surfactant proteins in preterm rabbits and found that
reconstituted surfactants containing SP-B gave results
comparable to those found with natural sheep surfactant.
Addition of SP-B to the surfactant lipids restored sur-
factant function, improved static and dynamic lung
mechanism, and also reduced protein leak into airspaces.
This effect was not reproduced with SP-A, and was
partially reproduced by SP-C. Kobayashi and cowork-
ers did a similar experiment in which they found that
mechanical properties of the lungs improved in a dose
related manner as increased concentrations of SP-B
were added to surfactant
[9]
. Animals that are given an-
tibodies to SP-B have immediate lung dysfunction indi-
cating surfactant inadequacy and mice with the SP-B
gene knocked out have lethal congenital lung dis-
ease
[10,11]
. Walther et al concluded from a study in a
surfactant-deficient rat model that addition of synthetic
SP-B SP-C to Survanta or to deproteinated Survanta
improved oxygenation and mechanical properties
[12]
.
Therefore surfactant proteins are critical for optimal
surfactant function under conditions of insufficiency
and inactivation.
FUTURE DIRECTIONS
A variety of ways in which surfactant treatment
may be improved are under study. Perfluorocarbons,
introduced into the lungs by aerosol or in liquid form,
with and without surfactant therapy, have been studied
for over twenty years. Fluorocarbons improve gas
exchange in some forms of acute lung injury by virtue
of both their oxygen and carbon dioxide carrying ca-
pacity and because of their surface properties. As yet,
a clear superiority of treatment with fluorocarbons over
surfactant treatment of clinical disease has not been
demonstrated
[13]
.
Other investigators are investigating the use of ei-
ther synthetic or recombinant surfactant proteins or
analogues. Control of protein concentration and the
ability to generate proteins that are free of animal con-
taminants are the goals for this approach. rSP-C sur-
factant is undergoing clinical trials of ARDS in this coun-
try and Europe
[14]
.
Still another path is the development of phospho-
lipid analogues that may improve surface activity of
surfactant as well as being resistant to phospholipase
degradation. Such formulations have been shown in
animal studies to be as effective as native surfactants
[5]
.
We and others have found that addition of non-
ionic polymers to various modified surfactants prevent
their inactivation (Column 4 in Tab 4), that is to say,
Taeusch HW et al / Acta Pharmacol Sin 2002 Oct; 23 Supplement: 11-15 14
shelf polymers can mimic some of the effects of sur-
factant proteins in improving surface activity and pre-
venting surfactant inactivation
[15-17]
. Animals with a
variety of lung injuries have improved gas exchange
and mechanical properties when treated with surfac-
tants containing these polymers
[18-20]
. Like SP-A and
calcium, polymers cause aggregation of surfactant lipids.
This mechanism may exclude soluble inhibitors from
interactions with surfactant lipids. Aggregation caused
by polymers may occur via osmotic effects and/or by
the ability of polymers to bind water. The osmolarity
of lower molecular weight polymers added to surfac-
tants may cause problems by reducing clearance of
pulmonary edema. Questions as to the best type of the
polymer (ionic or nonionic), concentration, molecular
weight, metabolism, and other effects (in vitro and in
vivo) all remain to be studied in detail. Nonetheless,
addition of polymers to surfactants affects surfactant
function offering one of several directions for improv-
ing treatment of diseases wherein surfactant inactiva-
tion plays a role.
ACKOWLEDGMENT Supported by NIH grant HL
66410.
NOTE This paper adapted from Choosing a Surfactant,
by Daniela Ramierez-Schrempp and H. William
Taeusch, published in Surfactant Therapy: Promoting
Best Practice, a symposium summary published by
Associates in Medical Marketing, 2002, for Ross
Laboratories, Columbus, Ohio, USA
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Surfactant Phospholipid Mimimum Minimum Minimum
Concentration surface surface surface
for optimal tension** tension tension
surface with with
activity* inactivation inactiation
** and with
polymer
***
Curosurf 1.3 41 180.5 2-9
Exosurf **** **** **** ****
Infasurf Not tested 51.0 161 5-11
Survanta 0.4 4 0.6 202 1-2
Natural 0.2 20.7 140.5 11-14
Surfactant
* g/L of phospholipid in 1.5 mmol/L calcium required to achieve
a surface tension of <10 mN/m after 5 min in a pulsating bubble
surfactometer (24).
** (mN/m) (32) Inactivating substance was serum.
*** Either dextran or polyethylene glycol (5 % w/w) was added
to the surfactant (32).
**** Surface tension did not become less than 10 mN/m.
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