U.S. patent application number 13/366144 was filed with the patent office on 2012-08-30 for surfactant protein d for the treatment of disorders associated with lung injury.
This patent application is currently assigned to CINCINNATI CHILDREN'S HOSPITAL MEDICAL CENTER. Invention is credited to Machiko Ikegami, Jeffrey A. Whitsett.
Application Number | 20120220531 13/366144 |
Document ID | / |
Family ID | 46719410 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120220531 |
Kind Code |
A1 |
Whitsett; Jeffrey A. ; et
al. |
August 30, 2012 |
SURFACTANT PROTEIN D FOR THE TREATMENT OF DISORDERS ASSOCIATED WITH
LUNG INJURY
Abstract
Surfactant protein D (SP-D) is a member of the collectin family
of collagenous lectin domain-containing proteins that is expressed
in epithelial cells of the lung. Described herein are methods and
compositions for the treatment of disorders associated with lung
injury, including methods and compositions for the treatment of
bronchopulmonary disorder (BPD).
Inventors: |
Whitsett; Jeffrey A.;
(Cincinatti, OH) ; Ikegami; Machiko; (Cincinatti,
OH) |
Assignee: |
CINCINNATI CHILDREN'S HOSPITAL
MEDICAL CENTER
Cincinatti
OH
|
Family ID: |
46719410 |
Appl. No.: |
13/366144 |
Filed: |
February 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61439760 |
Feb 4, 2011 |
|
|
|
Current U.S.
Class: |
514/15.5 |
Current CPC
Class: |
A61K 38/395 20130101;
A61K 38/1732 20130101; A61P 11/00 20180101; A61K 38/395 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/15.5 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61P 11/00 20060101 A61P011/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0003] This invention was made with government support under
HL08561 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for reducing the risk of developing bronchopulmonary
dysplasia (BPD), comprising administering a composition comprising
recombinant human SP-D (rhSP-D) and a pulmonary surfactant to a
subject in need thereof in an amount effective to reduce the risk
of developing BPD.
2. The method of claim 1, wherein the BPD is associated with lung
injury from mechanical ventilation.
3. The method of claim 1, wherein the composition further comprises
at least one protein selected from the group consisting of
surfactant protein A (SP-A), surfactant protein B (SP-B),
surfactant protein C (SP-C), and fragments and mimics thereof.
4. The method of claim 1, wherein the pulmonary surfactant is a
synthetic surfactant protein.
5. The method of claim 1, wherein the dose of the rhSP-D is about
0.1 mg to about 10 mg/kg body weight of the subject.
6. The method of claim 1, wherein the dose of the rhSP-D is about 2
mg/kg body weight.
7. The method of claim 6, wherein the total dose of the composition
is about 100 mg/kg body weight of the subject.
8. The method of claim 1, wherein the composition is administered
intratracheally.
9. The method of claim 1, further comprising selecting a subject at
risk for developing BPD prior to administering the composition to
the subject.
10. The method of claim 1, wherein the subject is an infant.
11. A method of reducing pulmonary injury associated with
ventilation in a subject, comprising administering recombinant
human surfactant protein D (rhSP-D) and a pulmonary surfactant to
the subject in an amount effective to reduce pulmonary injury
associated with the ventilation.
12. The method of claim 11, wherein the ventilation is mechanical
ventilation.
13. The method of claim 11, wherein the composition further
comprises at least one protein selected from the group consisting
of surfactant protein A (SP-A), surfactant protein B (SP-B),
surfactant protein C (SP-C), and fragments and mimics thereof
14. The method of claim 11, wherein the pulmonary surfactant is a
synthetic surfactant protein.
15. The method of claim 11, wherein the dose of the rhSP-D is about
0.1 mg to about 10 mg/kg body weight of the subject.
16. The method of claim 11, wherein the dose of the rhSP-D is about
2 mg/kg body weight of the subject.
17. The method of claim 16, wherein the total dose of the
composition is about 100 mg/kg body weight of the subject.
18. The method of claim 11, wherein the composition is introduced
intratracheally.
19. The method of claim 11, further comprising selecting a subject
at risk for developing BPD prior to administering the composition
to the subject.
20. The method of claim 11, wherein the subject is an infant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/439,760, filed on Feb. 4, 2011, which is
incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CHMC7.sub.--003A, created Feb. 3, 2012, which is 12
KB in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
BACKGROUND
[0004] Surfactant protein D (SP-D) is a member of the collectin
family of collagenous lectin domain-containing proteins that is
expressed in epithelial cells of the lung.
SUMMARY OF THE INVENTION
[0005] Described herein are methods and compositions for the
treatment of disorders associated with lung injury, including
methods and compositions for the treatment of bronchopulmonary
disorder (BPD).
[0006] In one embodiment, a method for reducing the risk of
developing bronchopulmonary dysplasia (BPD) is provided, comprising
administering a composition comprising recombinant human SP-D
(rhSP-D) and a pulmonary surfactant to a subject in need thereof in
an amount effective to reduce the risk of developing BPD. In some
aspects, the BPD is associated with lung injury from mechanical
ventilation. In some aspects, the composition further comprises at
least one protein selected from the group consisting of surfactant
protein A (SP-A), surfactant protein B (SP-B), surfactant protein C
(SP-C), and fragments and mimics thereof. In some aspects, the
pulmonary surfactant is a synthetic surfactant protein. In some
aspects, the dose of the rhSP-D is about 0.1 mg to about 10 mg/kg
body weight of the subject. In some aspects, the dose of the rhSP-D
is about 2 mg/kg body weight. In some aspects, the total dose of
the composition is about 100 mg/kg body weight of the subject. In
some aspects, the composition is administered intratracheally. In
some aspects, the method further comprises selecting a subject at
risk for developing BPD prior to administering the composition to
the subject. In some aspects, the subject is an infant.
[0007] In another embodiment, a method of reducing pulmonary injury
associated with ventilation in a subject is provided, comprising
administering recombinant human surfactant protein D (rhSP-D) and a
pulmonary surfactant to the subject in an amount effective to
reduce pulmonary injury associated with the ventilation. In some
aspects, the ventilation is mechanical ventilation. In some
aspects, the composition further comprises at least one protein
selected from the group consisting of surfactant protein A (SP-A),
surfactant protein B (SP-B), surfactant protein C (SP-C), and
fragments and mimics thereof. In some aspects, the pulmonary
surfactant is a synthetic surfactant protein. In some aspects, the
dose of the rhSP-D is about 0.1 mg to about 10 mg/kg body weight of
the subject. In some aspects, the dose of the rhSP-D is about 2
mg/kg body weight of the subject. In some aspects, the total dose
of the composition is about 100 mg/kg body weight of the subject.
In some aspects, the composition is introduced intratracheally. In
some aspects, the method further comprises selecting a subject at
risk for developing BPD prior to administering the composition to
the subject. In some aspects, the subject is an infant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows that treatment with recombinant human
surfactant protein D (rhSP-D) does not alter lung physiology in
premature lambs. Premature newborn lambs were resuscitated after
birth by ventilation with a peak inspiratory pressure (PIP) of
H.sub.2O, resulting in a mean Pco.sub.2 of 40 mm Hg (B) and a mean
V.sub.T of 11 ml/kg (C) for rhSP-D treated lambs and controls.
Surfactant was given at 20 minutes of age and ventilation was
changed to regulate V.sub.T at 8 to 9 ml/kg (C), requiring a mean
PIP of 27 cm H.sub.2O (A) for rhSP-D treated lambs and
controls.
[0009] FIG. 2 shows the effects of rhSP-D treatment on lung
function. (A) The modified ventilation index (MVI) was calculated
as peak inspiratory pressure.times.P.sub.CO2.times.respiratory
rate/1,000. Although not significant, MVI tends to be better
(lower) for the group treated with rhSP-D (+rhSP-D) at later times.
(B) Po.sub.2/Fio.sub.2 was higher in the +rhSP-D group compared
with the control group (*P<0.01 by two-way repeated measures
analysis of variance (ANOVA) (overall comparison of control versus
+rhSP-D group)). Po.sub.2/Fio.sub.2 was significantly decreased
after 210 minutes in the control group (P<0.05 vs. 18 min by
one-way ANOVA).
[0010] FIG. 3 shows that treatment with rhSP-D does not alter
pressure-volume curves or lung histology in premature lambs. (A)
The deflation limbs of pressure-volume curves were not different
between the rhSP-D treated lambs and controls. (B, C) Lung
histology assessed after staining with hematoxylin and eosin was
similar for both groups. Histology was typical of immature lung,
including thickened alveolar septal walls and patchy atelectasis.
More alveolar fluid was observed in control lambs than in lambs
treated with rhSP-D (+rhSP-D). Scale bar: 100 .mu.m.
[0011] FIG. 4 shows that treatment with rhSP-D decreases the number
of inflammatory cells in BALF and decreases neutrophil elastase
(NE) activity. (A) Increased total inflammatory cells and
neutrophils in BALF induced by ventilation were suppressed by
rhSP-D. (B) NE activity was assessed by a spectrophotometric assay
using a chromogenic substrate specific for NE. Treatment with
rhSP-D-containing SURVANTA.RTM. decreased NE activity (*P<0.05
versus the control group).
[0012] FIG. 5 shows that treatment with rhSP-D decreases the number
of proinflammatory markers in lung homogenates. (A) Increased
expression of interleukin-8 (IL-8) mRNA was significantly
suppressed by rhSP-D treatment. Although not significant (due to
the large variation in the control group), mean values of
interleukin-6 (IL-6), interleukin-1.beta. (IL-1.beta.),
keratinocyte-derived chemokine (KC), and monocyte chemotactic
protein 1 (MCP1) were generally lower in the +rhSP-D group. (B)
IL-8 protein in lung homogenates was significantly decreased by
rhSP-D treatment (*P<0.05 versus the control group), while
IL-1.beta. was not influenced by rhSP-D treatment.
[0013] FIG. 6 shows that the addition of rhSP-D to surfactant
increased resistance against surfactant inhibition. (A) Surface
tension was measured by a captive bubble surfactometer.
SURVANTA.RTM.+buffer had high surface activity, and minimum surface
tension was low and was not influenced by addition of rhSP-D.
Plasma protein inhibited the surface tension-lowering properties of
SURVANTA.RTM., and minimum surface tension was increased. The
addition of rhSP-D rendered the SURVANTA.RTM. more resistant to
plasma protein inhibition, with low minimum surface tension in the
presence of plasma protein (n=3, *P<0.05 versus
SURVANTA.RTM.+buffer with plasma). (B, C) Representative electron
micrographs of SURVANTA.RTM. mixed with buffer or rhSP-D. Addition
of rhSP-D changed the ultrastructure of SURVANTA.RTM. from simple
lipid layers to the mixture of multiple lipid layers and lipid
aggregates (n=3 per group). Scale bar: 500 nm.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Premature newborns are routinely resuscitated by manual
ventilation in the delivery room, followed by mechanical
ventilation and surfactant treatment in the neonatal intensive care
unit. The premature lung requires high inflating pressures and
oxygen for adequate ventilation and oxygenation and is highly
susceptible to injury because of its structural immaturity,
surfactant deficiency, presence of fetal lung fluid, and immature
immune system--factors that are likely to contribute to the
development of the chronic lung disease bronchopulmonary dysplasia
(BPD). Surfactant treatment is routinely given to very low birth
weight (i.e., <1,500 g) preterm infants as early as possible
after birth for the purpose of resuscitation in an effort to
prevent and/or treat neonatal respiratory distress.
[0015] As described herein, rhSP-D can be added to resuscitation
surfactant to improve surfactant distribution, minimize inhibition
of surfactant function by leaked proteins, and prevent
bronchopulmonary dysplasia (BPD)--a frequent consequence of the
resuscitation process. Some embodiments relate to methods and
compositions for the treatment of disorders associated with lung
injury, including BPD. In one embodiment, recombinant human
surfactant protein D (rhSP-D) is given in combination with a
surfactant formulation to a mammal in need of treatment for a lung
disorder. In some embodiments, the methods described herein reduce
the risk of developing BPD. In some embodiments, the methods
described herein reduce pulmonary injury associated with
ventilation. In some embodiments, the methods described herein
reduce pulmonary injury associated with the administration of
oxygen, including high levels of oxygen. In some embodiments, the
methods described herein reduce pulmonary injury associated with
infection, such as intra-uterine infection.
[0016] In some embodiments, the mammal is a human. The human can
be, e.g., an adult, a child, or an infant. In some embodiments, the
infant is a newborn infant or a premature newborn infant. In some
embodiments, the premature newborn infant is born at about 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 weeks
gestational age. In some embodiments, the newborn infant has a low
birth weight. For example, in some embodiments, the birth weight of
the newborn infant is less than about 500, 600, 700, 800, 900,
1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200, 2300, 2400, or 2500 grams. In a preferred embodiment,
the mammal is a premature newborn infant with respiratory disease
(such as neonatal respiratory distress syndrome (nRDS)), or an
infant at risk of developing or with symptoms of BPD.
[0017] In the embodiments described herein, a surfactant
formulation can encompass one or more proteins, protein fragments,
or mimics thereof. For example, in some embodiments, the surfactant
formulation contains surfactant protein A (SP-A), and/or surfactant
protein B (SP-B), and/or surfactant protein C (SP-C). In some
embodiments, the surfactant formulation contains a combination of
surfactant proteins, such as the combination of SP-B and SP-C. In
some embodiments, the surfactant formulation contains a fragment of
a surfactant protein. In some embodiments, the surfactant
formulation contains lipids. For example, in some embodiments, the
surfactant formulation contains dipalmitoylphosphatidylcholine
(DPPC). In some embodiments, the formulation contains DPPC and at
least one of phosphatidylglycerol (PG) and phosphatidylinositol
(PI). In a preferred embodiment, the surfactant formulation
contains SP-B, SP-C, and DPPC.
[0018] In some embodiments, the surfactant formulation contains an
animal derived surfactant. In some embodiments, the animal derived
surfactant is a commercially available surfactant, such as
ALVEOFACT.RTM., CUROSURF.RTM., INFASURF.RTM., or SURVANTA.RTM.. In
some embodiments, the animal derived surfactant is BLES.RTM.,
SURFACEN.RTM., or CLSE.RTM.. In some embodiments, the surfactant
formulation contains a synthetic surfactant. In some embodiments,
the synthetic surfactant is a commercially available synthetic
surfactant, such as EXOSURF.RTM., PUMACTANT.RTM., SURFAXIN.RTM.,
AEROSURF.RTM., VENTICUTE.RTM., or CHF 5633. In some embodiments, a
combination treatment of rhSP-D and an animal surfactant is
provided. In some of these embodiments, the animal surfactant
contains at least one surfactant protein and at least one lipid. In
some embodiments, a combination treatment of rhSP-D and a synthetic
surfactant is provided. In some of these embodiments, the synthetic
surfactant contains at least one recombinant protein, at least one
surfactant protein fragment or mimic of a surfactant protein, and
at least one lipid.
[0019] In some embodiments, the surfactant formulation contains a
purified surfactant protein. In some embodiments, the surfactant
formulation contains a recombinant protein. In some embodiments,
the surfactant formulation contains a surfactant protein that is
not SP-D. In some embodiments, the surfactant formulation does not
contain a surfactant protein. In some embodiments, the surfactant
formulation contains a lipoprotein complex. For example, in some
embodiments, the surfactant formulation contains a
phospholipoprotein complex.
[0020] The compositions described herein can be administered by any
suitable route, including orally, intratracheally, parenterally, by
inhalation spray, rectally, or topically in dosage unit
formulations containing conventional pharmaceutically acceptable
carriers, adjuvants, and vehicles. As used herein, the term
"parenteral" includes subcutaneous, intravenous, intraarterial,
intramuscular, intrasternal, intratendinous, intraspinal,
intracranial, intrathoracic, infusion, or intraperitoneal
administration. In a preferred embodiment, a composition described
herein is administered intratracheally. In another preferred
embodiment, a composition described herein is administered in an
aerosolized form. For example, in one embodiment, rhSP-D and a
surfactant formulation are administered as an aerosolized product
that is administered by mask or continuous positive airway pressure
(CPAP).
[0021] The compositions described herein can be administered as a
single dose or in multiple doses. In some embodiments, the
composition is administered once. In some embodiments, the
composition is administered more than once. In a preferred
embodiment, the composition is administered to a premature newborn
infant in one or two doses. In some embodiments, rhSP-D and a
surfactant formulation are each administered once per day. In some
embodiments, rhSP-D and a surfactant formulation are administered
together once per day. In some embodiments, rhSP-D and a surfactant
formulation are administered together more than once per day.
[0022] In some embodiments, one or both of rhSP-D and a surfactant
formulation is administered one, two, three, four, or more times
per day. However, either or both can be administered less than once
per day, e.g., about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, or 14 days.
[0023] Suitable dose ranges vary, but in general, the rhSP-D can be
administered in a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight. In a
preferred embodiment, the rhSP-D is provided in a dose of about 1
mg/kg to about 2 mg/kg body weight. In general, the surfactant
formulation can be administered in a dose of about 25, 50, 75, 100,
125, 150, 175, 200, 300, 400, or 500 mg/kg body weight. In a
preferred embodiment, the surfactant formulation is provided in a
dose of about 100 mg/kg to about 200 mg/kg body weight. For
example, in a preferred embodiment, the rhSP-D is provided in a
dose of about 2 mg/kg body weight, and the surfactant formulation
is provided in a dose of about 100 mg/kg body weight. In another
preferred embodiment, the rhSP-D is provided in a dose of about 7
mg/kg body weight, and the surfactant formulation is provided in a
dose of about 100 mg/kg body weight. In some embodiments, the dose
of rhSP-D is about 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%,
2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%,
or 5% of the total dose of a composition. For example, in a
preferred embodiment, the dose of rhSP-D is 2 mg/kg body weight in
a total dose of 100 mg/kg body weight (i.e., 2%) of rhSP-D and a
surfactant formulation.
[0024] The selection of a particular dose may be based on the
weight or identity of a mammal, the dose, and/or the dosing
schedule of another co-administered compound. However, in some
embodiments, it may be necessary to use doses outside the ranges
described above. In some embodiments, the daily dosage of rhSP-D
and a surfactant formulation are the same, and in some embodiments,
the daily dosages vary. In some embodiments, the rhSP-D and
surfactant formulation are administered together. In some
embodiments, the rhSP-D and surfactant formulation are administered
separately. In some embodiments, the daily dosage of a composition
described herein is administered in a single dosage form. In some
embodiments, the daily dosage of a composition described herein is
administered in multiple dosage forms.
[0025] In some embodiments, at least one of rhSP-D and a surfactant
formulation is administered in consistent daily dosages throughout
the period of treatment. In some embodiments, at least one of
rhSP-D and a surfactant formulation is administered in varying
daily dosages during the period of treatment. In some of these
embodiments, the daily dosages comprise increasing daily dosages
over time. In some of these embodiments, the daily dosages comprise
decreasing daily dosages over time.
[0026] In some embodiments, the dosage is adjusted so that the
mammal maintains or exhibits reduced symptoms of a disorder. For
example, in some embodiments, the dosage is adjusted so that a
patient exhibits a reduction in symptoms of BPD. However, the
dosage may also be adjusted by a treating physician based on a
patient's particular needs. Further, the exact formulation, route
of administration, and dosage can be chosen by a physician in view
of the patient's condition.
[0027] In some embodiments, at least one of rhSP-D and a surfactant
formulation is administered with varying frequency during
treatment. In some of these embodiments, the varying frequency
comprises a decreased frequency over time. For example, one or both
of rhSP-D and the surfactant formulation can be initially
administered more than once per day, followed by administration
only once per day at a later point in treatment. In some
embodiments, the daily dosage of at least one of rhSP-D and a
surfactant formulation is consistent despite the varying frequency
of administration.
[0028] In some embodiments, rhSP-D and a surfactant formulation are
administered in a single pharmaceutical composition, such as a
pharmaceutical composition comprising rhSP-D, a purified surfactant
protein, a lipid, and a pharmaceutically acceptable carrier.
[0029] In some embodiments, administration is continued for a
certain amount of time or until a particular outcome is achieved.
For example, in some embodiments, administration of the
compositions provided herein is continued for a period of at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, 48,
72, 84, 96, 108, or 120 hours. In some embodiments, administration
of rhSP-D and a surfactant formulation is continued until the
reduction in symptoms of respiratory distress is stabilized for a
period of at least about 12, 16, 20, 24, 36, 48, 72, 84, 96, 108,
or 120 hours. In a preferred embodiment, symptoms of respiratory
distress are stabilized for a period of about 72 hours to about 96
hours. In some embodiments, administration is continued for the
duration of the life of a mammal. For example, in some embodiments,
administration is continued daily, weekly, or monthly for the life
of a human.
[0030] The compositions described herein may be accompanied by
instructions for administration. Such notice, for example, may be
the labeling approved by the U.S. Food and Drug Administration for
prescription drugs, or an approved product insert. Compositions
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
[0031] Instructions and/or information may be present in a variety
of forms, including printed information on a suitable medium or
substrate (e.g., a piece or pieces of paper on which the
information is printed), computer readable medium (e.g., diskette,
CD, etc., on which the information has been recorded), or a website
address that may be accessed via the internet. Printed information
may, for example, be provided on a label associated with a drug
product, on the container for a drug product, packaged with a drug
product, or separately provided apart from a drug product, or
provided in a manner in which a patient can independently obtain
the information (e.g., a website). Printed information may also be
provided to a medical caregiver involved in treatment of a
patient.
[0032] The compositions described herein can be provided prior to,
simultaneously with, or subsequent to ventilation and/or oxygen
treatment. In some embodiments, the mammal receives ventilation
and/or oxygen treatment for a period of time prior to receiving a
composition. For example, in some embodiments, the mammal receives
ventilation and/or oxygen treatment for about 1, 2, 3, 4, 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 minutes
prior to receiving a composition. In some embodiments, the mammal
receives ventilation and/or oxygen treatment for about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, or 48 hours prior to receiving a
composition. In a preferred embodiment, a premature infant receives
ventilation immediately following birth, and treatment with rhSP-D
and a surfactant formulation within about 20 minutes of birth. For
example, a premature infant can be intubated with an endotracheal
tube and placed on a ventilator at birth, then receive rhSP-D and a
surfactant formulation through the endotracheal tube about 20
minutes following birth. In some embodiments, the ventilation is
manual ventilation. In some embodiments, the ventilation is
mechanical ventilation. In some embodiments, the ventilation is
both manual and mechanical. For example, in some embodiments, a
premature infant is resuscitated by manual ventilation in the
delivery room, followed by mechanical ventilation and treatment
with rhSP-D and a surfactant formulation in the neonatal intensive
care unit. In some embodiments, the mammal receives a composition
about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
75, 90, 105, or 120 minutes prior to ventilation and/or oxygen
treatment for a lung disorder. In some embodiments, the mammal
receives a composition about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to
treatment for a lung disorder. For example, in some embodiments, a
patient with cystic fibrosis receives a composition within an hour
of undergoing ventilation treatment. However, this timeframe can be
adjusted by a treating physician based on a patient's particular
needs.
[0033] In some embodiments, the mammal receives alternating
treatment with a composition and ventilation and/or oxygen. For
example, in some embodiments, the mammal receives ventilation,
followed by a composition, followed by ventilation. In some
embodiments, the mammal receives alternating and simultaneous
treatment with a composition and ventilation and/or oxygen. For
example, in some embodiments, the mammal receives a composition,
followed by ventilation, followed by the composition and
ventilation.
[0034] In some embodiments, the mammal is administered a
composition described herein within a defined period of time
following birth. In some embodiments, the mammal is administered a
composition immediately following birth. In some embodiments, the
mammal is administered a composition within about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105,
or 120 minutes of birth.
[0035] The term "treatment" can include any intervention performed
with the intention of preventing the development or altering the
pathology of a disorder. Accordingly, "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with a disorder,
as well as those in which the disorder is to be prevented. In some
embodiments, the compositions described herein are useful to reduce
the risk of developing BPD. In some embodiments, the compositions
described herein are useful for reducing pulmonary inflammation
associated with lung injury. In some embodiments, the compositions
described herein are useful for reducing the symptoms of BPD
resulting from lung injury.
[0036] The terms "protein," "polypeptide," and "peptide" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms can apply to amino acid polymers in which one
or more amino acid residue is an analog or mimic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be produced via several
methods known in the art. For example, polypeptide products can be
biochemically synthesized by employing standard solid phase
techniques. Such methods include, but are not limited to, exclusive
solid phase synthesis, partial solid phase synthesis methods,
fragment condensation, and classical solution syntheses known to
those of skill in the art. Polypeptides can also be generated using
recombinant techniques known to those of skill in the art. For
example, polypeptides can be synthesized by cloning a
polynucleotide comprising the cDNA of a gene into an expression
vector and culturing the cell harboring the vector to express the
encoded polypeptide. In addition, polypeptides can be purified
using methods known to those of skill in the art, including
preparative high performance liquid chromatography. As used herein,
the term "purified" does not require absolute purity; rather, it is
intended as a relative definition. In some embodiments, a
polypeptide is about 75%, 80%, 85%, 90%, 95%, or 99% pure.
Polypeptides can also be modified, e.g., by the addition of
carbohydrate residues to form glycoproteins. The terms "protein,"
"polypeptide," and "peptide" include glycoproteins, as well as
non-glycoproteins.
[0037] In some embodiments, the rhSP-D has the amino acid sequence
of SEQ ID NO: 1. In some embodiments, the rhSP-D has an amino acid
sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some
embodiments, the rhSP-D comprises the amino acid sequence of SEQ ID
NO: 2 (NP.sub.--003010.4). In some embodiments, the rhSP-D
comprises an amino acid sequence that is at least 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to
SEQ ID NO: 2 (precursor sequence of NP.sub.--003010.4). In some
embodiments, the rhSP-D is encoded by the nucleic acid sequence of
SEQ ID NO: 3 (NM.sub.--003019). In some embodiments, the rhSP-D is
encoded by a nucleic acid sequence that is at least 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to
SEQ ID NO: 3. In some embodiments, the rhSP-D is expressed from a
vector containing the nucleic acid sequence of SEQ ID NO: 4. In
some embodiments, the rhSP-D is expressed from a vector containing
a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
4. As will be appreciated by one of skill in the art, particular
motifs and the degree of conservation between species provides
guidance as to areas of rhSP-D that are important for structure and
function (and are therefore not good candidates for varying from
the sequences provided herein), and areas of variation that do not
need to be conserved in the same manner.
[0038] The compositions described herein can include
pharmaceutically acceptable carriers, such as adjuvants,
excipients, and/or stabilizers that are nontoxic to the cell or
mammal being exposed thereto at the doses and concentrations
employed. In some embodiments, the pharmaceutically acceptable
carrier is an aqueous pH buffered solution. For example, in some
embodiments, compositions are pH adjusted with sodium bicarbonate.
Examples of pharmaceutically acceptable carriers include, but are
not limited to, adjuvants, lipids, preservatives, stabilizers,
wetting agents, emulsifiers, and buffers. In some embodiments, the
compositions contain a lipid. For example, in some embodiments, the
compositions contain phosphatidylcholine (PC),
dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG),
phosphatidylethanolamine (PE), phosphatidylinositol (PI),
sphingomyelin, tripalmitoylglycerol, palmitic acid, or mixtures
thereof, In a preferred embodiment, compositions contain a rhSP-D,
a surfactant protein, DPPC, and PI. Further, compositions can be
prepared in solid form (including granules, powders or
suppositories) or liquid form (e.g., solutions, suspensions, or
emulsions). For example, in some embodiments, compositions are
suspended in sodium chloride solution. In a preferred embodiment,
the composition is in an aerosolized formulation.
EXAMPLE 1
rhSP-D Treatment in Premature Lambs
[0039] Premature lambs were delivered by cesarean section at 130
days' gestational age (GA) (full term is 150 days GA) and
tracheostomized as previously described (Kramer B W, et al.
Surfactant protein A recruits neutrophils into the lungs of
ventilated preterm lambs. Am J Respir Crit Care Med 2001;
163:158-165; Ikegami M, et al. Intratracheal recombinant surfactant
protein D prevents endotoxin shock in the newborn preterm lamb. Am
J Respir Crit Care Med 2006; 173:1342-1347). Premature newborn
lambs were resuscitated with 100% O.sub.2, a peak inspiratory
pressure (PIP) of 40 cm H.sub.2O, 4 cm H.sub.2O positive
end-expiratory pressure (PEEP), and a respiratory rate of 40/min
using a pressure-limited ventilator (Sechrist Industries, Anaheim,
Calif.). To avoid overstretch of the premature newborn infant lung
during manual ventilation, the clinical resuscitation bag has a
pressure relief valve set at 40 cm H.sub.2O, and therefore PIP for
resuscitation was limited to 40 cm H.sub.2O.
[0040] Premature lambs at 130 days GA require surfactant treatment
to survive. At 20 minutes of age, two groups of lambs were treated
with SURVANTA.RTM. (Abbott Laboratories, Columbus, Ohio) mixed with
rhSP-D (SEQ ID NO: 1) (+rhSP-D group) or buffer (control group)
using two boluses for instillation (Ueda T, et al. Distribution of
surfactant and ventilation in surfactant-treated preterm lambs. J
Appl Physiol 1994; 76:45-55). rhSP-D was synthesized as previously
described (Ikegami M, et al. Intratracheal recombinant surfactant
protein D prevents endotoxin shock in the newborn preterm lamb. Am
J Respir Crit Care Med 2006; 173:1342-1347; Ikegami M, et al.
Surfactant protein-D and surfactant inhibit endotoxin induced
pulmonary inflammation. Chest 2007; 132:1447-1454; Ikegami M, et
al. Surfactant protein-D regulates the postnatal maturation of
pulmonary surfactant lipid pool sizes. J Appl Physiol 2009;
106:1545-1552). Seven milligrams of rhSP-D in 5 ml buffer (20 mM
Tris, 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid, pH 7.4) or
5 ml buffer alone were mixed with a clinical treatment dose of
SURVANTA.RTM. (100 mg/4 ml/kg)--amounts that are similar to both
SP-D and surfactant lipid pool sizes in the normal term newborn
lung (Ikegami M, et al. Surfactant protein-D regulates the
postnatal maturation of pulmonary surfactant lipid pool sizes. J
Appl Physiol 2009; 106:1545-1552; Ikegami M, Jobe A H. Surfactant
metabolism. Semin Perinatol 1993; 17:233-240). After surfactant
treatment, the PIP was decreased to regulate tidal volume (V.sub.T)
at 8 to 9 ml/kg (Bicore Monitoring Systems, Anaheim, Calif.), and
F.sub.iO2 was adjusted to maintain a target P.sub.02 of 100 to 150
mm Hg. Ventilatory rate, inspiratory time of 0.6 seconds, and PEEP
were not changed.
[0041] A five hour study period was chosen to detect changes in
proinflammatory cytokine mRNAs induced by initial ventilation (Naik
A S, et al. Effects of ventilation with different positive
end-expiratory pressures on cytokine expression in the preterm lamb
lung. Am J Respir Crit Care Med 2001; 164:494-498). After five
hours, lambs were ventilated with F.sub.iO2=1 for five minutes,
then given 100 mg pentobarbital intravascularly, after which the
endotracheal tube was clamped to permit oxygen absorption
atelectasis (Ikegami M, et al. Intratracheal recombinant surfactant
protein D prevents endotoxin shock in the newborn preterm lamb. Am
J Respir Crit Care Med 2006; 173:1342-1347; Ikegami M, Jobe A.
Postnatal lung inflammation increased by ventilation of preterm
lambs exposed antenatally to E.coli endotoxin. Pediatr Res 2002;
52:356-362). After the thorax was opened, the deflation limb of
pressure-volume curve was measured (Kramer B W, et al. Surfactant
protein A recruits neutrophils into the lungs of ventilated preterm
lambs. Am J Respir Crit Care Med 2001; 163:158-165; Ikegami M, et
al. Intratracheal recombinant surfactant protein D prevents
endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care
Med 2006; 173:1342-1347). Lung tissue of the right lower lobe was
frozen in liquid nitrogen for RNA isolation and measurement of
neutrophil elastase (NE) activity (Lowry O H, et al. Protein
measurement with the Folin phenol reagent. J Blot Chem 1951;
193:265-275; Watterberg K L, et al. Secretory leukocyte protease
inhibitor and lung inflammation in developing bronchopulmonary
dysplasia. J Pediatr 1994; 125:264-269). NE is a potent serine
proteinase, responsible for tissue destruction in the adult lung
with emphysema (Shapiro S D, et al. Neutrophil elastase contributes
to cigarette smoke-induced emphysema in mice. Am J Pathol 2003;
163:2329-2335). In the preterm newborn lung, increased NE activity
affects lung remodeling and increases alveolar epithelial apoptosis
and the development of BPD (Watterberg K L, et al. Secretory
leukocyte protease inhibitor and lung inflammation in developing
bronchopulmonary dysplasia. J Pediatr 1994; 125:264-269; Yasumatsu
R, et al. SERPINB1 upregulation is associated with in vivo complex
formation with neutrophil elastase and cathepsin G in a baboon
model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol
Physiol 2006;291:L619-L627). NE activity was assessed by a
spectrophotometric assay using a chromogenic substrate specific for
NE, N-methoxy-succinyl-Ala-Ala-Pro-Val pNA (Yasumatsu R, et al.
SERPINB1 upregulation is associated with in vivo complex formation
with neutrophil elastase and cathepsin G in a baboon model of
bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol
2006; 291:L619-L627).
[0042] Sequences of primers for quantitative reverse
transcriptase-polymerase chain reaction were: IL-8:
5'-TGGCCAGGATTCACGAGTTC (SEQ ID NO:5) and
5'-TCTGTGAGGTAGAAAGATGACTGAGATATT (SEQ ID NO:6); IL-6:
5'-GGAGGAAAAAGATGGATGCTTCCAA (SEQ ID NO:7) and
5'-CAGCAGTGGTTTTGATCAAGCAA (SEQ ID NO:8); IL-113:
5'-GGCTCTCCACCTCCTCTCA (SEQ ID NO:9) and 5'-AGCTCATGCAGAACACCTT
(SEQ ID NO:10); tumor necrosis factor (TNF)-.alpha.:
5'-GCCGGAATACCTGGACTATGC (SEQ ID NO:11) and
5'-CAGGGCGATGATCCCAAAGTAG (SEQ ID NO:12); keratinocyte-derived
chemokine (KC): 5'-TGCCAGTGCCTGCAGAC (SEQ ID NO:13) and
5'-AGTGGCTATGACTTCGGTTTGG (SEQ ID NO:14); monocyte chemotactic
protein 1 (MCP1): 5'-CCCCGACTATCTGTTTCCACAAC (SEQ ID NO:15) and
5'-CCTGGAAGGGCTTCTGATCTG (SEQ ID NO:16); and ovine ribosomal
protein L32 5'-GCAGAAGATTCAAGGGCCAGATC (SEQ ID NO:17) and
5'-GGTTTTCTTGTTGCTCCCGTAAC (SEQ ID NO:18).
[0043] Lung tissue of the right middle lobe was homogenized in 0.9%
NaCl and supernatant after centrifugation at 1,000.times.g for 15
minutes and frozen for ELISA of proinflammatory cytokine proteins
(Kramer B W, et al. Surfactant protein A recruits neutrophils into
the lungs of ventilated preterm lambs. Am J Respir Crit Care Med
2001; 163:158-165; Ikegami M, et al. Intratracheal recombinant
surfactant protein D prevents endotoxin shock in the newborn
preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347). BALF
was recovered from the left lung (Ikegami M, et al. Whitsett J A.
Intratracheal recombinant surfactant protein D prevents endotoxin
shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006;
173:1342-1347) for further analyses. Total proteins were analyzed
(Lowry O H, et al. Protein measurement with the Folin phenol
reagent. J Biol Chem 1951; 193:265-275) in the supernatant of BALF
after 10 minutes of centrifugation at 284.times.g. The right upper
lobe was inflation-fixed at 30 cm H.sub.2O for morphology (Ikegami
M, et al. Surfactant protein-D regulates the postnatal maturation
of pulmonary surfactant lipid pool sizes. J Appl Physiol 2009;
106:1545-1552). The amount of rhSP-D in aliquots of BALF was
analyzed by ELISA (Ikegami M, et al. Whitsett J A. Intratracheal
recombinant surfactant protein D prevents endotoxin shock in the
newborn preterm lamb. Am J Respir Crit Care Med 2006;
173:1342-1347).
[0044] Results are given as means.+-.SEM. Comparisons between
+rhSP-D and control groups were made with two-tailed unpaired t
tests. For multiple groups, one-way analysis of variance (ANOVA)
followed by Bonferroni-Dunn test, or two-way repeated measures
ANOVA were used. Significance was accepted at a P
value<0.05.
EXAMPLE 2
Lung Function Following rhSP-D Treatment
[0045] Six control and six rhSP-D-treated lambs were studied. Sex
(three male and three female per group), cord blood pH
(7.36.+-.0.04 [control], 7.36.+-.0.05 [+SP-D]), body weight
(3.0.+-.0.3 [control], 3.0.+-.0.1 kg [+SP-D]), and lung weight
(116.+-.15 [control], 115.+-.5 g [+SP-D]) were similar between
+rhSP-D and control groups. Blood pressure, heart rate, hematocrit,
glucose, sodium, potassium, and calcium in the blood samples were
recorded every 30 minutes and were normal throughout the study
period (data not shown). Rectal temperature was maintained at the
normal body temperature for sheep (38.5.degree. C.) by means of
heating pads, radiant heat, and plastic bodycovering wrap.
Ventilation was regulated well for both groups. Lambs were
resuscitated with PIP 40 cm H.sub.2O for 20 minutes after birth
(FIG. 1A), which resulted in mean Pco.sub.2 of 40 mm Hg (FIG. 1B)
and V.sub.T of 11 ml/kg (FIG. 1C) for both groups. After surfactant
treatment was given at 20 minutes of age, ventilation was changed
to regulate V.sub.T at 8 to 9 ml/kg (FIG. 1C) and required a mean
PIP of 27 cm H.sub.2O (FIG. 1A) for both groups. These results
indicate that lung immaturity, as well as ventilatory stress used
to support premature lambs, were comparable between the groups.
[0046] A modified ventilation index was calculated as
PIP.times.Pco.sub.2.times.respiratory rate/1,000 (Norden M A, et
al. Predictors of survival for infants with congenital
diaphragmatic hernia. J Pediatr Surg 1994; 29:1442-1446). Although
it did not reach statistical significance, the mean modified
ventilation index was better for the +rhSP-D group after 240
minutes (FIG. 2A). High Fi.sub.O2 (0.75-1.0) was used for both
groups to maintain P.sub.02 at the target. Premature lambs at this
GA have patent ductus arteriosis, and P.sub.02/F.sub.lo2 may not be
directly associated with lung function. Nevertheless,
P.sub.02/F.sub.lo2 was higher in the +rhSP-D group than the control
group (P<0.01 by two-way repeated measures ANOVA) (FIG. 2B).
P02/Flo2 was significantly decreased after 210 minutes (P<0.05
by one-way ANOVA) in the control group.
[0047] The deflation limb of pressure-volume curves was not
different between the groups (FIG. 3A). Likewise, lung morphology
was similar for both groups, with typical findings consistent with
immaturity, including thickened alveolar septal walls and patchy
atelectasis. More fluid was noted in alveoli of the control lambs
compared with the +rhSP-D lambs (FIGS. 3B and 3C).
EXAMPLE 3
Pulmonary Inflammation Following rhSP-D Treatment
[0048] Previous studies indicated a lack of inflammation as
detected in BALF and lung tissue from 130-day GA lambs killed at
delivery without ventilation (Naik A S, et al. Effects of
ventilation with different positive end-expiratory pressures on
cytokine expression in the preterm lamb lung. Am J Respir Crit Care
Med 2001; 164:494-498; Ikegami M, Jobe A. Postnatal lung
inflammation increased by ventilation of pre term lambs exposed
antenatally to E. coli endotoxin. Pediatr Res 2002;
52:356-362).
[0049] Despite the use of a lung-protective ventilatory strategy,
five hours of ventilation was associated with lung inflammation in
the control lambs. In contrast, lung inflammation was decreased in
all the lambs treated with rhSP-D. Inflammatory cells in the
pellets collected by centrifugation were counted using trypan blue
and differential cell counts were performed on the stained cytospin
preparation (Kramer B W, et al. Surfactant protein A recruits
neutrophils into the lungs of ventilated preterm lambs. Am J Respir
Crit Care Med 2001; 163:158-165; Naik A S, et al. Effects of
ventilation with different positive end-expiratory pressures on
cytokine expression in the preterm lamb lung. Am J Respir Crit Care
Med 2001; 164:494-498). Total inflammatory cell numbers and
neutrophils were significantly decreased by rhSP-D (FIG. 4A).
Increased NE activity has been associated with the development of
BPD (Watterberg K L, et al. Secretory leukocyte protease inhibitor
and lung inflammation in developing bronchopulmonary dysplasia. J
Pediatr 1994; 125:264-269; Yasumatsu R, et al. SERPINB1
upregulation is associated with in vivo complex formation with
neutrophil elastase and cathepsin G in a baboon model of
bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol
2006; 291:L619-L627). The addition of rhSP-D to SURVANTA.RTM.
decreased NE activity in the lung (FIG. 4B). Expression of IL-8,
IL-6, IL-1.beta., TNF-.alpha., KC, and MCP1 mRNA were analyzed by
reverse transcriptase-polymerase chain reaction (FIG. 5A), and
IL-8, IL-6, and IL-1.beta. proteins in the supernatants of lung
homogenates were measured by ELISA (FIG. 5B). Ovine ribosomal
protein L32 was used as a reference RNA. Proinflammatory cytokine
IL-8 (mRNA and protein), which plays a major role in neutrophil
recruitment, was significantly decreased in the lung of
rhSP-D-treated lambs. Although not statistically significant, mean
IL-6 mRNA expression (P=0.06), and IL-6 protein (P=0.1) in the lung
were lower in the +rhSP-D group. IL-1.beta. protein and mRNA were
not significantly influenced by rhSP-D treatment. Expression of
TNF-.alpha. mRNA was similarly present at low levels in both groups
(data not shown). KC, a functional homolog of IL-8, is critical for
neutrophil recruitment and known to increase in ventilation-induced
lung injury in adults (Belperio J A, et al. Critical role for CXCR2
and CXCR2 ligands during the pathogenesis of ventilator-induced
lung injury. J Clin Invest 2002; 110:1703-1716). MCP1 possesses
potent chemotactic activity for monocytes. Because of the large
variation in lung inflammation in the control lambs, KC and MCP1
mRNA in the lung were not significantly different between the two
groups, although mean levels were decreased by rhSP-D
treatment.
[0050] The CD45 antibody recognizes the leukocyte common antigen
and is present on cells of hematopoietic origin, except for
erythroid cells and platelets. CD45-positive cells were isolated
from BALF using magnetic cell separation (Miltenyi Biotech Inc.,
Auburn, Calif.) and CD14, CD11b, and CD44 were analyzed by flow
cytometry (data not shown). CD14-positive cells were not detected
in either group, suggesting that lung inflammation was not
associated with infection. Both CD11b and CD44 influence
vascular-to-tissue migration of neutrophils and monocytes to the
sites of inflammation (Weirich E, et al. Neutrophil CD11b
expression as a diagnostic marker for early-onset neonatal
infection. J Pediatr 1998; 132:445-451). Treatment with rhSP-D did
not influence expression of CD11b or CD44, suggesting that
suppression of neutrophil recruitment by rhSP-D in the lung was
independent of changes in CD11b and CD44.
[0051] After instillation of 7 mg of rhSP-D, 6.7.+-.0.2 mg rhSP-D
was recovered in BALF 4.7 hours after treatment. The slow clearance
of exogenous rhSP-D from the lung is consistent with previous
findings (Ikegami M, Jobe A H. Surfactant metabolism. Semin
Perinatol 1993; 17:233-240), supporting the low rate of surfactant
clearance in the preterm lung. Human SP-D was not detected in BALF
from control lambs.
EXAMPLE 4
Increased Resistance Against Surfactant Inhibition Following rhSP-D
Treatment
[0052] Surface tension was measured by captive bubble surfactometer
(Schoel M, et al. The captive bubble method for the evaluation of
pulmonary surfactant: surface tension, area, and volume
calculations. Biochim Biophys Acta 1994; 1200:281-290) on 3 .mu.L
of samples containing 15 .mu.g/.mu.L SURVANTA.RTM. and 2% rhSP-D or
buffer in the presence or absence of surfactant inhibitor (21
.mu.g/.mu.L plasma protein) (Ikegami M, et al. Characteristics of
surfactant from SP-A deficient mice. Am J Physiol Lung Cell Mol
Physiol 1998; 275:L247-L25). This amount of plasma protein relative
to SURVANTA.RTM. was 30% lower than the concentration that is known
to inhibit the activity of SURVANTA.RTM. in the ventilated
premature newborn lamb lung in vivo (Wada K, et al. Tidal volume
effects on surfactant treatment responses with the initiation of
ventilation in preterm lambs. J Appl Physiol 1997; 83:1054-1061).
The influence of rhSP-D on the ultrastructure of SURVANTA.RTM. was
studied as previously described (Schmiedl A, et al. Influence of
plasma and inflammatory proteins on the ultrastructure of exogenous
surfactant. J Electron Microsc (Tokyo) 2004; 53:407-416).
[0053] Minimum surface tension of SURVANTA.RTM. with (+rhSP-D) or
without rhSP-D (+buffer) in the presence or absence of a surfactant
inhibitor (plasma protein) was measured with a captive bubble
surfactometer (FIG. 6A). The minimum surface tension was low with
or without rhSP-D--consistent with the high surface activity of
SURVANTA.RTM. and similarity of lung function and pressure-volume
curves seen in both +rhSP-D-treated and control lambs. Immediately
after mixing with plasma, surfactant mixtures were applied to the
bubble. Plasma proteins inhibited the surface tension-lowering
properties of SURVANTA.RTM., with the minimum surface tension being
increased to greater than 15 mN/m. The addition of 2% rhSP-D to
SURVANTA.RTM. rendered the SURVANTA.RTM. more resistant to plasma
protein inhibition, the minimum surface tension remaining low in
the presence of plasma proteins. Because SP-D influences surfactant
ultrastructure in the alveolus by causing lysis of surfactant lipid
layers (Ikegami M, et al. Surfactant protein-D regulates the
postnatal maturation of pulmonary surfactant lipid pool sizes. J
Appl Physiol 2009; 106:1545-1552), ultrastructure of the surfactant
mixtures used for treatment was assessed. The simple lipid layers
formed by SURVANTA.RTM. (FIG. 6B) were changed by the addition of
rhSP-D, causing the formation of lipid aggregates and multilayers
(FIG. 6C). These changes in the ultrastructure of SURVANTA.RTM.
caused by rhSP-D may be related to its resistance to inhibition of
surface activity by plasma protein. Although proteins in BALF in
both the control group (60.+-.7 mg/kg) and +rhSP-D group (57.+-.11
mg/kg) were threefold higher than that in non-ventilated premature
lambs seen in previous studies (Naik A S, et al. Effects of
ventilation with different positive end-expiratory pressures on
cytokine expression in the preterm lamb lung. Am J Respir Crit Care
Med 2001; 164:494-498; Ikegami M, Jobe A. Postnatal lung
inflammation increased by ventilation of preterm lambs exposed
antenatally to E. coli endotoxin. Pediatr Res 2002; 52:356-362),
they were not high enough to inhibit the function of the large
amount of SURVANTA.RTM. given to the lambs. Inhibition of
surfactant function by plasma protein occurs when alveolar proteins
are increased above 200 mg/kg (Wada K, et al. Tidal volume effects
on surfactant treatment responses with the initiation of
ventilation in preterm lambs. J Appl Physiol 1997;
83:1054-1061).
EXAMPLE 5
rhSP-D and Animal Surfactant Treatment in a Premature Infant
[0054] rhSP-D and natural animal surfactants, including
SURVANTA.RTM., INFASURF.RTM., and CUROSURF.RTM., are administered
to premature infants. The combinations of rhSP-D and each of the
natural animal surfactants are tested clinically against the
natural surfactants alone (i.e., a test for superior effects). The
premature infants are assessed clinically for the prevention and/or
treatment of neonatal respiratory distress syndrome (RDS), and for
the prevention of bronchopulmonary dysplasia (as these conditions
are defined by one of skill in the art at the time of the study).
Study endpoints include the percentage of infants recovering from
RDS and the incidence of BPD in the study population.
EXAMPLE 6
rhSP-D and Synthetic Surfactant Treatment in a Premature Infant
[0055] rhSP-D and a synthetic surfactant, with or without any
component of surfactant protein, are administered to premature
infants. The combination of rhSP-D and the synthetic surfactant is
tested clinically against the synthetic surfactant alone (i.e., a
test for superiority), and/or a natural surfactant alone (i.e., a
test for superiority), and/or the combination of rhSP-D and a
natural surfactant (i.e., a test for equivalence or superiority).
The premature infants are assessed clinically for the prevention
and/or treatment of neonatal respiratory distress syndrome (RDS),
and for the prevention of bronchopulmonary dysplasia (as these
conditions are defined by one of skill in the art at the time of
the study). Study endpoints include the percentage of infants
recovering from RDS and the incidence of BPD in the study
population.
Sequence CWU 1
1
181375PRTHomo sapien 1Met Leu Leu Phe Leu Leu Ser Ala Leu Val Leu
Leu Thr Gln Pro Leu1 5 10 15Gly Tyr Leu Glu Ala Glu Met Lys Thr Tyr
Ser His Arg Thr Met Pro 20 25 30Ser Ala Cys Thr Leu Val Met Cys Ser
Ser Val Glu Ser Gly Leu Pro 35 40 45Gly Arg Asp Gly Arg Asp Gly Arg
Glu Gly Pro Arg Gly Glu Lys Gly 50 55 60Asp Pro Gly Leu Pro Gly Ala
Ala Gly Gln Ala Gly Met Pro Gly Gln65 70 75 80Ala Gly Pro Val Gly
Pro Lys Gly Asp Asn Gly Ser Val Gly Glu Pro 85 90 95Gly Pro Lys Gly
Asp Thr Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly 100 105 110Val Pro
Gly Pro Ala Gly Arg Glu Gly Pro Leu Gly Lys Gln Gly Asn 115 120
125Ile Gly Pro Gln Gly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys
130 135 140Gly Glu Val Gly Ala Pro Gly Met Gln Gly Ser Ala Gly Ala
Arg Gly145 150 155 160Leu Ala Gly Pro Lys Gly Glu Arg Gly Val Pro
Gly Glu Arg Gly Val 165 170 175Pro Gly Asn Thr Gly Ala Ala Gly Ser
Ala Gly Ala Met Gly Pro Gln 180 185 190Gly Ser Pro Gly Ala Arg Gly
Pro Pro Gly Leu Lys Gly Asp Lys Gly 195 200 205Ile Pro Gly Asp Lys
Gly Ala Lys Gly Glu Ser Gly Leu Pro Asp Val 210 215 220Ala Ser Leu
Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His225 230 235
240Leu Gln Ala Ala Phe Ser Gln Tyr Lys Lys Val Glu Leu Phe Pro Asn
245 250 255Gly Gln Ser Val Gly Glu Lys Ile Phe Lys Thr Ala Gly Phe
Val Lys 260 265 270Pro Phe Thr Glu Ala Gln Leu Leu Cys Thr Gln Ala
Gly Gly Gln Leu 275 280 285Ala Ser Pro Arg Ser Ala Ala Glu Asn Ala
Ala Leu Gln Gln Leu Val 290 295 300Val Ala Lys Asn Glu Ala Ala Phe
Leu Ser Met Thr Asp Ser Lys Thr305 310 315 320Glu Gly Lys Phe Thr
Tyr Pro Thr Gly Glu Ser Leu Val Tyr Ser Asn 325 330 335Trp Ala Pro
Gly Glu Pro Asn Asp Asp Gly Gly Ser Glu Asp Cys Val 340 345 350Glu
Ile Phe Thr Asn Gly Lys Trp Asn Asp Arg Ala Cys Gly Glu Lys 355 360
365Arg Leu Val Val Cys Glu Phe 370 3752355PRTHomo sapien 2Ala Glu
Met Lys Thr Tyr Ser His Arg Thr Met Pro Ser Ala Cys Thr1 5 10 15Leu
Val Met Cys Ser Ser Val Glu Ser Gly Leu Pro Gly Arg Asp Gly 20 25
30Arg Asp Gly Arg Glu Gly Pro Arg Gly Glu Lys Gly Asp Pro Gly Leu
35 40 45Pro Gly Ala Ala Gly Gln Ala Gly Met Pro Gly Gln Ala Gly Pro
Val 50 55 60Gly Pro Lys Gly Asp Asn Gly Ser Val Gly Glu Pro Gly Pro
Lys Gly65 70 75 80Asp Thr Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly
Val Pro Gly Pro 85 90 95Ala Gly Arg Glu Gly Pro Leu Gly Lys Gln Gly
Asn Ile Gly Pro Gln 100 105 110Gly Lys Pro Gly Pro Lys Gly Glu Ala
Gly Pro Lys Gly Glu Val Gly 115 120 125Ala Pro Gly Met Gln Gly Ser
Ala Gly Ala Arg Gly Leu Ala Gly Pro 130 135 140Lys Gly Glu Arg Gly
Val Pro Gly Glu Arg Gly Val Pro Gly Asn Thr145 150 155 160Gly Ala
Ala Gly Ser Ala Gly Ala Met Gly Pro Gln Gly Ser Pro Gly 165 170
175Ala Arg Gly Pro Pro Gly Leu Lys Gly Asp Lys Gly Ile Pro Gly Asp
180 185 190Lys Gly Ala Lys Gly Glu Ser Gly Leu Pro Asp Val Ala Ser
Leu Arg 195 200 205Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His
Leu Gln Ala Ala 210 215 220Phe Ser Gln Tyr Lys Lys Val Glu Leu Phe
Pro Asn Gly Gln Ser Val225 230 235 240Gly Glu Lys Ile Phe Lys Thr
Ala Gly Phe Val Lys Pro Phe Thr Glu 245 250 255Ala Gln Leu Leu Cys
Thr Gln Ala Gly Gly Gln Leu Ala Ser Pro Arg 260 265 270Ser Ala Ala
Glu Asn Ala Ala Leu Gln Gln Leu Val Val Ala Lys Asn 275 280 285Glu
Ala Ala Phe Leu Ser Met Thr Asp Ser Lys Thr Glu Gly Lys Phe 290 295
300Thr Tyr Pro Thr Gly Glu Ser Leu Val Tyr Ser Asn Trp Ala Pro
Gly305 310 315 320Glu Pro Asn Asp Asp Gly Gly Ser Glu Asp Cys Val
Glu Ile Phe Thr 325 330 335Asn Gly Lys Trp Asn Asp Arg Ala Cys Gly
Glu Lys Arg Leu Val Val 340 345 350Cys Glu Phe 35531299DNAHomo
sapien 3agtttgcttg gagctcctgg ggcctaacaa aaagaaacct gccatgctgc
tcttcctcct 60ctctgcactg gtcctgctca cacagcccct gggctacctg gaagcagaaa
tgaagaccta 120ctcccacaga acaatgccca gtgcttgcac cctggtcatg
tgtagctcag tggagagtgg 180cctgcctggt cgcgatggac gggatgggag
agagggccct cggggcgaga agggggaccc 240aggtttgcca ggagctgcag
ggcaagcagg gatgcctgga caagctggcc cagttgggcc 300caaaggggac
aatggctctg ttggagaacc tggaccaaag ggagacactg ggccaagtgg
360acctccagga cctcccggtg tgcctggtcc agctggaaga gaaggtcccc
tggggaagca 420ggggaacata ggacctcagg gcaagccagg cccaaaagga
gaagctgggc ccaaaggaga 480agtaggtgcc ccaggcatgc agggctcggc
aggggcaaga ggcctcgcag gccctaaggg 540agagcgaggt gtccctggtg
agcgtggagt ccctggaaac acaggggcag cagggtctgc 600tggagccatg
ggtccccagg gaagtccagg tgccagggga cccccgggat tgaaggggga
660caaaggcatt cctggagaca aaggagcaaa gggagaaagt gggcttccag
atgttgcttc 720tctgaggcag caggttgagg ccttacaggg acaagtacag
cacctccagg ctgctttctc 780tcagtataag aaagttgagc tcttcccaaa
tggccaaagt gtcggggaga agattttcaa 840gacagcaggc tttgtaaaac
catttacgga ggcacagctg ctgtgcacac aggctggtgg 900acagttggcc
tctccacgct ctgccgctga gaatgccgcc ttgcaacagc tggtcgtagc
960taagaacgag gctgctttcc tgagcatgac tgattccaag acagagggca
agttcaccta 1020ccccacagga gagtccctgg tctattccaa ctgggcccca
ggggagccca acgatgatgg 1080cgggtcagag gactgtgtgg agatcttcac
caatggcaag tggaatgaca gggcttgtgg 1140agaaaagcgt cttgtggtct
gcgagttctg agccaactgg ggtgggtggg gcagtgcttg 1200gcccaggagt
ttggccagaa gtcaaggctt agaccctcat gctgccaata tcctaataaa
1260aaggtgacca tctgtgccgg gaaaaaaaaa aaaaaaaaa 129941221DNAHomo
sapien 4gcgaattcaa acctgccatg ctgctcttcc tcctctctgc actggtcctg
ctcacacagc 60ccctgggcta cctggaagca gaaatgaaga cctactccca cagaacaatg
cccagtgctt 120gcaccctggt catgtgtagc tcagtggaga gtggcctgcc
tggtcgcgat ggacgggatg 180ggagagaggg ccctcggggc gagaaggggg
acccaggttt gccaggagct gcagggcaag 240cagggatgcc tggacaagct
ggcccagttg ggcccaaagg ggacaatggc tctgttggag 300aacctggacc
aaagggagac actgggccaa gtggacctcc aggacctccc ggtgtgcctg
360gtccagctgg aagagaaggt cccctgggga agcaggggaa cataggacct
cagggcaagc 420caggcccaaa aggagaagct gggcccaaag gagaagtagg
tgccccaggc atgcagggct 480cggcaggggc aagaggcctc gcaggcccta
agggagagcg aggtgtccct ggtgagcgtg 540gagtccctgg aaacacaggg
gcagcagggt ctgctggagc catgggtccc cagggaagtc 600caggtgccag
gggacccccg ggattgaagg gggacaaagg cattcctgga gacaaaggag
660caaagggaga aagtgggctt ccagatgttg cttctctgag gcagcaggtt
gaggccttac 720agggacaagt acagcacctc caggctgctt tctctcagta
taagaaagtt gagctcttcc 780caaatggcca aagtgtcggg gagaagattt
tcaagacagc aggctttgta aaaccattta 840cggaggcaca gctgctgtgc
acacaggctg gtggacagtt ggcctctcca cgctctgccg 900ctgagaatgc
cgccttgcaa cagctggtcg tagctaagaa cgaggctgct ttcctgagca
960tgactgattc caagacagag ggcaagttca cctaccccac aggagagtcc
ctggtctatt 1020ccaactgggc cccaggggag cccaacgatg atggcgggtc
agaggactgt gtggagatct 1080tcaccaatgg caagtggaat gacagggctt
gtggagaaaa gcgtcttgtg gtctgcgagt 1140tctgagccaa ctggggtggg
tggggcagtg cttggcccag gagtttggcc agaagtcaag 1200gcttagaccc
tcaggatcct c 1221520DNAOvis aries 5tggccaggat tcacgagttc
20630DNAOvis aries 6tctgtgaggt agaaagatga ctgagatatt 30725DNAOvis
aries 7ggaggaaaaa gatggatgct tccaa 25823DNAOvis aries 8cagcagtggt
tttgatcaag caa 23919DNAOvis aries 9ggctctccac ctcctctca
191019DNAOvis aries 10agctcatgca gaacacctt 191121DNAOvis aries
11gccggaatac ctggactatg c 211222DNAOvis aries 12cagggcgatg
atcccaaagt ag 221317DNAOvis aries 13tgccagtgcc tgcagac
171422DNAOvis aries 14agtggctatg acttcggttt gg 221523DNAOvis aries
15ccccgactat ctgtttccac aac 231621DNAOvis aries 16cctggaaggg
cttctgatct g 211723DNAOvis aries 17gcagaagatt caagggccag atc
231823DNAOvis aries 18ggttttcttg ttgctcccgt aac 23
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