U.S. patent application number 13/689341 was filed with the patent office on 2013-07-18 for pulmonary surfactant formulations.
This patent application is currently assigned to DISCOVERY LABORATORIES, INC.. The applicant listed for this patent is Discovery Laboratories, Inc.. Invention is credited to Rom E. Eliaz, Mark E. Johnson, Ralph Niven, Maithili Rairkar.
Application Number | 20130184198 13/689341 |
Document ID | / |
Family ID | 36615453 |
Filed Date | 2013-07-18 |
United States Patent
Application |
20130184198 |
Kind Code |
A1 |
Rairkar; Maithili ; et
al. |
July 18, 2013 |
Pulmonary Surfactant Formulations
Abstract
Synthetic pulmonary surfactant compositions comprising
dipalmitoyl phosphatidylcholine, phosphatidylglycerol, and
essentially neutral lipid, and having essentially no 1-palmitoyl
2-oleoyl phosphatidylglycerol and essentially no palmitic acid are
provided. Methods for treating respiratory disease are also
provided comprising administering a therapeutically effective
amount of a synthetic pulmonary surfactant comprising dipalmitoyl
phosphatidylcholine, phosphatidylglycerol, and essentially neutral
lipid, and having essentially no 1-palmitoyl 2-oleoyl
phosphatidylglycerol and essentially no palmitic acid.
Inventors: |
Rairkar; Maithili; (San
Jose, CA) ; Eliaz; Rom E.; (Lehavim, IL) ;
Niven; Ralph; (San Carlos, CA) ; Johnson; Mark
E.; (Napa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Discovery Laboratories, Inc.; |
Warrington |
PA |
US |
|
|
Assignee: |
DISCOVERY LABORATORIES,
INC.
Warrington
PA
|
Family ID: |
36615453 |
Appl. No.: |
13/689341 |
Filed: |
November 29, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11316308 |
Dec 21, 2005 |
8337815 |
|
|
13689341 |
|
|
|
|
60638665 |
Dec 23, 2004 |
|
|
|
Current U.S.
Class: |
514/1.5 ;
514/15.5; 514/77 |
Current CPC
Class: |
A61K 31/685 20130101;
A61K 31/685 20130101; A61K 45/06 20130101; A61K 31/683 20130101;
A61K 31/575 20130101; A61K 38/16 20130101; A61K 31/683 20130101;
C07K 14/785 20130101; A61K 31/575 20130101; A61K 9/0082 20130101;
A61P 11/00 20180101; A61K 38/395 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/1.5 ; 514/77;
514/15.5 |
International
Class: |
A61K 31/685 20060101
A61K031/685; A61K 38/16 20060101 A61K038/16 |
Claims
1. A synthetic pulmonary surfactant comprising dipalmitoyl
phosphatidylcholine, phosphatidylglycerol, and essentially neutral
lipid, and having essentially no 1-palmitoyl 2-oleoyl
phosphatidylglycerol and essentially no palmitic acid.
2. The pulmonary surfactant of claim 1, wherein the
phosphatidylglycerol is saturated, non-saturated or
semi-saturated.
3. The pulmonary surfactant of claim 2, wherein the saturated
phosphatidylglycerol is dipalmitoyl phosphatidylglycerol.
4. The pulmonary surfactant of claim 1, wherein each of 1-palmitoyl
2-oleoyl phosphatidylglycerol and palmitic acid are present in an
amount less than about five mole percent of total phospholipid.
5. The pulmonary surfactant of claim 1, wherein essentially neutral
lipid comprises cholesterol.
6. The pulmonary surfactant of claim 1, wherein essentially neutral
lipid comprises a fatty acid, a fatty acid ester, a fatty acid
alcohol, cholesterol, corticosteroid, glucocorticosteroid,
trifluorinate glucocorticoid, agonist, plant sterol, phospholipid,
phosphatidylcholine, phosphatidyl ethanolamine,
phosphatidylinositol, sphingomyelin, diglycerides, diolein,
dipalmitolein, mixed caprylin-caprin, triglycerides, triolein,
tripalmitolein, trilinolein, tricaprylin, or trilaurin.
7. The pulmonary surfactant of claim 5, wherein the cholesterol
concentration is from about 0.1 mol % to about 50 mol %.
8. The pulmonary surfactant of claim 5, wherein the cholesterol
concentration is from about 1 mol % to about 20 mol %.
9. The pulmonary surfactant of claim 5, wherein the cholesterol
concentration is cholesterol concentration from about 8 mol % to
about 15 mol %.
10. The pulmonary surfactant of claim 3, wherein the molar ratio of
dipalmitoyl phosphatidylcholine to dipalmitoyl phosphatidylglycerol
is 4 to 1.
11. The pulmonary surfactant of claim 3, wherein the molar ratio of
dipalmitoyl phosphatidylcholine to dipalmitoyl phosphatidylglycerol
is 7 to 3.
12. The pulmonary surfactant of claim 3, wherein the molar ratio of
dipalmitoyl phosphatidylcholine to dipalmitoyl phosphatidylglycerol
is 3 to 1.
13. The pulmonary surfactant of claim 1, wherein the total
concentration of dipalmitoyl phosphatidylcholine and
phosphatidylglycerol is from about 10 mg/ml to about 150 mg/ml.
14. The pulmonary surfactant of claim 13, wherein the total
concentration of dipalmitoyl phosphatidylcholine and
phosphatidylglycerol is from about 50 mg/ml to about 125 mg/ml.
15. The pulmonary surfactant of claim 1, wherein a dynamic surface
tension of the surfactant as measured by pulsating bubble surface
tensionometry is about 10 mN or less.
16. The pulmonary surfactant of claim 1, further comprising a
surfactant polypeptide.
17. The pulmonary surfactant of claim 16, wherein the surfactant
polypeptide comprises at least 10 amino acid residues and no more
than about 60 amino acid residues, the polypeptide including a
sequence having alternating hydrophobic and hydrophilic amino acid
residue regions represented by the formula (Z.sub.a U.sub.b).sub.c
Z.sub.a, wherein: Z is a hydrophilic amino acid residue
independently selected from the group consisting of R and K; U is a
hydrophobic amino acid residue independently selected from the
group consisting of L and C; a is 1 or 2; b has an average value of
about 3 to about 8; c is 1 to 10; and d is 0 to 2.
18. The pulmonary surfactant of claim 17, having an amino acid
residue sequence represented by the formula:
KLLLLKLLLLKLLLLKLLLLK.
19. A synthetic pulmonary surfactant consisting essentially of
dipalmitoyl phosphatidylcholine, non-saturated
phosphatidylglycerol, essentially neutral lipid and surfactant
polypeptide.
20. The pulmonary surfactant of claim 19, wherein the non-saturated
phosphatidylglycerol is palmitoyl oleyl phosphatidylglycerol.
21. The pulmonary surfactant of claim 19, wherein the essentially
neutral lipid is cholesterol.
22. The pulmonary surfactant of claim 19, wherein the surfactant
polypeptide has an amino acid residue sequence represented by the
formula: KLLLLKLLLLKLLLLKLLLLK.
23. A synthetic pulmonary surfactant consisting essentially of
dipalmitoyl phosphatidylcholine, nonsaturated phosphatidylglycerol,
palmitic acid, essentially neutral lipid and surfactant
polypeptide.
24. The surfactant of claim 23, wherein the nonsaturated
phosphatidylglycerol is palmitoyl oleyl phosphatidylglycerol
25. The pulmonary surfactant of claim 23, wherein the neutral lipid
is cholesterol.
26. The pulmonary surfactant of claim 23, wherein the surfactant
polypeptide has an amino acid residue sequence represented by the
formula: KLLLLKLLLLKLLLLKLLLLK.
27. A synthetic pulmonary surfactant consisting essentially of
dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol,
essentially neutral lipid and surfactant polypeptide.
28. The pulmonary surfactant of claim 27, wherein essentially
neutral lipid is cholesterol.
29. The pulmonary surfactant of claim 27 wherein essentially
neutral lipid comprises a fatty acid, a fatty acid ester, a fatty
acid alcohol, cholesterol, corticosteroid, glucocorticosteroid,
trifluorinate glucocorticoid, .beta.2 agonist, plant sterol,
phospholipid, phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, sphingomyelin, diglycerides, fatty alcohols,
diolein, dipalmitolein, mixed caprylin-caprin, triglycerides,
triolein, tripalmitolein, trilinolein, tricaprylin, or
trilaurin.
30. The pulmonary surfactant of claim 27 wherein the surfactant
polypeptide comprises at least 10 amino acid residues and no more
than about 60 amino acid residues, the polypeptide including a
sequence having alternating hydrophobic and hydrophilic amino acid
residue regions represented by the formula (Z.sub.a U.sub.b).sub.c
Z.sub.d, wherein: Z is a hydrophilic amino acid residue
independently selected from the group consisting of R and K; U is a
hydrophobic amino acid residue independently selected from the
group consisting of L and C; a is 1 or 2; b has an average value of
about 3 to about 8; c is 1 to 10; and d is 0 to 2.
31. The pulmonary surfactant of claim 30, having an amino acid
residue sequence represented by the formula:
KLLLLKLLLLKLLLLKLLLLK.
32. A synthetic pulmonary surfactant consisting of dipalmitoyl
phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and
essentially neutral lipid.
33. The pulmonary surfactant of claim 32, wherein essentially
neutral lipid comprises cholesterol.
34. The pulmonary surfactant of claim 32, wherein essentially
neutral lipid comprises a fatty acid, a fatty acid ester, a fatty
acid alcohol, cholesterol, corticosteroid, glucocorticosteroid,
trifluorinate glucocorticoid, .beta.2 agonist, plant sterol,
phospholipid, phosphatidylcholine, phosphatidyl ethanolamine,
phosphatidylinositol, sphingomyelin, diglycerides, diolein,
dipalmitolein, mixed caprylin-caprin, triglycerides, triolein,
tripalmitolein, trilinolein, tricaprylin, or trilaurin.
35. A method of treating infant respiratory distress syndrome
comprising administering a therapeutically effective amount of a
synthetic pulmonary surfactant comprising dipalmitoyl
phosphatidylcholine, phosphatidylglycerol, and essentially neutral
lipid, and having essentially no 1-palmitoyl 2-oleoyl
phosphatidylglycerol and essentially no palmitic acid.
36. The method of claim 35, wherein the phosphatidylglycerol is
saturated, non-saturated or semi-saturated.
37. The method of claim 36, wherein the saturated
phosphatidylglycerol is dipalmitoyl phosphatidylglycerol.
38. The method of claim 35, wherein essentially neutral lipid
comprises cholesterol.
39. The method of claim 35, wherein essentially neutral lipid
comprises a fatty acid, a fatty acid ester, a fatty acid alcohol,
cholesterol, corticosteroid, plant sterol, phospholipid,
phosphatidylcholine, phosphatidyl ethanolamine,
phosphatidylinositol, sphingomyelin, diglycerides, diolein,
dipalmitolein, mixed caprylin-caprin, triglycerides, triolein,
tripalmitolein, trilinolein, tricaprylin, or trilaurin.
40. A method of treating infant respiratory distress syndrome
comprising administering a therapeutically effective amount of a
synthetic pulmonary surfactant, the surfactant consisting
essentially of dipalmitoyl phosphatidylcholine,
phosphatidylglycerol, essentially neutral lipid and a surfactant
polypeptide, the polypeptide having alternating hydrophobic and
hydrophilic amino acid residue regions, represented by the formula
(Z.sub.a U.sub.b).sub.c Z.sub.d, wherein: Z is a hydrophilic amino
acid residue independently selected from the group consisting of R
and K; U is a hydrophobic amino acid residue independently selected
from the group consisting of L and C; a is 1 or 2; b has an average
value of about 3 to about 8; c is 1 to 10; and d is 0 to 2.
41. The method of claim 40, wherein the phosphatidylglycerol is
saturated, non-saturated or semi-saturated.
42. The method of claim 41, wherein the saturated
phosphatidylglycerol is dipalmitoyl phosphatidylglycerol.
43. The method of claim 40, wherein essentially neutral lipid
comprises cholesterol.
44. The method of claim 40, wherein the polypeptide has an amino
acid residue sequence represented by the formula:
KLLLLKLLLLKLLLLKLLLLK.
45. A method of treating respiratory distress syndrome comprising
administering a therapeutically effective amount of a synthetic
pulmonary surfactant comprising dipalmitoyl phosphatidylcholine,
dipalmitoyl phosphatidylglycerol, and essentially neutral lipid,
and having essentially no 1-palmitoyl 2-oleoyl phosphatidylglycerol
and essentially no palmitic acid.
46. A method of treating respiratory distress syndrome comprising
administering a therapeutically effective amount of a synthetic
pulmonary surfactant, the surfactant comprising one or more
pharmaceutically acceptable phospholipids admixed with a
polypeptide having alternating hydrophobic and hydrophilic amino
acid residue regions, represented by the formula (Z.sub.a
U.sub.b).sub.c Z.sub.d, wherein: Z is a hydrophilic amino acid
residue independently selected from the group consisting of R and
K; U is a hydrophobic amino acid residue independently selected
from the group consisting of L and C; a is 1 or 2; b has an average
value of about 3 to about 8; c is 1 to 10; and d is 0 to 2.
47. The method of claim 46, wherein the polypeptide has an amino
acid residue sequence represented by the formula:
KLLLLKLLLLKLLLLKLLLLK.
48. A method of manufacturing a synthetic pulmonary surfactant
comprising terminally sterilizing the surfactant of claim 1 by
autoclaving.
49. A method for drug delivery to the pulmonary system comprising
administering to a patient in need of treatment an effective amount
of a synthetic pulmonary surfactant of claim 1, wherein the
microparticles have a diameter between 0.5 microns and 5 microns
and viscosity less than 6 cp at a cholesterol concentration from
about 8 mole % to about 15 mole %, in a pharmaceutically acceptable
carrier for administration to the lungs.
50. A method for reducing viscosity of a drug delivery formulation
to the pulmonary system comprising adding cholesterol at a
concentration from about 8 mol % to about 15 mol % to the synthetic
pulmonary surfactant of claim 1.
51. A method for increasing aerosolization of a drug delivery
formulation to the pulmonary system comprising adding cholesterol
at a concentration from about 8 mol % to about 15 mol % to the
synthetic pulmonary surfactant of claim 1.
52. A method for increasing storage stability of a drug delivery
formulation to the pulmonary system comprising adding cholesterol
at a concentration from about 8 mol % to about 15 mol % to the
synthetic pulmonary surfactant of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority, under 35 U.S.C.
.sctn.119(e), to application Ser. No. 60/638,665 filed Dec. 23,
2004, the disclosure of which is incorporated by reference in its
entirety.
FIELD
[0002] The invention relates to a novel lung surfactant composition
comprising a pulmonary surfactant and a neutral lipid that exhibits
a reduction in surface tension, low viscosity characteristics and
enhanced storage stability.
BACKGROUND
[0003] Pulmonary surfactants are complex lipid and protein
compositions that can be extracted from animals, purified and used
to treat neonatal respiratory distress syndrome. Animal-derived
surfactants are difficult to purify, limited by the scale to which
they can be manufactured and have the potential to cause
immunogenic reactions on repeat use, and thus are not indicated for
use in other clinical indications. To capitalize on the potential
use of surfactants in a range of pulmonary disease states requires
the availability of a functional surfactant that can both be
manufactured at the scale required and which inherently has the
flexibility to be modified for use with specific delivery systems
and for specific disease states. One such surfactant under
development is synthetically produced and has a composition of
dipalmitoyl phosphatidylcholine (DPPC), or 1-palmitoyl 2-oleoyl
phosphatidylglycerol (POPG), palmitic acid (PA) and an engineered
mimic of surfactant protein B, called sinapultide (KL4 or KL.sub.4)
dispersed within an isotonic aqueous Tris-saline buffer of pH 7.7.
The composition is currently under evaluation for use in RDS,
meconium aspiration syndrome and in acute respiratory distress
syndrome at various concentrations. The composition can also be
modified for use by reducing, increasing or substituting one or
more of the components.
[0004] Previous work has shown that formulations having reduced
concentrations of either palmitic acid (PA) or cetyl alcohol (CA),
relative to that of 30 mg/ml Surfaxin.RTM. (i.e., 4.05 mg/ml PA),
exhibit lower viscosity when compared side by side with
Surfaxin.RTM.. However, this is at the expense of loss of
functional surface tension activity as the content of the palmitic
acid or cetyl alcohol is reduced as measured by in vitro
techniques. However, by using DPPG instead of POPG and adding
cholesterol, palmitic acid could be completely omitted from the
formulation with the resulting compositions exhibiting low
viscosity while retaining good surface activity.
[0005] Recently it was found that cholesterol at low concentrations
contribute significantly to the termination of phase separation
during compression of interfacial films of the pulmonary surfactant
(Discher et al., 1999, Biochemistry 38:374-83). Other studies
suggested that at low concentration cholesterol contributes to the
elastic response of the neighboring lipids in the lipid bilayer.
This elastic response is expressed by a tendency of the surrounding
lipids to adapt to the hydrophobic shape of cholesterol (Kessel et
al., 2001, Biophys J. 81:643-58). In fact, cholesterol can modulate
physical properties of lipid bilayers to induce liquid phase
coexistence and corresponding domain formation (Radhakrishnan and.
McConnell, 1999, Biophys J. 77:1507-17.).
[0006] Due to these findings, and the existence of cholesterol in
natural lung surfactant this report is related to the inclusion of
various concentrations of cholesterol, 1-30 mol %, focusing on 2-15
mol %, to KL.sub.4-containing formulations to improve their
properties. In particular (but not limited to) it can help in
aerosolization of KL.sub.4-containing formulations by decreasing
the viscosity of the formulations and otherwise modifying the
structure of the concentrated lipid-based dispersions, such that an
increase in the amount of surfactant that can be aerosolized is
achieved. In addition, inclusion of cholesterol with saturated
lipids in the surfactant formulation could facilitate and enhance
an increase in lateral stability of the monolayer essential for
alveolar expansion. Moreover, since the transition temperature of
DPPC is close to body temperature (41.degree. C.) cholesterol will
"soften" the bilayer, increase its permeability and eliminate
phase-separation during alveolar compression and expansion.
Furthermore, cholesterol will contribute to membrane perturbation
effects, thus will decrease the energy of interfacial tension and
lower line tension (as in the critical point, when the
characteristics of two phases become similar). Similarly it may
improve lipid-peptide interaction, particularly KL.sub.4-DPPC/DPPG
interactions.
[0007] The current standard method of delivery does not
instantaneously deliver the surfactant over the surface of the
airways and alveoli. The procedure involves the introduction of a
bolus of surfactant to a patient who has been intubated (an
invasive procedure). In order to distribute throughout the lungs,
the surfactant must be aspirated deeper into the lungs during
breathing maneuvers while simultaneously flowing and spreading
across the lung surfaces. Accordingly, a formulation that can be
effectively delivered as an aerosol in sufficient quantity and of
appropriate aerosol size and size characteristics should distribute
throughout the lungs and exert a therapeutic response without the
need to employ an invasive delivery procedure.
BRIEF SUMMARY
[0008] The present invention is related to novel lung surfactant
compositions and methods of use related thereto. The invention
relates, in part, to the discovery that formulations containing a
surfactant, 1,2 dipalmitoyl phosphatidylcholine (DPPC),
phosphatidylglycerol (DPPG), essentially neutral lipid, and
essentially no palmitic acid (PA) or 1-palmitoyl 2-oleoyl
phosphatidylglycerol (POPG) exhibit higher aerosol output rates
from various aerosol generators and low viscosity compared to
formulations containing PA and/or POPG. Thus, the present invention
relates to a pulmonary surfactant comprising dipalmitoyl
phosphatidylcholine, phosphatidylglycerol, and essentially neutral
lipid, and having essentially no 1-palmitoyl 2-oleoyl
phosphatidylglycerol and essentially no palmitic acid.
[0009] The present invention also provides a synthetic pulmonary
surfactant comprising dipalmitoyl phosphatidylcholine,
phosphatidylglycerol, and essentially neutral lipid, and having
essentially no 1-palmitoyl 2-oleoyl phosphatidylglycerol and
essentially no palmitic acid. In some aspects, the
phosphatidylglycerol is saturated, non-saturated or semi-saturated.
In some such aspects, the pulmonary surfactant the saturated
phosphatidylglycerol is dipalmitoyl phosphatidylglycerol. In some
aspects, the pulmonary surfactant each of 1-palmitoyl 2-oleoyl
phosphatidylglycerol and palmitic acid are present in an amount
less than about five mole percent of total phospholipid. In some
aspects, the essentially neutral lipid comprises cholesterol. In
other aspects, the pulmonary surfactant the essentially neutral
lipid comprises a fatty acid, a fatty acid ester, a fatty acid
alcohol, cholesterol, corticosteroid, glucocorticosteroid,
trifluorinate glucocorticoid, .beta.2 agonist, plant sterol,
phospholipid, phosphatidylcholine, phosphatidyl ethanolamine,
phosphatidylinositol, sphingomyelin, diglycerides, diolein,
dipalmitolein, mixed caprylin-caprin, triglycerides, triolein,
tripalmitolein, trilinolein, tricaprylin, or trilaurin In some such
aspects, the cholesterol concentration is from about 0.1 mol % to
about 50 mol %. In some such aspects, the cholesterol concentration
is from about 1 mol % to about 20 mol %. In some such aspects, the
cholesterol concentration is cholesterol concentration from about 8
mol % to about 15 mol %. In some such aspects, the molar ratio of
dipalmitoyl phosphatidylcholine to dipalmitoyl phosphatidylglycerol
is 4 to 1. In some such aspects, the molar ratio of dipalmitoyl
phosphatidylcholine to dipalmitoyl phosphatidylglycerol is 7 to 3.
In some such aspects, the molar ratio of dipalmitoyl
phosphatidylcholine to dipalmitoyl phosphatidylglycerol is 3 to 1.
In some such aspects, the total concentration of dipalmitoyl
phosphatidylcholine and phosphatidylglycerol is from about 10 mg/ml
to about 150 mg/ml. In some aspects, the total concentration of
dipalmitoyl phosphatidylcholine and phosphatidylglycerol is from
about 50 mg/ml to about 125 mg/ml. In other aspects, a dynamic
surface tension of the surfactant as measured by pulsating bubble
surface tensionometry is about 10 mN or less. In some such aspects,
the pulmonary surfactant further comprises a surfactant
polypeptide. In certain aspects, the surfactant polypeptide
comprises at least 10 amino acid residues and no more than about 60
amino acid residues, the polypeptide including a sequence having
alternating hydrophobic and hydrophilic amino acid residue regions
represented by the formula (Za Ub)c Zd, wherein: Z is a hydrophilic
amino acid residue independently selected from the group consisting
of R and K; U is a hydrophobic amino acid residue independently
selected from the group consisting of L and C; a is 1 or 2; b has
an average value of about 3 to about 8; c is 1 to 10; and d is 0 to
2. In some such aspects, the pulmonary surfactant has an amino acid
residue sequence represented by the formula:
KLLLLKLLLLKLLLLKLLLLK.
[0010] The present invention also provides a synthetic pulmonary
surfactant consisting essentially of dipalmitoyl
phosphatidylcholine, non-saturated phosphatidylglycerol,
essentially neutral lipid and surfactant polypeptide. In some
aspects, the non-saturated phosphatidylglycerol is palmitoyl oleyl
phosphatidylglycerol. In some such aspects, the essentially neutral
lipid is cholesterol. In some such aspects, the surfactant
polypeptide has an amino acid residue sequence represented by the
formula: KLLLLKLLLLKLLLLKLLLLK.
[0011] The present invention provides a synthetic pulmonary
surfactant consisting essentially of dipalmitoyl
phosphatidylcholine, nonsaturated phosphatidylglycerol, palmitic
acid, essentially neutral lipid and surfactant polypeptide. In some
such aspects, the nonsaturated phosphatidylglycerol is palmitoyl
oleyl phosphatidylglycerol. In some such aspects, the neutral lipid
is cholesterol. In some such aspects, the surfactant polypeptide
has an amino acid residue sequence represented by the formula:
KLLLLKLLLLKLLLLKLLLLK.
[0012] The present invention also provides a synthetic pulmonary
surfactant consisting essentially of dipalmitoyl
phosphatidylcholine, dipalmitoyl phosphatidylglycerol, essentially
neutral lipid and surfactant polypeptide. In some such aspects, the
essentially neutral lipid is cholesterol. In some such aspects, the
essentially neutral lipid comprises a fatty acid, a fatty acid
ester, a fatty acid alcohol, cholesterol, corticosteroid,
glucocorticosteroid, trifluorinate glucocorticoid, .beta.2 agonist,
plant sterol, phospholipid, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylinositol, sphingomyelin,
diglycerides, fatty alcohols, diolein, dipalmitolein, mixed
caprylin-caprin, triglycerides, triolein, tripalmitolein,
trilinolein, tricaprylin, or trilaurin. In some such aspects, the
surfactant polypeptide comprises at least 10 amino acid residues
and no more than about 60 amino acid residues, the polypeptide
including a sequence having alternating hydrophobic and hydrophilic
amino acid residue regions represented by the formula (Za Ub)c Zd,
wherein: Z is a hydrophilic amino acid residue independently
selected from the group consisting of R and K; U is a hydrophobic
amino acid residue independently selected from the group consisting
of L and C; a is 1 or 2; b has an average value of about 3 to about
8; c is 1 to 10; and d is 0 to 2. In some such methods, the
pulmonary surfactant has an amino acid residue sequence represented
by the formula: KLLLLKLLLLKLLLLKLLLLK.
[0013] The present invention provides a synthetic pulmonary
surfactant consisting of dipalmitoyl phosphatidylcholine,
dipalmitoyl phosphatidylglycerol, and essentially neutral lipid. In
some aspects, the essentially neutral lipid comprises cholesterol.
In some such aspects, the essentially neutral lipid comprises a
fatty acid, a fatty acid ester, a fatty acid alcohol, cholesterol,
corticosteroid, glucocorticosteroid, trifluorinate glucocorticoid,
.beta.2 agonist, plant sterol, phospholipid, phosphatidylcholine,
phosphatidyl ethanolamine, phosphatidylinositol, sphingomyelin,
diglycerides, diolein, dipalmitolein, mixed caprylin-caprin,
triglycerides, triolein, tripalmitolein, trilinolein, tricaprylin,
or trilaurin.
[0014] The present invention further provides methods of treating
respiratory disease, such as infant respiratory distress syndrome,
comprising administering a therapeutically effective amount of a
synthetic pulmonary surfactant comprising dipalmitoyl
phosphatidylcholine, phosphatidylglycerol, and essentially neutral
lipid, and having essentially no 1-palmitoyl 2-oleoyl
phosphatidylglycerol and essentially no palmitic acid. In some such
methods, the phosphatidylglycerol is saturated, non-saturated or
semi-saturated. In some such methods, the saturated
phosphatidylglycerol is dipalmitoyl phosphatidylglycerol. In other
methods, the essentially neutral lipid comprises cholesterol. In
some methods, the essentially neutral lipid comprises a fatty acid,
a fatty acid ester, a fatty acid alcohol, cholesterol,
corticosteroid, plant sterol, phospholipid, phosphatidylcholine,
phosphatidyl ethanolamine, phosphatidylinositol, sphingomyelin,
diglycerides, diolein, dipalmitolein, mixed caprylin-caprin,
triglycerides, triolein, tripalmitolein, trilinolein, tricaprylin,
or trilaurin.
[0015] The present invention further provides methods of treating
respiratory disease, such as infant respiratory distress syndrome,
comprising administering a therapeutically effective amount of a
synthetic pulmonary surfactant, the surfactant consisting
essentially of dipalmitoyl phosphatidylcholine,
phosphatidylglycerol, essentially neutral lipid and a surfactant
polypeptide, the polypeptide having alternating hydrophobic and
hydrophilic amino acid residue regions, represented by the formula
(Za Ub)c Zd, wherein: Z is a hydrophilic amino acid residue
independently selected from the group consisting of R and K; U is a
hydrophobic amino acid residue independently selected from the
group consisting of L and C; a is 1 or 2; b has an average value of
about 3 to about 8; c is 1 to 10; and d is 0 to 2. In some such
methods, the phosphatidylglycerol is saturated, non-saturated or
semi-saturated. In some such methods, the saturated
phosphatidylglycerol is dipalmitoyl phosphatidylglycerol. In some
such methods, the essentially neutral lipid comprises cholesterol.
In some such methods, the polypeptide has an amino acid residue
sequence represented by the formula: KLLLLKLLLLKLLLLKLLLLK.
[0016] The present invention further provides methods of treating
respiratory disease, such as respiratory distress syndrome,
comprising administering a therapeutically effective amount of a
synthetic pulmonary surfactant comprising dipalmitoyl
phosphatidylcholine, dipalmitoyl phosphatidylglycerol, and
essentially neutral lipid, and having essentially no 1-palmitoyl
2-oleoyl phosphatidylglycerol and essentially no palmitic acid.
[0017] The present invention further provides methods of treating
respiratory disease, such as respiratory distress syndrome,
comprising administering a therapeutically effective amount of a
synthetic pulmonary surfactant, the surfactant comprising one or
more pharmaceutically acceptable phospholipids admixed with a
polypeptide having alternating hydrophobic and hydrophilic amino
acid residue regions, represented by the formula (Za Ub)c Zd,
wherein: Z is a hydrophilic amino acid residue independently
selected from the group consisting of R and K; U is a hydrophobic
amino acid residue independently selected from the group consisting
of L and C; a is 1 or 2; b has an average value of about 3 to about
8; c is 1 to 10; and d is 0 to 2. In some such methods, the
polypeptide has an amino acid residue sequence represented by the
formula: KLLLLKLLLLKLLLLKLLLLK.
[0018] The present invention also provides a method of
manufacturing a synthetic pulmonary surfactant comprising
terminally sterilizing the surfactant of the present invention by
autoclaving.
[0019] The present invention further provides a method for drug
delivery to the pulmonary system comprising administering to a
patient in need of treatment an effective amount of a synthetic
pulmonary surfactant of the present invention, wherein the
microparticles have a diameter between 0.5 microns and 5 microns
and viscosity less than 30 cP at a cholesterol concentration from
about 8 mole % to about 15 mole %, in a pharmaceutically acceptable
carrier for administration to the lungs.
[0020] The present invention further provides a method for reducing
viscosity of a drug delivery formulation to the pulmonary system
comprising adding cholesterol at a concentration from about 8 mol %
to about 15 mol % to the synthetic pulmonary surfactant of the
present invention as described above.
[0021] The present invention further provides a method for
increasing the aerosolization rate of a drug delivery formulation
to the pulmonary system comprising adding cholesterol at a
concentration from about 8 mol % to about 15 mol % to the synthetic
pulmonary surfactant of the present invention as described
above.
[0022] The present invention further provides a method for
increasing storage stability of a drug delivery formulation to the
pulmonary system comprising adding cholesterol at a concentration
from about 8 mol % to about 15 mol % to the synthetic pulmonary
surfactant of claim 1 as described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1. Surface activities of DPPG formulations measured at
3 mg/ml TPL
[0024] FIG. 2. Surface activities of formulations measured at 3
mg/ml TPL
[0025] FIG. 3. Plot of cholesterol concentration vs. apparent
viscosity for formulations after 1 and 4 days refrigerated
storage.
[0026] FIG. 4. Particle size analysis. Ten measurements of the
volume median diameter (D50) were made and averaged for each
formulation lot. The average of the D50's for the lots of each
formulation are shown, along with the standard deviations. To show
the correlation to viscosity, the viscosity 4 days post hydration
was plotted as well.
[0027] FIG. 5. Aerosol output rate of formulations measured at 60
mg/ml (mg-TPL/min)
[0028] FIG. 6. Long-term apparent viscosity.
[0029] FIG. 7. Particle size analysis. Ten measurements of the
volume median diameter (D50) were made and averaged for each
formulation lot. The average of the D50's for the three lots of
each formulation are shown, along with the standard deviations.
Little to no difference in apparent particle size was observed,
with the exception of the 2 mol % cholesterol formulations that
exhibited larger apparent particle sizes at this 60 mg/ml
concentration.
[0030] FIG. 8. Apparent viscosity and surface activity after 30
days at 25.degree. C. and 60% humidity.
[0031] FIG. 9. The change in compliance of fetal rabbit lungs after
treatment with a 30 mg/ml DPPC, DPPG and cholesterol (10 mol %)
composition (n=6) compared with control animals treated with
vehicle (n=6).
[0032] FIG. 10. Apparent viscosity data for formulations placed on
thermal stability.
[0033] FIG. 11. Aerosol output rate measurements for formulations
placed on thermal stability.
[0034] FIG. 12. Percent Loss of KL.sub.4 for the formulations
placed on thermal stability at 5.degree. C. storage.
[0035] FIG. 13. Percent Loss of KL.sub.4 for the formulations
placed on thermal stability at 25.degree. C. storage.
DETAILED DESCRIPTION
[0036] A. General Overview
[0037] It is to be understood that this invention is not limited to
particular methods, reagents, compounds, compositions or biological
systems, which can, of course, vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to be limiting. As
used in this specification and the appended claims, the singular
forms "a", "an" and "the" include plural referents unless the
content clearly dictates otherwise. Thus, for example, reference to
"a cell" includes a combination of two or more cells, and the
like.
[0038] The term "about" as used herein when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of .+-.20% or .+-.10%, more
preferably .+-.5%, even more preferably .+-.1%, and still more
preferably .+-.0.1% from the specified value, as such variations
are appropriate to perform the disclosed methods.
[0039] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0040] The present invention delivers mixtures of active agents in
a media. As used herein the term "mixture" means a solution,
suspension, dispersion or emulsion. "Emulsion" refers to a mixture
of two or more generally immiscible liquids, and is generally in
the form of a colloid. The mixture can be of lipids, for example,
which can be homogeneously or heterogeneously dispersed throughout
the emulsion. Alternatively, the lipids can be aggregated in the
form of, for example, clusters or layers, including monolayers or
bilayers. "Suspension" or "dispersion" refers to a mixture,
preferably finely divided, of two or more phases (solid, liquid or
gas), such as, for example liquid in liquid, solid in solid, gas in
liquid, and the like which preferably can remain stable for
extended periods of time. Preferably the dispersion of this
invention is a fluid dispersion.
[0041] The mixture comprises the active agent at desired
concentration and a medium. Preferably, the concentration of the
active agent in the medium is selected to ensure that the patient
is receiving an effective amount of active agent and is typically
about 1 to about 100/mg/ml. Based on the active agent chosen and
the medium, one of skill in the art is readily able to determine
the proper concentration. Mixtures often include one or more
wetting agents. The term "wetting agent" means a material that
reduces the surface tension of a liquid and therefore increases its
adhesion to a solid surface. Preferably, a wetting agent comprises
a molecule with a hydrophilic group at one end and a hydrophobic
group at the other. The hydrophilic group is believed to prevent
beading or collection of a material on a surface, such as the nasal
prongs. Suitable wetting agents are soaps, alcohols, fatty acids,
combinations thereof and the like.
[0042] "Composition" and "formulation" are used interchangeably to
refer to a product which results by combining or mixing more than
one element or ingredient.
[0043] "Storage stability" refers to the stability of a drug
product under the anticipated storage conditions. A formulation
with enhanced or improved storage stability would exhibit less
chemical degradation and less changes in key physical properties
(e.g., loss in in vitro surface activity in the case of lung
surfactants) during storage and over time.
[0044] "Active agent" as used herein refers to a substance or
combination of substances that can be used for therapeutic purposes
(e.g., a drug), diagnostic purposes or prophylactic purposes via
pulmonary delivery. For example, an active agent can be useful for
diagnosing the presence or absence of a disease or a condition in a
patient and/or for the treatment of a disease or condition in a
patient. "Active agent" thus refers to substances or combinations
of substances that are capable of exerting a biological effect when
delivered by pulmonary routes. The bioactive agents can be neutral,
positively or negatively charged. Exemplary agents include, for
example, insulins, autocoids, antimicrobials., antipyretics,
anti-inflammatories, surfactants, antibodies, antifungals,
antibacterials, analgesics, anorectics, antiarthritics,
antispasmodics, antidepressants, antipsychotics, antiepileptics,
antimalarials, antiprotozoals, anti-gout agents, tranquilizers,
anxiolytics, narcotic antagonists, antiparkinsonisms, cholinergic
agonists, antithyroid agents, antioxidants, antineoplastics, anti
virals, appetite suppressants, antiemetics, anticholinergics,
antihistaminics, antimigraines, bone modulating agents,
bronchodilators and anti-asthma drugs, chelators, antidotes and
antagonists, contrast media, corticosteroids, mucolytics, cough
suppressants and nasal decongestants, lipid regulating drugs,
general anesthetics, local anesthetics, muscle relaxants,
nutritional agents, parasympathomimetics, prostaglandins,
radio-pharmaceuticals, diuretics, antiarrhythmics, antiemetics,
immunomodulators, hematopoietics, anticoagulants and thrombolytics,
coronary, cerebral or peripheral vasodilators, hormones,
contraceptives, diuretics, antihypertensives, cardiovascular agents
such as cardiotonic agents, narcotics, vitamins, vaccines, and the
like.
[0045] Preferably, the active agent is a high-dose therapeutic.
Such high dose therapeutics include antibiotics, such as amikacin,
gentamicin, colistin, tobramycin, amphotericin B. Others include
mucolytic agents such as N-acetylcysteine, Nacystelyn, alginase,
mercaptoethanol and the like. Antiviral agents such as ribavirin,
gancyclovir, and the like, diamidines such as pentamidine and the
like and proteins such as antibodies are also contemplated.
[0046] Currently, at least three classes of lung surfactant
replacements for the treatment of respiratory diseases are
contemplated: (1) "natural"; (2) "synthetic"; and "biomimetic".
Natural surfactant replacements are prepared from animal lungs by
lavage or extraction with organic solvents, and purified by
chromatography (see, e.g., Creuwels et al., 1997 Lung 175:1-39;
Notter and Wang, 1997, Reviews in Chemical Engineering 13:1-118;
and Kattwinkel, 1998, Clinics in Perinatology 25:17-32). A number
of animal-derived surfactant replacements are FDA-approved (see,
e.g., Kattwinkel, 1998, Clinics in Perinatology 25:17-32; Gortner,
1990, Lung 168 (Suppl):864-869; Kendig et al., 1991, N. Engl. J.
Med. 324:865-871; Collaborative European Multicenter Study Group.
Surfactant replacement therapy in severe neonatal respiratory
distress syndrome: An international randomized clinical trial. 1988
Pediatrics 82:683-691. Synthetic surfactant replacements are by
definition protein-free, and are made from synthetic phospholipids
with added chemical agents (lipids or detergents) to facilitate
adsorption and spreading (see, e.g., Morley et al., 1981 Lancet
i:64-68 and Phibbs. et al., 1991, Pediatrics 88:1-9). A third class
of formulations is the "biomimetic lung surfactants." Biomimetic
surfactants are designed to mimic the biophysical characteristics
of natural lung surfactant while not sharing its precise molecular
composition. These formulations contain synthetic phospholipid
mixtures in combination with recombinantly-derived or
chemically-synthesized peptide analogs to SP-B and/or SP-C (for a
review of biomimetic surfactants, see, e.g., McLean and Lewis,
1995, Life Sciences 56:363-378). All three classes of surfactants
are contemplated for use in the formulations of the present
invention, unless otherwise noted. Therefore "surfactant" as used
herein includes natural, synthetic and biomimetic surfactants.
[0047] The preferred active agent is a substance or combination of
substances that is used for pulmonary prophylactic or rescue
therapy, such as a surfactant. A particularly preferred surfactant
is "KL.sub.4" (also referred to as "KL.sub.4 surfactant" which
incorporates a peptide mimetic of surfactant protein B (see WO
89/06657; WO 92/22315; WO 98/49191; U.S. Pat. Nos. 5,260,273;
5,164,369; 5,407,914; 5,789,381; 5,952,303; 6,013,619; 6,013,764;
6,120,795; and 6,613,734; all of which are expressly incorporated
by reference in their entirety for all purposes). KL.sub.4 is based
on Surfaxin.RTM. and preferably is present in the instant invention
as an aqueous dispersion of dipalmitoyl phosphatidylcholine,
phosphatidylglycerol, and essentially neutral lipid (e.g.,
cholesterol), and having essentially no 1-palmitoyl 2-oleoyl
phosphatidylglycerol and essentially no palmitic acid. Such
surfactant peptides are cationic peptides that can be derived from
animal sources or can be derived synthetically as discussed above.
When an animal-derived surfactant is employed, the surfactant is
often bovine or porcine-derived.
[0048] Preferably the peptide is present within an aqueous
dispersion of phospholipids and free fatty acids or fatty alcohols,
in this case 1,2 dipalmitoyl phosphatidylcholine (DPPC),
phosphatidylglycerol (DPPG), and cholesterol, and essentially no
palmitic acid (PA) or 1-palmitoyl 2-oleoyl phosphatidylglycerol
(POPG).
[0049] This invention contemplates the use of other cationic
peptides beyond KL.sub.4 surfactant. Preferably, cationic peptides
consist of at least about 10, preferably at least 11 amino acid
residues, and no more than about 60, more usually fewer than about
35 and preferably fewer than about 25 amino acid residues.
[0050] Many cationic peptides have been disclosed in the art. See,
for example, U.S. Pat. No. 5,164,369, which is hereby incorporated
by reference in its entirety for all purposes. Examples of cationic
peptides include KLLLLKLLLLKLLLLK (KL.sub.4), DLLLLDLLLLDLLLLDLLLLD
(DL4), RLLLLRLLLLRLLLLRLLLLR (RL4), RLLLLLLLLRLLLLLLLLRLL (RL8),
RRLLLLLLLRRLLLLLLLRRL (R2L7), RLLLLCLLLRLLLLLCLLLR,
RLLLLLCLLLRLLLLCLLLRLL, and
RLLLLCLLLRLLLLCLLLRLLLLCLLLRDLLLDLLLDLLLDLLLDLLLD, and polylysine,
magainans, defensins, polymyxins, iseganan, histatin and the like.
Preferably, the cationic peptide is KL.sub.4.
[0051] "Surfactant activity" for a protein or polypeptide is
defined as the ability, when combined with lipids, either alone or
in combination with other proteins, to exhibit activity in the in
vivo assay of Robertson1980, Lung 158:57-68. In this assay, the
sample to be assessed is administered through an endotracheal tube
to fetal rabbits or lambs delivered prematurely by Caesarian
section. (These "preemies" lack their own PS, and are supported on
a ventilator.) Measurements of lung compliance, blood gases and
ventilator pressure provide indices of activity. Preliminary
assessment of activity may also be made by an in vitro assay, for
example that of King and Clements, 1972, Am. J. Physiol.
223:715-726, or that illustrated below which utilizes a measurement
of surface tension at a air-water interface when a protein or
polypeptide is admixed with a phospholipid and a neutral lipid.
[0052] Examples of phospholipids useful in surfactant compositions
include native and/or synthetic phospholipids. Phospholipids that
can be used include phosphatidylcholines, phospatidylglycerols,
phosphatidylethanolamines, phosphatidylserines, phosphatidic acids,
and phosphatidylethanolamines. Exemplary phospholipids include 1,2
dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol
(DPPG), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl
phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine
(DSPC), diphytanoyl phosphatidylcholine, nonadecanoyl
phosphatidylcholine, arachidoyl phosphatidylcholine, dioleoyl
phosphatidylcholine (DOPC) (C18:1), dipalmitoleoyl
phosphatidylcholine (C16:1), linoleoyl phosphatidylcholine (C18:2),
dipalmitoyl phosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine (DOPE), dioleoyl
phosphatidylglycerol (DOPG), palmitoyloleoyl phosphatidylglycerol
(POPG), distearoylphosphatidylserine (DSPS) soybean lecithin, egg
yolk lecithin, sphingomyelin, phosphatidylinositols,
diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidic
acids, Egg phosphatidylcholine (EPC).
[0053] In one preferred embodiment, the phospholipids useful in
surfactant compositions of the invention include 1,2 dipalmitoyl
phosphatidylcholine (DPPC) and phosphatidylglycerol and essentially
no palmitoyloleoyl phosphatidylglycerol (POPG). In another
preferred embodiment, the phosphatidylglycerol is saturated,
non-saturated or semi-saturated. In another preferred embodiment,
the saturated phosphatidylglycerol is dipalmitoyl
phosphatidylglycerol (DPPG).
[0054] Examples of neutral lipids useful in surfactant compositions
of the invention include native and/or synthetic neutral lipids.
Neutral lipids can include a fatty acid, a fatty acid ester, a
fatty acid alcohol, cholesterol, corticosteroid,
glucocorticosteroid, trifluorinate glucocorticoid, .beta.2 agonist,
plant sterol, phospholipid, phosphatidylcholine, phosphatidyl
ethanolamine, phosphatidylinositol, sphingomyelin, diglycerides,
diolein, dipalmitolein, mixed caprylin-caprin, triglycerides,
triolein, tripalmitolein, trilinolein, tricaprylin, or trilaurin.
In a preferred embodiment, the essentially neutral lipid is
cholesterol.
[0055] Examples of fatty acids, fatty acid esters and fatty acid
alcohols useful in surfactant compositions include methyl
palmitate, cholesteryl palmitate and the like, palmitic acid, cetyl
alcohol, lauric acid, myristic acid, stearic acid, phytanic acid,
dipalmitic acid, and the like. In a preferred embodiment, the
surfactant comprises essentially no palmitic acid.
[0056] The term "medium" refers to both aqueous and non-aqueous
mediums. The preferred medium is chosen so as not cause any adverse
effect on the biological activity of the active agent being
delivered.
[0057] Preferably, the non-aqueous mediums can include, for
example, hydrogen-containing chlorofluorocarbons, fluorocarbons
certain organic compounds and admixtures thereof. To provide some
adjunctive respiratory support, and to provide efficient lung
filling in the degassed state, the perfluorocarbon liquid should
have an oxygen solubility greater than about 40 ml/100 ml.
Representative perfluorocarbon liquids include, but are note
limited to, FC-84, FC-72, RM-82, FC-75 (3M Company, Minneapolis,
Minn.), RM-101 (MDI Corporation, Bridgeport, Conn.),
dimethyladamantane (Sun Tech, Inc.), trimethylbicyclononane (Sun
Tech, Inc.), and perfluorodecalin (Green Cross Corp., Japan),
combinations thereof and the like.
[0058] Preferably, when an aqueous medium is employed, the medium
is a water-containing liquid. Suitable mediums include isotonic
ionic solutions preferably buffered to within 1 pH unit of
physiologic pH (7.3). The medium should be free of pathogens and
other deleterious materials and can be composed of pure water but
also optionally can include up to about 20% by volume and
preferably up to about 5% of nontoxic organic liquids such as
oxy-group containing liquids such as alcohols, esters, ethers,
ketones and the like. In selecting organic components it is
important to avoid materials which are likely to give rise to
undesired reactions such as intoxication, sedation, and the like.
Preferably, the medium is saline or tromethamine buffer.
[0059] "Respiratory" refers to the process by which oxygen is taken
into the body and carbon dioxide is discharged, through the bodily
system including the nose, throat, larynx, trachea, bronchi and
lungs.
[0060] "Respiratory disease", "respiratory disorder", "respiratory
condition", and "respiratory syndrome" refer to any one of several
ailments that involve inflammation and affect a component of the
respiratory system including especially the trachea, bronchi and
lungs. Examples of such ailments include acute alveolar disease,
obstructive respiratory disease (e.g., asthma; bronchitis; and
chronic obstructive pulmonary disease, referred to as COPD), upper
airway disease (e.g., such as otitis media, rhinitis/sinusitis and
sleep apnea), insterstitial lung disease, allergy, and respiratory
infection (e.g., pneumonia, pneyumocystis carinii, and respiratory
syncitial virus (RSV)).
[0061] Specific examples of acute alveolar disease include acute
lung injury (ALI), acute respiratory distress syndrome (ARDS),
meconium aspiration syndrome (MAS) and respiratory distress
syndrome (RDS). ALI is associated with conditions that either
directly or indirectly injure the air sacs of the lung, the
alveoli. ALI is a syndrome of inflammation and increased
permeability of the lungs with an associated breakdown of the
lungs' surfactant layer. The most serious manifestation of ALI is
ARDS. Among the causes of ALI are complications typically
associated with certain major surgeries, mechanical ventilator
induced lung injury (often referred to as VILI), smoke inhalation,
pneumonia, and sepsis.
[0062] ARDS in adults is a life-threatening disorder for which no
approved therapies exist anywhere in the world. It is characterized
by an excess of fluid in the lungs and decreased oxygen levels in
the patient. One prominent characteristic of this disorder is the
destruction of surfactants naturally present in lung tissue. The
conditions are caused by illnesses including pneumonia and septic
shock (a toxic condition caused by infection) and events such as
smoke inhalation, near drowning, industrial accidents and other
traumas.
[0063] MAS in full term infants is a condition in which full-term
infants are born with meconium in their lungs that depletes the
natural surfactant in their lungs. Meconium is an infant's first
bowel movement in its mother's womb and when inhaled, MAS can
occur. MAS can be life-threatening as a result of the failure of
the lungs.
[0064] RDS in premature infants is a condition in which premature
infants are born with an insufficient amount of their own natural
surfactant. Premature infants born prior to 32 weeks gestation have
not fully developed a natural lung surfactant and therefore need
treatment to sustain life. This condition often results in the need
for mechanical ventilation.
[0065] COPD is an umbrella term as used herein to describe lung
disease associated with airflow obstruction. Most generally,
emphysema, chronic bronchitis and chronic asthma either alone or in
combinations fall into this category.
[0066] Asthma is a common disease characterized by sudden
constriction and inflammation of the lungs. Constriction of the
upper airway system occurs when the airway muscles tighten, while
inflammation is a swelling of the airways usually due to an
allergic reaction caused by an airborne irritant. Both of these
events cause airways to narrow and can result in wheezing,
shortness of breath and chest tightness. Several studies have shown
that surfactant damage and dysfunction is a significant component
of asthma. Airway constriction occurs when there is a surfactant
dysfunction in the airways of the deep lung of the type that
develops during an asthma attack.
[0067] In addition, the compositions and methods of the present
invention are useful in the treatment of other respiratory diseases
and disorders, such acute bronchitis, bronchiectasis, pneumonia
(including ventilator-associated pneumonia, nosocomial pneumonia,
viral pneumonia, bacterial pneumonia, mycobacterial pneumonia,
fungal pneumonia, eosinophilic pneumonia, and Pneumocystis carinii
pneumonia), tuberculosis, cystic fibrosis (CF), emphysema radiation
pneumonitis, inflammation caused by smoking, pulmonary edema,
pneumoconiosis, sarcoidiosis, silicosis, asbestosis, berylliosis,
coal worker's pneumonoconiosis (CWP), byssinosis, interstitial lung
diseases (ILD) such as idiopathic pulmonary fibrosis, ILD
associated with collagen vascular disorders, systemic lupus
erythematosus, rheumatoid arthritis, ankylosing spondylitis,
systemic sclerosis, and pulmonary inflammation that is a result of
or is secondary to another disorder such as influenza.
[0068] "Inflammation" or "inflammatory response" refer to an innate
immune response that occurs when tissues are injured by bacteria,
trauma, toxins, heat, or any other cause. The damaged tissue
releases compounds including histamine, bradykinin, and serotonin.
Inflammation refers to both acute responses (i.e., responses in
which the inflammatory processes are active) and chronic responses
(i.e., responses marked by slow progression and formation of new
connective tissue). Acute and chronic inflammation can be
distinguished by the cell types involved. Acute inflammation often
involves polymorphonuclear neutrophils; whereas chronic
inflammation is normally characterized by a lymphohistiocytic
and/or granulomatous response. Inflammation includes reactions of
both the specific and non-specific defense systems. A specific
defense system reaction is a specific immune system reaction
response to an antigen (possibly including an autoantigen). A
non-specific defense system reaction is an inflammatory response
mediated by leukocytes incapable of immunological memory. Such
cells include granulocytes, macrophages, neutrophils and
eosinophils. Examples of specific types of inflammation are diffuse
inflammation, focal inflammation, croupous inflammation,
interstitial inflammation, obliterative inflammation,
parenchymatous inflammation, reactive inflammation, specific
inflammation, toxic inflammation and traumatic inflammation.
[0069] "Polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0070] "Amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics
that function in a manner similar to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0071] Amino acids can be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission
(see Table 1 below). Nucleotides, likewise, can be referred to by
their commonly accepted single-letter codes.
TABLE-US-00001 TABLE 1 SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr
L-tyrosine G Gly L-glycine F Phe L-phenylalanine M Met L-methionine
A Ala L-alanine S Ser L-serine I He L-isoleuine L Leu L-leucine T
Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine H His
L-histidine Q Gln L-glutamine E Glu L-glutamic acid W Trp
L-tryptohan R Arg L-arginine D Asp L-aspartic acid N Asn
L-asparagine C Cys L-cysteine
[0072] It should be noted that all amino acid residue sequences are
represented herein by formulae whose left to right orientation is
in the conventional direction of amino-terminus to
carboxy-terminus. Furthermore, it should be noted that a dash at
the beginning or end of an amino acid residue sequence indicates a
bond to a radical such as H and OH (hydrogen and hydroxyl) at the
amino- and carboxy-termini, respectively, or a further sequence of
one or more amino acid residues.
[0073] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0074] "Recombinant" when used with reference, e.g., to a cell, or
nucleic acid, protein, or vector, indicates that the cell, nucleic
acid, protein or vector, has been modified by the introduction of a
heterologous nucleic acid or protein or the alteration of a native
nucleic acid or protein, or that the cell is derived from a cell so
modified. Thus, for example, recombinant cells express genes that
are not found within the native (non-recombinant) form of the cell
or express native genes that are otherwise abnormally expressed,
under expressed or not expressed at all.
[0075] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic, and desirable, and includes excipients
that are acceptable for veterinary use as well as for human
pharmaceutical use. Such excipients can be solid, liquid,
semisolid, or, in the case of an aerosol composition, gaseous.
[0076] "Pharmaceutically acceptable salts and esters" means salts
and esters that are pharmaceutically acceptable and have the
desired pharmacological properties. Such salts include salts that
can be formed where acidic protons present in the compounds are
capable of reacting with inorganic or organic bases. Suitable
inorganic salts include those formed with the alkali metals, e.g.
sodium and potassium, magnesium, calcium, and aluminum. Suitable
organic salts include those formed with organic bases such as the
amine bases, e.g. ethanolamine, diethanolamine, triethanolamine,
tromethamine, N methylglucamine, and the like. Such salts also
include acid addition salts formed with inorganic acids (e.g.
hydrochloric and hydrobromic acids) and organic acids (e.g., acetic
acid, citric acid, maleic acid, and the alkane- and arene-sulfonic
acids such as methanesulfonic acid and benzenesulfonic acid).
Pharmaceutically acceptable esters include esters formed from
carboxy, sulfonyloxy, and phosphonoxy groups present in the
compounds, e.g., C1-6 alkyl esters. When there are two acidic
groups present, a pharmaceutically acceptable salt or ester can be
a mono-acid-mono-salt or ester or a di-salt or ester; and similarly
where there are more than two acidic groups present, some or all of
such groups can be salified or esterified. Compounds named in this
invention can be present in unsalified or unesterified form, or in
salified and/or esterified form, and the naming of such compounds
is intended to include both the original (unsalified and
unesterified) compound and its pharmaceutically acceptable salts
and esters. Also, certain compounds named in this invention can be
present in more than one stereoisomeric form, and the naming of
such compounds is intended to include all single stereoisomers and
all mixtures (whether racemic or otherwise) of such
stereoisomers.
[0077] "Pharmaceutically acceptable", "physiologically tolerable"
and grammatical variations thereof, as they refer to compositions,
carriers, diluents and reagents, are used interchangeably and
represent that the materials are capable of administration to or
upon a human without the production of undesirable physiological
effects to a degree that would prohibit administration of the
composition.
[0078] Except when noted, "subject" or "patient" are used
interchangeably and refer to mammals such as human patients and
non-human primates, as well as experimental animals such as
rabbits, dogs, cats, rats, mice, and other animals. Accordingly,
"subject" or "patient" as used herein means any mammalian patient
or subject to which the compositions of the invention can be
administered. In some embodiments of the present invention, the
patient will be suffering from a condition that causes a
respiratory disease or condition, e.g., ARDS or RDS. In an
exemplary embodiment of the present invention, to identify subject
patients for treatment with a pharmaceutical composition comprising
one or more surfactant compositions according to the methods of the
invention, accepted screening methods are employed to determine the
status of an existing disease or condition in a subject or risk
factors associated with a targeted or suspected disease or
condition. These screening methods include, for example,
examinations to determine whether a subject is suffering from a
respiratory disease. These and other routine methods allow the
clinician to select subjects in need of therapy.
[0079] "Treating" refers to any indicia of success in the treatment
or amelioration or prevention of, for example, a respiratory
disease such as ARDS or RDS, including any objective or subjective
parameter such as abatement; remission; diminishing of symptoms or
making the disease condition more tolerable to the patient; slowing
in the rate of degeneration or decline; or making the final point
of degeneration less debilitating. The treatment or amelioration of
symptoms can be based on objective or subjective parameters;
including the results of an examination. Accordingly, the term
"treating" includes the administration of the compounds or agents
of the present invention to prevent or delay, to alleviate, or to
arrest or inhibit development of the symptoms or conditions
associated with respiratory disease.
[0080] "Therapeutic effect" refers to the reduction, elimination,
or prevention of the disease, symptoms of the disease, or side
effects of the disease in the subject. A "therapeutically effective
amount" means the amount that, when administered to a subject for
treating a disease, condition or disorder, is sufficient to effect
treatment for that disease.
[0081] "Therapeutic compound" as used herein refers to a compound
useful in the prophylaxis or treatment of a respiratory disease or
condition.
[0082] "Therapeutically effective" as used herein refers to a
characteristic of an amount of a therapeutic compound, or a
characteristic of amounts of combined therapeutic compounds in
combination therapy. The amount or combined amounts achieve the
goal of preventing, avoiding, reducing or eliminating the
respiratory disease or condition.
[0083] "Pro-drug" refers to a compound that is a drug precursor
which, following administration to a subject and subsequent
absorption, is converted to an active species in vivo via some
process, such as a metabolic process. Other products from the
conversion process are easily disposed of by the body. The more
preferred pro-drugs are those involving a conversion process that
produces products that are generally accepted as safe.
[0084] "Concomitant administration", "concurrent administration",
or "co-administration" as used herein includes administration of
the active agents (e.g., the KL.sub.4 composition), in conjunction
or combination, together, or before or after each other. The
multiple agent(s) may be administered by the same or by different
routes, simultaneously or sequentially, as long as they are given
in a manner sufficient to allow all agents to achieve effective
concentrations at the site of action. A person of ordinary skill in
the art, would have no difficulty determining the appropriate
timing, sequence and dosages of administration for particular drugs
and compositions of the present invention.
[0085] B. Pulmonary Surfactants--Overview
[0086] Naturally-occurring pulmonary surfactant is a complex
mixture of lipids and proteins that promotes the formation of a
monolayer at the alveolar air-water interface and, by reducing the
surface tension and stabilizing the monolayer to withstand high
surface pressures generated during compression, prevents collapse
of the alveolus during expiration. Premature infants, and
occasionally full term neonates, can lack sufficient endogenous
surfactant, or lack a fully functional surfactant for normal lung
function. This can give rise to a condition termed respiratory
distress syndrome (RDS) which can necessitate mechanical
ventilation and administration of hyperbaric oxygen. Such
intervention, unfortunately, can produce permanent damage to lung
tissue and can cause retinopathy of prematurity (ROP) leading to
blindness.
[0087] Pulmonary surfactant (PS) lines the alveolar epithelium of
mature mammalian lungs. Natural PS has been described as a
"lipoprotein complex" because it contains both phospholipids and
apoproteins that interact to reduce surface tension at the lung
air-liquid interface. Natural surfactant contains several lipid
species of which dipalmitoyl phosphatidylcholine (DPPC) is the
major component together with phosphatidylglycerol (PG) and
palmitic acid (PA). At least four specific proteins are associated,
termed SP-A, SP-B, SP-C and SP-D. Of these four, SP-B and SP-C are
distinct, low molecular weight, relatively hydrophobic proteins
that have been shown to enhance the surface-active properties of
surfactant phospholipid mixtures. It is believed that they
facilitate transfer of lipids from the bulk phase lamellar
organization to the air-water interface and also stabilize the
lipid monolayer during expiration. The structure of SP-B (which is
alternatively referred to as SP18) is unusual in that charged amino
acids (predominantly basic) are located at fairly regular intervals
within stretches of otherwise hydrophobic residues. For the domain
consisting of residues 59-80 of the native SP-B sequence, these
charged groups have been shown to be necessary for biological
activity. In addition, natural and synthetic peptides which are
modeled on this hydrophobic-hydrophilic domain when combined with
DPPC and PG, exhibit good surfactant activity.
[0088] Surfactant is stored in lung epithelial cells in the form of
lamellar bodies and, following export, it undergoes a structural
transition to form tubular myelin before giving rise to a monolayer
at the air-water interface. It has been proposed that surfactant
proteins SP-A, -B and -C can facilitate these structural
transitions and stabilize the lipid monolayer during expansion and
contraction of the alveolus; however, an understanding of
lipid-protein interactions at the molecular level is presently
lacking. The present invention, therefore, has important
implications not only with respect to the treatment of RDS in
infants as well as adults, but also because of the insight it can
provide into lipid-protein interactions in general.
[0089] Several exogenous surfactant formulations are currently used
in the treatment of infant RDS. While these have reduced morbidity
and mortality, continual improvements are needed. In particular,
because of the complications that can arise due to mechanical
ventilation and administration of hyperbaric oxygen, the sooner
normal lung function can be established in a premature infant the
more favorable will be the clinical outcome.
[0090] Consistent with the foregoing, important characteristics in
an exogenous surfactant include the ability to spread rapidly to
the alveoli following administration and the ability to maintain a
stable monolayer at the alveolar air-water interface so that
repeated treatment is not required. Thus, various compounds and
compositions that are useful in the preparation of superior
exogenous surfactants are disclosed herein.
[0091] C. Surfactant Compositions
[0092] A surfactant composition of the present invention can
contain any of a variety of pharmaceutically acceptable compounds
having surfactant activity to form a pulmonary surfactant (PS)
useful in the treatment of respiratory distress syndrome. Typically
a surfactant composition has admixed therein one or more
phospholipids. Phospholipids useful in forming alveolar surfactants
are well known in the art. See Notter, R. H. and D. L. Shapiro,
1987, Clin. Perinatol 14:433-79, for a review of the use of both
native and synthetic phospholipids for surfactants.
[0093] The surfactant compositions of this invention that are
prepared using a protein, a polypeptide, an amino acid
residue-containing molecule, or another organic molecule of the
present invention having surfactant activity (collectively,
"surfactant molecules"), that can include one or more phospholipids
and neutral lipids, are well suited for the treatment of
respiratory diseases such as RDS and ARDS. Such surfactant
compositions typically range from dilute to concentrated, depending
upon the intended use as described further herein.
[0094] Thus a surfactant composition can contain from as little as
about 0.05 to almost 100 weight percent lipid, so long as the
resulting composition has surfactant activity. By weight percent is
meant the percentage of a compound by weight in a composition by
weight. Thus, a composition having 50 weight percent lipid
contains, for example, 50 grams lipid per 100 grams total
composition. Typically, a surfactant composition contains 0.1 to 50
weight percent lipid, although higher concentrations of lipid can
be used for "bolus" methods and for preparing more dilute
surfactant compositions from a concentrated stock.
[0095] Exemplary surfactant compositions containing phospholipid
and a surfactant molecule can contain, therefore, 0.1, 1, 10, 50,
80, to almost 100 weight percent lipid and about 50, 20, 10, to
less than 1 weight percent surfactant molecule. Similarly,
exemplary surfactant compositions containing neutral lipid and a
surfactant molecule can contain, therefore 0.1, 1, 10, 50, 80, to
almost 100 weight percent lipid and about 50, 20, 10, to less than
1 weight percent surfactant molecule The surfactant composition is
prepared by admixing a solution of a surfactant molecule with a
dispersion of lipid components, or by admixing the surfactant
molecule with a dispersion of lipids, or by admixing the surfactant
molecule and phospholipids directly in the presence of organic
solvent.
[0096] Lipid-based surfactant compositions of the present invention
are generally sterile colloidal dispersions containing a surfactant
molecule of the present invention that has been combined with the
lipids and a free fatty acid, alcohol or neutral lipid in an
organic solvent system, dried, and then rehydrated. Because of the
large variety of compounds and substances that have surfactant
activity, it is to be understood that a surfactant composition
useful in the present invention can be free from detectable protein
or polypeptide, and contains only phospholipids, aqueous medium
and/or buffers. In various preferred embodiments of the present
invention, pulmonary surfactants that are effective in treating
respiratory diseases, such as RDS or ARDS, comprising an effective
amount of a surfactant molecule admixed with a pharmaceutically
acceptable phospholipids and neutral lipid are disclosed. In one
preferred embodiment, the surfactant molecule is a polypeptide or
protein; in other preferred embodiments, the surfactant molecule is
an organic molecule displaying surfactant activity which can
comprise amino acid residues, modified amino acids, amino acid
derivatives, amino acid analogs, and the like molecules, or other
organic molecules mimicking that activity.
[0097] Methods for determining the optimal polypeptide:lipid weight
ratios for a given polypeptide-phospholipid-neutral lipid
combination are well known. Therapeutically effective ratios are in
the range of about 1:5 to about 1:10,000, preferably about 1:7 to
about 1:5,000, more preferably about 1:10 to about 1:1000, and more
preferably about 1:15 to about 1:100. The lipid portion of a
surfactant composition of the present invention is preferably about
50 to about 90, more preferably about 50 to about 75, weight
percent dipalmitoylphosphatidylcholine (DPPC) with the remainder
comprising, phosphatidylglycerol (PG), essentially neutral lipid or
admixtures thereof.
[0098] Phospholipids useful in forming the present surfactant
compositions are well known in the art. (See, e.g., Notter, R. H.
and D. L. Shapiro 1988, for a review of the use of both native and
synthetic phospholipids for surfactants.). Methods and materials
useful in the preparation of preferred surfactant compositions as
disclosed herein are also described in the Examples that follow.
See also WO 89/06657; WO 92/22315; WO 98/49191; U.S. Pat. Nos.
5,260,273; 5,164,369; 5,407,914; 5,789,381; 5,952,303; 6,013,619;
6,013,764; 6,120,795; and 6,613,734.
[0099] A pulmonary surfactant of the present invention is generally
prepared by admixing a solution of a subject polypeptide with a
suspension of liposomes or by admixing the subject polypeptide (or
other organic surfactant molecule) and lipids directly in the
presence of organic solvent. The solvent is then removed by
dialysis or evaporation under nitrogen and/or exposure to vacuum or
by other appropriate techniques.
[0100] A pulmonary surfactant composition is preferably formulated
for endotracheal administration, e.g., typically as a liquid
suspension, as a dry powder "dust", or as an aerosol. Those. of
skill in the art will appreciate that surfactant compositions of
the present invention can be formulated for a variety of uses and
methods of administration including, without limitation, liquid
suspensions or aerosols which can be used for lavage.
[0101] For example, a surfactant (surfactant molecule-lipid
micelle) can be suspended in a liquid with a pharmaceutically
acceptable excipient such as water, saline, dextrose, glycerol and
the like. A surfactant-containing therapeutic composition can also
contain small amounts of non-toxic auxiliary substances such as pH
buffering agents, including tromethamine acetate (TRIS acetate),
sodium acetate, sodium phosphate, and the like. To prepare a
surfactant in dust form, a surfactant is prepared as described
herein, then lyophilized and recovered as a dry powder. Surfactants
can be prepared by processes known per se and familiar to the
person skilled in the art, for example as described in WO95/32992.
Lyophilized preparations are disclosed, for example, in WO
97/35882, WO 91/00871 and DE 3229179. WO 97/26863 describes a
process for the preparation of powdered pulmonary surfactant
formulations by spray drying. Other approaches such as
supercritical fluid extraction can also be envisioned as means to
produce dry powders.
[0102] If it is to be used in aerosol administration, a subject
surfactant is supplied in finely divided form along with an
additional surfactant and propellant. Typical surfactants which can
be administered are phospholipids and esters. However, it is
preferred, in the present case, to utilize the other components of
the surfactant complex, DPPC and PG together with essentially
neutral lipid. Useful propellants are typically gases at ambient
conditions, and are condensed under pressure. Lower alkane and
fluorinated alkane, such as Freon, can be used. The aerosol is
packaged in a container equipped with a suitable valve so that the
ingredients can be maintained under pressure until released.
[0103] To prepare a surfactant composition, the surfactant molecule
or polypeptide molecule is dissolved in an organic solvent that
maintains the molecule in its monomeric, substantially
aggregate-free form. Preferred such solvents can be polar or
non-polar and exhibit solubility parameter delta (8) values in the
range of about 9 to about 15 (calcm.sup.3).sup.1/2 or about 9
Hildebrand units (H) to about 15H.
[0104] Particularly preferred solvents are the hydrogen bonded
solvents such as the C.sub.1 to C.sub.4 alipathic alcohols, i.e.,
methanol (.delta.=14.5H), ethanol (.delta.=12.7H), n-propanol
(.delta.=11.9H), iso-propanol (.delta.=11.5H), n-butanol
(.delta.=11.4H), iso-butanol (.delta.=10.8H) and t-butanol (?).
Among halogenated solvents particularly preferred are
trifluoroethanol (TFE) and chloroform (.delta.=9.3H). Mixtures or
blends of aliphatic alcohols and halogenated solvents can be
utilized as well. In a preferred method for producing a surfactant
composition, the polypeptide or other surfactant molecule is
dissolved in the organic solvent together with the phospholipids,
and the resulting solution is combined with an aqueous buffer
solution. The resulting dispersion is then dialyzed or evaporated
to remove the organic solvent leaving an aqueous dispersion.
Alternatively, the organic and aqueous solvent components can be
removed by evaporation and vacuum. The dried, or partially dried
lipid/polypeptide mixture thus produced is rehydrated in an aqueous
buffer system to produce the surfactant dispersion.
[0105] The present invention also contemplates a variety of
surfactant compositions, particularly lipid-based surfactants.
Thus, in one preferred embodiment, the invention discloses a
lipid-based surfactant composition prepared from a polypeptide
comprising about 10 amino acid residues and no more than about 60
amino acid residues and is constituted by alternating groupings of
charged amino acid residues and uncharged amino acid residues, and
a pharmaceutically acceptable phospholipid, wherein the polypeptide
is present in an amount sufficient to increase the surfactant
activity of the composition above that of the phospholipid.
[0106] In another preferred variation, a surfactant composition of
the present invention comprises a surfactant molecule constituted
by alternating groupings of charged and uncharged residues; the
residues can be amino acids, modified amino acids, amino acid
analogs or derivatives, and the like. Molecules having surfactant
activity as disclosed herein are especially preferred for use in
compositions of the present invention.
[0107] In various preferred embodiments of the present invention,
as noted previously, surfactant compositions also comprise one or
more phospholipids. The polypeptide:lipid weight ratio is in the
range of about 1:7 to about 1:1,000 in various preferred surfactant
compositions of the present invention. Suitable phospholipids are
preferably selected from the following group:
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(dipalmitoylphosphatidylcholine, DPPC); phosphatidyl glycerol (PG);
and an admixture of DPPC and PG in a weight ratio of about 3:1.
[0108] The surfactant compositions of the present invention can
have essentially no 1-palmitoyl 2-oleoyl phosphatidylglycerol and
no palmitic acid, in various preferred embodiments.
[0109] In one preferred embodiment, the phospholipids useful in
surfactant compositions of the invention include 1,2 dipalmitoyl
phosphatidylcholine (DPPC) and phosphatidylglycerol and essentially
no palmitoyloleoyl phosphatidylglycerol (POPG). In another
preferred embodiment, the phosphatidylglycerol is saturated,
non-saturated or semi-saturated. In another preferred embodiment,
the saturated phosphatidylglycerol is dipalmitoyl
phosphatidylglycerol (DPPG). If an admixture of DPPC and PG is
selected, it is preferable that DPPC and PG be present in a weight
ratio of preferably about 4:1; more preferably about 7:3; and most
preferably 3:1.
[0110] For example, in one embodiment of the present invention, a
30 mg/ml surfactant composition of the present invention comprises,
in each ml of composition, 0.801 mg KL.sub.4 peptide, 22.5 mg DPPC,
7.5 mg DPPG, 1.575 mg cholesterol, and essentially no PA or POPG.
In various embodiments, the surfactant is prepared aseptically and
is supplied in vials containing a sufficient volume to deliver
either various volumes of the dispersion.
[0111] Thus, in one exemplary formulation, a preparation having a
phospholipid concentration of about 30 mg/mL administered at a
dosage volume of about 5 mL/kg would result in a dose of about 150
mg/kg. Similarly, an exemplary preparation having a lipid
concentration of about 60 mg/mL administered at a dosage volume of
about 5 mL/kg would result in a dose of about 300 mg/kg.
[0112] One preferred final surfactant composition comprises a
sterile colloidal dispersion containing surfactant polypeptide (or
other surfactant molecules according to the present invention). By
way of illustration, a drug product/surfactant composition
containing KL.sub.4 peptide is described as exemplary.
[0113] Peptide is preferably combined with lipids in an organic
solvent system which is then added to an aqueous buffer system or
vice a versa. The organic solvent is removed from the resulting
aqueous-organic mixture by thin film evaporation allowing the
colloidal dispersion to form. All processing is performed
aseptically.
[0114] One exemplary composition comprises surfactant peptide and a
lipid component. In one embodiment, the lipid component comprises
1,2 dipalmitoyl phosphatidylcholine (DPPC) and
phosphatidylglycerol, essentially neutral lipid, and having
essentially no palmitoyloleoyl phosphatidylglycerol (POPG). In
another preferred embodiment, the phosphatidylglycerol is
saturated, non-saturated or semi-saturated. In another preferred
embodiment, the saturated phosphatidylglycerol is dipalmitoyl
phosphatidylglycerol (DPPG).
[0115] For example, a surfactant composition including KL.sub.4
peptide can be prepared from an admixture of DPPC and PG in a 3:1
ratio by weight with cholesterol, 5.25% by weight compared with the
phospholipids, in an organic solvent. KL.sub.4 peptide is prepared
in the surfactant dispersion as 2.7% by weight of the phospholipid
concentration. Organic solvents can be removed from the
lipid/peptide mixture by evaporation under nitrogen and vacuum or
related means. A Tris buffer solution can be added to form
colloidal dispersions of the peptide-containing surfactant.
[0116] A Tham buffer system can also be included in a surfactant
composition of the present invention. (Tham is a buffering agent
also known as Tris, tromethamine, and
tris(hydroxymethyl)aminomethane.) In various preferred embodiments,
the compositions have a pH range of about 6.5-8.0.
[0117] A wide variety of surfactant molecules, proteins, and
polypeptides which are preferred for use according to the disclosed
methods are described above and in the sections that follow. Other
preferred components of surfactant compositions used as disclosed
herein include a variety of phospholipids and neutral lipid as
further described herein.
[0118] For example, currently there are a variety of known
surfactants described that have been used in related methods. These
surfactants are all suitable for use in the present invention
according to the discovery that dilute surfactant lavages are
beneficial. These surfactants include natural surfactants derived
from aqueous lavages of lungs of mammals, including but not limited
to bovine, porcine or ovine species, such as BLES, Infasurf or CLSE
(Calf Lung Surfactant, Forest Products), Alveofact (Thomae,
Germany); surfactant material extracted from animal lungs by, but
not limited to, organic solvents, such as Surfactant TA (Tokyo
Tanabe, Japan), Survanta (Beractant, Abbott Laboratories, Abbott
Park, Curosurf (Chiesi Farmaceutici, Parma, Italy). Surfactants can
comprise mixtures of phospholipids, spreading agents and proteins
or peptides. The phospholipids can be phosphatidyl choline (e.g.,
DPPC) and phosphatidylglycerol (e.g., DPPG). The spreading agents
increase the rate of spreading along an air-water interface and can
include cholesterol, detergents and the like. The proteins and
peptides can be any of those described herein or which otherwise
augment surfactant activity of phospholipids, and can be isolated
from natural sources, synthesized chemically or produced by
recombinant DNA methodologies, such as SP-C.
[0119] Details regarding the composition and methods of preparation
of these and other surfactants can be found in the following U.S.
Pat. Nos. 4,603,124, 5,013,720, 5,024,995, 5,171,737, 5,185,154,
5,238,920, 5,302,581, 5,547,937, 5,552,161, and 5,614,216, the
disclosures of which are hereby incorporated by reference.
[0120] A surfactant of the present invention is administered, as
appropriate to the dosage form, by endotracheal tube, by
bronchoscope, by cannula, by spray administration, or by
aerosolization (atomization, nebulization, dispersion and
deaggregation) of the suspension or dust into the inspired gas.
Amounts of PS between about 1.0 and about 500 mg/kg, and preferably
about 50 mg to about 500 mg/kg, and typically a dose of about 50
mg/kg, 100 mg/kg, 133 mg/kg, or 200 mg/kg, measured in terms of
total phospholipid content, are administered in one dose. For use
in newly born infants, one or two administrations are generally
sufficient. For adults, sufficient reconstituted surfactant complex
is preferably administered to produce a PO.sub.2 within the normal
range (see, e.g., Hallman et al., 1982, J Clin Inves 70:673-682).
It must be appreciated that the treatment regimen can vary from
individual to individual, depending on the severity of the
respiratory disease, the symptoms present, and other relevant
variables; thus, single or multiple doses can be administered to an
individual.
[0121] As disclosed herein, the invention contemplates the use of
both concentrated and dilute surfactant compositions, depending
upon the particular use, as described further herein. Concentrated
surfactant compositions are typically used for "bolus" type
administrations, whereas dilute surfactant compositions are
typically used for "lavage" type administrations.
[0122] Typically, a concentrated surfactant has from 20 to 200
milligrams (mg) of active surfactant compound per milliliter (ml),
more preferably about 25 to 100 mg/ml. A typical dilute surfactant
has active surfactant compound at a concentration of from about 0.1
to 20 mg/ml, and more preferably about 0.5 to 10 mg/ml.
[0123] Polypeptides suitable for preparing surfactants in
accordance with the present invention are further described in
Section D below.
[0124] D. Proteins and Polypeptides
[0125] A protein or polypeptide of the present invention (subject
protein or polypeptide) is characterized by its amino acid residue
sequence and novel functional properties. A subject protein or
polypeptide when admixed with a pharmaceutically acceptable
phospholipid forms a pulmonary surfactant having a surfactant
activity greater than the surfactant activity of the phospholipid
or phospholipid and lipid alone. For example, a protein or
polypeptide having a surfactant activity exhibits a lower .DELTA.P
when measured in a surfactant.
[0126] It is also to be understood that molecules comprising 60 or
more amino acid residues, i.e., protein molecules, can be useful in
surfactant compositions according to the present invention. While
the present disclosure focuses primarily upon polypeptide molecules
and molecules including amino acid residues, analogs, and/or other
organic molecules, proteins having alternating hydrophobic and
hydrophilic amino acid residue regions and proteins having
surfactant ability as described herein are also contemplated by,
and encompassed by, the present disclosures.
[0127] Molecules demonstrating surfactant activity which comprise
10 or fewer amino acid residues are also contemplated by the
present invention. For example, a molecule comprising five amino
acid residues linked to five amino acid derivatives or analogs can
be useful as disclosed herein, particularly if it has alternating
hydrophobic and hydrophilic amino acid residue regions and has
surfactant ability, as defined herein. Thus, molecules comprising
two to 100 amino acid residues having a configuration that
maximizes their interaction with the alveoli are contemplated by
the present invention. While larger molecules are somewhat more
difficult to synthesize, it should be appreciated by those of skill
in the relevant art that, as disclosed herein, even molecules
containing 60 or more amino acid residues (or their analogs) can be
excellent surfactants, provided they possess the disclosed
characteristics.
[0128] Polypeptides suitable for preparing lipid-based surfactants
in accordance with the present invention can be synthesized from
amino acids by techniques that are known to those skilled in the
polypeptide art. Summary of the many techniques available can be
found, for example, in Steward and Young, 1969, Solid Phase Peptide
Synthesis, W.H. Freeman Co., San Francisco, 1969, and Meienhofer,
1983, Hormonal Proteins and Peptides, Vol. 2, p. 46, Academic
Press, New York, for solid phase peptide synthesis, and Schroder
and Kubke, 1965, The Peptides, Vol. 1, Academic Press, New York,
for classical solution synthesis.
[0129] In general, these methods comprise the sequential addition
of one or more amino acid residues or suitably protected amino acid
residues to a growing peptide chain. Normally, either the amino or
carboxyl group of the first amino acid residue is protected by a
suitable, selectively removable protecting group. A different,
selectively removable protecting group is utilized for amino acids
containing a reactive side group (e.g., lysine).
[0130] Using a solid phase synthesis as exemplary, the protected or
derivatized amino acid is attached to an inert solid support
through its unprotected carboxyl or amino group. The protecting
group of the amino or carboxyl group is then selectively removed
and the next amino acid in the sequence having the complementary
(amino or carboxyl) group suitably protected is admixed and reacted
under conditions suitable for forming the amide linkage with the
residue already attached to the solid support. The protecting group
of the amino or carboxyl group is then removed from this newly
added amino acid residue, and the next amino acid (suitably
protected) is then added, and so forth. After all the desired amino
acids have been linked in the proper sequence, any remaining
terminal and side group protecting groups (and any solid support)
are removed sequentially or concurrently, to afford the final
polypeptide. That polypeptide is then washed by dissolving in a
lower aliphatic alcohol, and dried. The dried surfactant
polypeptide can be further purified by known techniques, if
desired. (Various methods. of preparing polypeptides of the present
invention are also described in WO 89/06657; WO 92/22315; WO
98/49191; U.S. Pat. Nos. 5,260,273; 5,164,369; 5,407,914;
5,789,381; 5,952,303; 6,013,619; 6,013,764; 6,120,795; and
6,613,734; all of which are expressly incorporated by reference in
their entirety for all purposes). In some embodiments, the
surfactant polypeptides are polypeptides that include amino acid
residue sequences having alternating charged and uncharged amino
acid residue regions. Polypeptides including amino acid residue
sequences having alternating hydrophobic and hydrophilic amino acid
residue regions are also preferred according to the present
invention. Particularly preferred surfactant polypeptides within
these groupings are further characterized as having at least about
4, more preferably at least about 8, and even more preferably at
least about 10, amino acid residues, and are generally not more
than about 60 amino acid residues in length.
[0131] In other embodiments, surfactant polypeptides of the present
invention are constituted by alternating groupings of charged amino
acid residues and uncharged amino acid residues as represented by
the formula [(Charged).sub.a
(Uncharged).sub.b].sub.C(Charged).sub.d, wherein a has an average
value of about 1 to about 5; b has an average value of about 3 to
about 20; c is 1 to 10; and d is 0 to 3. Organic surfactant
molecules not comprised solely of amino acid residues alone
preferably have a similar structure constituted by alternating
groupings of charged and uncharged (or hydrophilic/hydrophobic)
constituent molecules.
[0132] In one preferred embodiment, surfactant polypeptides include
a sequence having alternating groupings of amino acid residues as
represented by the formula (Z.sub.aJ.sub.b).sub.cZ.sub.d, wherein Z
is an amino acid residue independently selected from the group
consisting of R, D, E, and K; J is an .alpha.-aminoaliphatic
carboxylic acid; a has an average value of about 1 to about 5; b
has an average value of about 3 to about 20; c is 1 to 10; and d is
0 to 3.
[0133] In another embodiment, preferred polypeptides of the present
invention have alternating groupings of amino acids residue regions
as represented by the formula (B.sub.aU.sub.b).sub.cB.sub.d,
wherein B is an amino acid residue independently selected from the
group consisting of H, 5-hydroxylysine, 4-hydroxyproline, and
3-hydroxyproline; and U is an amino acid residue independently
selected from the group consisting of V, I, L, C, Y, and F. In one
preferred variation, B is an amino acid derived from collagen and
is preferably selected from the group consisting of
5-hydroxylysine, 4-hydroxyproline, and 3-hydroxyproline; a has an
average value of about 1 to about 5; b has an average value of
about 3 to about 20; c is 1 to 10; and d is 0 to 3.
[0134] In still another preferred embodiment, surfactant
polypeptides of the present invention include a sequence having
alternating groupings of amino acid residues as represented by the
formula (B.sub.aJ.sub.b).sub.cB.sub.d, wherein B is an amino acid
residue independently selected from the group consisting of H,
5-hydroxylysine, 4-hydroxyproline, and 3-hydroxyproline; and J is
an .alpha.-aminoaliphatic carboxylic acid; a has an average value
of about 1 to about 5; b has an average value of about 3 to about
20; c is 1 to 10; and d is 0 to 3.
[0135] In various embodiments including "J" in the relevant
formula, J is an .alpha.-aminoaliphatic carboxylic acid having four
to six carbons, inclusive. In other preferred variations, J is an
.alpha.-aminoaliphatic carboxylic acid having six or more carbons,
inclusive. In yet other variations, J is preferably selected from
the group consisting of .alpha.-aminobutanoic acid,
.alpha.-aminopentanoic acid, .alpha.-amino-2-methylpropanoic acid,
and .alpha.-aminohexanoic acid.
[0136] Another preferred embodiment discloses surfactant
polypeptides including a sequence having alternating groupings of
amino acid residues as represented by the formula
(Z.sub.aU.sub.b).sub.cZ.sub.d, wherein Z is an amino acid residue
independently selected from the group consisting of R, D, E, and K;
and U is an amino acid residue independently selected from the
group consisting of V, I, L, C, Y and F; from the group consisting
of V, I, L, C and F; or from the group consisting of L and C; a has
an average value of about 1 to about 5; b has an average value of
about 3 to about 20; c is 1 to 10; and d is 0 to 3.
[0137] In the foregoing formulae, Z and U, Z and J, B and U, and B
and J are amino acid residues that, at each occurrence, are
independently selected. In addition, in each of the aforementioned
formulae, a generally has an average value of about 1 to about 5; b
generally has an average value of about 3 to about 20; c is 1 to
10; and d is 0 to 3.
[0138] In one variation of the foregoing embodiments, Z and B are
charged amino acid residues. In other preferred embodiments, Z and
B are hydrophilic or positively charged amino acid residues. In one
variation, Z is preferably selected from the group consisting of R,
D, E and K. In a related embodiment, Z is preferably selected from
the group consisting of R and K. In yet another preferred
embodiment, B is selected from the group consisting of H,
5-hydroxylysine, 4-hydroxyproline, and 3-hydroxyproline. In one
preferred embodiment, B is H. In another preferred embodiment, B is
a collagen constituent amino acid residue and is selected from the
group consisting of 5-hydroxylysine, (6-hydroxylysine),
4-hydroxyproline, and 3-hydroxyproline.
[0139] In various disclosed embodiments, U and J are, preferably,
uncharged amino acid residues. In another preferred embodiment, U
and J are hydrophobic amino acid residues. In one embodiment, U is
preferably selected from the group consisting of V, I, L, C, Y, and
F. In another preferred embodiment, U is selected from the group
consisting of V, I, L, C, and F. In yet another preferred
embodiment, U is selected from the group consisting of L and C. In
various preferred embodiments, U is L.
[0140] Similarly, in various embodiments, B is an amino acid
preferably selected from the group consisting of H,
5-hydroxylysine, 4-hydroxyproline, and 3-hydroxyproline.
Alternatively, B can be selected from the group consisting of
collagen-derived amino acids, which includes 5-hydroxylysine,
4-hydroxyproline, and 3-hydroxyproline.
[0141] In another embodiment of the present invention, charged and
uncharged amino acids are selected from groups of modified amino
acids. For example, in one preferred embodiment, a charged amino
acid is selected from the group consisting of citrulline,
homoarginine, or ornithine, to name a few examples. Similarly, in
various preferred embodiments, the uncharged amino acid is selected
from the group consisting of .alpha.-aminobutanoic acid,
.alpha.-aminopentanoic acid, .alpha.-amino-2-methylpropanoic acid,
and .alpha.-aminohexanoic acid.
[0142] In preferred embodiments of the present invention, items
"a", "b", "c" and "d" are numbers which indicate the number of
charged or uncharged residues (or hydrophilic or hydrophobic
residues). In various embodiments, "a" has an average value of
about 1 to about 5, preferably about 1 to about 3, more preferably
about 1 to about 2, and even more preferably, 1.
[0143] In various embodiments, "b" has an average value of about 3
to about 20, preferably about 3 to about 12, more preferably about
3 to about 10, even more preferably in the range of about 4-8. In
one preferred embodiment, "b" is about 4.
[0144] In various embodiments, "c" is 1 to 10, preferably 2 to 10,
more preferably in the range of 3-8 or 4-8, and even more
preferably 3 to 6. In one preferred embodiment, "c" is about 4.
[0145] In various embodiments, "d" is 0 to 3 or 1 to 3. In one
preferred embodiment, "d" is 0 to 2 or 1 to 2; in another preferred
embodiment, "d" is 1.
[0146] By stating that an amino acid residue, e.g., a residue
represented by Z or U, is independently selected, it is meant that
at each occurrence, a residue from the specified group is selected.
That is, when "a" is 2, for example, each of the hydrophilic
residues represented by Z will be independently selected and thus
can include RR, RD, RE, RK, DR, DD, DE, DK, and the like. By
stating that "a" and "b" have average values, it is meant that
although the number of residues within the repeating sequence
(e.g., Z.sub.aU.sub.b) can vary somewhat within the peptide
sequence, the average values of "a" and "b" would be about 1 to
about 5 and about 3 to about 20, respectively.
[0147] For example, using the formula (Z.sub.aU.sub.b).sub.c
Z.sub.d for the peptide designated "KL8" in Table 2 below, the
formula can be rewritten as
K.sub.1L.sub.8K.sub.1L.sub.sK.sub.1L.sub.2, wherein the average
value of "b" is six [i.e., (8+8+2)/3=6], c is three and d is
zero.
[0148] Exemplary preferred polypeptides of the above formula are
shown in Table 2 below:
TABLE-US-00002 TABLE 2 SEQ ID Designation.sup.1 NO: Amino Acid
Sequence KL4 1 KLLLLKLLLLKLLLLKLLLLK KL8 2 KLLLLLLLLKLLLLLLLLKLL
KL7 3 KKLLLLLLLKKLLLLLLLKKL DL4 4 DLLLLDLLLLDLLLLDLLLLD RL4 5
RLLLLRLLLLRLLLLRLLLLR RL8 6 RLLLLLLLLRLLLLLLLLRLL RL7 7
RRLLLLLLLRRLLLLLLLRRL RCL1 8 RLLLLCLLLRLLLLCLLLR RCL2 9
RLLLLCLLLRLLLLCLLLRLL RCL3 10 RLLLLCLLLRLLLLCLLLRLLLLCLLLR HL4 11
HLLLLHLLLLIALLLLHLLLLH .sup.1The designation is an abbreviation for
the indicated amino acid residue sequence.
[0149] Also suitable are composite polypeptides of about 4 to 60
amino acid residues having a configuration that maximizes their
interaction with the alveoli. A composite polypeptide consists
essentially of an amino terminal sequence and a carboxy terminal
sequence. The amino terminal sequence has an amino acid sequence of
a hydrophobic region polypeptide or a hydrophobic peptide of this
invention, preferably hydrophobic polypeptide, as defined in the
above formula. The carboxy terminal sequence has the amino acid
residue sequence of a subject carboxy terminal peptide.
[0150] Proteins and polypeptides derived from or having
characteristics similar to those of natural Surfactant Protein (SP)
are useful in the present methods. As noted, SP isolated from any
mammalian species can be utilized, although bovine, porcine and
human surfactants are particularly preferred.
[0151] Natural surfactant proteins include SP-A, SP-B, SP-C or
SP-D, or fragments thereof, alone or in combination with lipids. A
preferred fragment is the amino-terminal residues 1-25 of SP-B.
[0152] A related peptide is the WMAP-10 peptide (Marion Merrell Dow
Research Institute) having the sequence
succinyl-Leu-Leu-Glu-Lys-Leu-Leu-Gln-Trp-Lys-amide. Alternative
peptides are polymers of lysine, arginine or histidine that induce
a lowering of surface tension in admixtures of phospholipids as
described herein.
[0153] In addition, human SP18 (SP-B) surfactant protein can be
utilized as described herein. See, e.g., WO 89/06657; WO 92/22315;
WO 98/49191; U.S. Pat. Nos. 5,260,273; 5,164,369; 5,407,914;
5,789,381; 5,952,303; 6,013,619; 6,013,764; 6,120,795; and
6,613,734; all of which are expressly incorporated by reference in
their entirety for all purposes)
[0154] Thus, in one preferred embodiment, a surfactant molecule of
the present invention comprises a polypeptide. In one variation, a
surfactant polypeptide comprises about 4, more preferably about 10,
amino acid residues. In various embodiments, a surfactant
polypeptide preferably comprises 60 or fewer amino acid residues,
more usually fewer than about 35, and even more preferably, fewer
than about 25 amino acid residues. In various preferred
embodiments, subject polypeptides correspond to the sequence of
SP18 monomer, e.g., a single group of contiguous residues in the
linear sequence of SP18. In other embodiments, subject polypeptides
preferably have alternating charged and uncharged amino acid
residue regions or have alternating hydrophobic and hydrophilic
amino acid residue regions.
[0155] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0156] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)). The term "conservative substitution" also includes the use
of a substituted amino acid in place of an unsubstituted parent
amino acid provided that such a polypeptide also displays the
requisite binding activity.
[0157] Additional residues can be added at either terminus of a
polypeptide of the present invention, such as for the purpose of
providing a "linker" by which such a polypeptide can be
conveniently affixed to a label or solid matrix, or carrier.
Labels, solid matrices and carriers that can be used with the
polypeptides of this invention are known in the art; some examples
are also described herein.
[0158] Amino acid residue linkers are usually at least one residue
and can be 40 or more residues, more often 1 to 10 residues.
Typical amino acid residues used for linking are tyrosine,
cysteine, lysine, glutamic and aspartic acid, or the like. In
addition, a polypeptide sequence of this invention can differ from
the natural sequence by the sequence being modified by
terminal-NH.sub.2 acylation, e.g., acetylation, or thioglycolic
acid amidation, terminal-carboxlyamidation, e.g., ammonia,
methylamine, and the like.
[0159] In another embodiment, a polypeptide of this invention has
amino acid residue sequence that has a composite hydrophobicity of
less than zero, preferably less than or equal to -1, more
preferably less than or equal to -2. These hydrophobic polypeptides
perform the function of the hydrophobic region of SP8. Thus, in one
preferred embodiment, the amino acid sequence mimics the pattern of
charged and uncharged--or hydrophobic and hydrophilic--residues of
SP18.
[0160] It should be understood, however, that polypeptides and
other surfactant molecules of the present invention are not limited
to molecules having sequences like that of native SP18. On the
contrary, some of the most preferred surfactant molecules of the
present invention have little resemblance to SP18 with respect to a
specific amino acid residue sequence, except that they have similar
surfactant activity and alternating charged/uncharged (or
hydrophobic/hydrophilic) residue sequences.
[0161] One disclosed embodiment of the present invention comprises
a peptide-containing preparation, the 21-residue peptide being a
mimic of human SP-B consisting of repeated units of four
hydrophobic leucine (L) residues, bounded by basic polar lysine (K)
residues. This exemplary peptide, which is abbreviated herein as
"KL.sub.4," (or "KL4") has the following amino acid residue
sequence:
TABLE-US-00003 (SEQ ID NO 1) KLLLLKLLLLKLLLLKLLLLK.
[0162] Combined with the phospholipids dipalmitoyl
phosphatidylcholine and palmitoyl-, oleoylphosphatidyl glycerol
(3:1) and palmitic acid, the phospholipid-peptide aqueous
dispersion has been named "KL.sub.4-Surfactant," and it is
generally referred to herein in that manner. The efficacy of
KL.sub.4-Surfactant in various experimental and clinical studies
has been previously reported. See, e.g., Cochrane and Revak, 1991,
Science 254: 566-568; Vincent et al., 1991, Biochemistry
30:8395-8401; Cochrane et al., 1996, Am J Resp & Crit Care Med,
152:404-410; and Revak et al., 1996, Ped. Res. 39:715-724.
[0163] E. Amino Acids, Natural Metabolites, Derivatives, Designed
Analogs, and Other Organic Molecules
[0164] Surfactant molecules of the present invention also include
organic molecules having surfactant activity, as defined above and
as further described herein. While polypeptides and proteins are
often described as exemplary, it should be understood that
surfactant molecules of the present invention are not limited to
those having either conventional amino acid side chains or a
polyamide backbone structure.
[0165] As noted previously, the present invention contemplates a
variety of surfactant molecules, including proteins, polypeptides,
and molecules including amino acid residues, as well as a variety
of surfactant compositions. While one tends to think of the
"common" natural amino acids (i.e., those listed in Table 1; see
Section A above) as being preferred for use in biological
compositions, it is also true that a wide variety of other
molecules, including uncommon but naturally occurring amino acids,
metabolites and catabolites of natural amino acids, substituted
amino acids, and amino acid analogs, as well as amino acids in the
"D" configuration, are useful in molecules and compositions of the
present invention. In addition, "designed" amino acid derivatives,
analogs and mimics are also useful in various compounds,
compositions and methods of the present invention, as well as
polymers including backbone structures composed of non-amide
linkages.
[0166] For example, in addition to the L-amino acids listed in
Section A above, amino acid metabolites such as homoarginine,
citrulline, ornithine, and .alpha.-aminobutanoic acid are also
useful in molecules and compositions of the present invention.
Thus, in the various formulas described above, "Charged", Z, or B
can comprise homoarginine, citrulline, or ornithine, as well as a
variety of other molecules as identified herein. Similarly, J can
comprise .alpha.-aminobutanoic acid (also known as
.alpha.-aminobutyric acid), .alpha.-aminopentanoic acid,
.alpha.-aminohexanoic acid, and a variety of other molecules
identified herein.
[0167] Further, substituted amino acids which are not generally
derived from proteins, but which are known in nature, are useful as
disclosed herein, include the following examples: L-canavanine;
1-methyl-L-histidine; 3-methyl-L-histidine; 2-methyl L-histidine;
.alpha.,.epsilon.-diaminopimelic acid (L form, meso form, or both);
sarcosine; L-ornithine betaine; betaine of histidine (herzynine);
L-citrulline; L-phosphoarginine; D-octopine; o-carbamyl-D-serine;
.gamma.-aminobutanoic acid; and .beta.-lysine. D-amino acids and
D-amino acid analogs, including the following, are also useful in
proteins, peptides and compositions of the present invention:
D-alanine, D-serine, D-valine, D-leucine, D-isoleucine,
D-alloisoleucine, D-phenylalanine, D-glutamic acid, D-proline, and
D-allohydroxyproline, and the like. The foregoing can also be used
in surfactant molecules according to the present invention;
particularly preferred for use accordingly are those corresponding
to the formula {(Charged).sub.a
(Uncharged).sub.b}.sub.c(Charged).sub.d.
[0168] The present invention also discloses that an extensive
variety of amino acids, including metabolites and catabolites
thereof, can be incorporated into molecules which display a
surfactant activity. For example, molecules such as ornithine,
homoarginine, citrulline, and .alpha.-aminobutanoic acid are useful
components of molecules displaying surfactant activity as described
herein. Surfactant molecules according to the present invention can
also comprise longer straight-chain molecules;
.alpha.-aminopentanoic acid and .alpha.-aminohexanoic acid are two
additional examples of such useful molecules.
[0169] It should also be appreciated that the present invention
encompasses a wide variety of modified amino acids, including
analogs, metabolites, catabolites, and derivatives, irrespective of
the time or location at which modification occurs. In essence, one
can place modified amino acids into three categories: (1)
catabolites and metabolites of amino acids; (2) modified amino
acids generated via posttranslational modification (e.g.,
modification of side chains); and (3) modifications made to amino
acids via non-metabolic or non-catabolic processes (e.g., the
synthesis of modified amino acids or derivatives in the
laboratory).
[0170] The present invention also contemplates that one can readily
design side chains of the amino acids of residue units that include
longer or shortened side chains by adding or subtracting methylene
groups in either linear, branched chain, or hydrocarbon or
heterocyclic ring arrangements. The linear and branched chain
structures can also contain non-carbon atoms such as S, O, or N.
Fatty acids can also be useful constituents of surfactant molecules
herein. The designed side chains can terminate with (R') or without
(R) charged or polar group appendages.
[0171] In addition, analogs, including molecules resulting from the
use of different linkers, are also useful as disclosed herein.
Molecules with side chains linked together via linkages other than
the amide linkage, e.g., molecules containing amino acid side
chains or other side chains (R- or R'-) wherein the components are
linked via carboxy- or phospho-esters, ethylene, methylene, ketone
or ether linkages, to name a few examples, are also useful as
disclosed herein. In essence, any amino acid side chain, R or R'
group-containing molecule can be useful as disclosed herein, as
long as the molecule includes alternating hydrophilic and
hydrophobic residues (i.e., component molecules) and displays
surfactant activity as described herein.
[0172] The present invention also contemplates molecules comprising
peptide dimers joined by an appropriate linker, e.g., peptide
dimers linked by cysteine molecules. (As those of skill in the art
are aware, two cysteine molecules can be linked together by a
disulfide bridge formed by oxidation of their thiol groups.). Such
linkers or bridges can thus cross-link different polypeptide
chains, dimers, trimers, and the like. Other useful linkers which
can be used to connect peptide dimers and/or other peptide
multimers include those listed above, e.g., carboxy- or
phospho-ester, ethylene, methylene, ketone or ether linkages, and
the like.
[0173] While it is appreciated that many useful polypeptides
disclosed herein, e.g., the KL.sub.4 polypeptide (SEQ ID NO:1),
comprise naturally-occurring amino acids in the "L" form which are
joined via peptide linkages, it should also be understood that
molecules including amino acid side chain analogs, non-amide
linkages (e.g., differing backbones) can also display a significant
surfactant activity and can possess other advantages, as well. For
example, if it is desirable to construct a molecule (e.g., for use
in a surfactant composition) that is not readily degraded, one can
wish to synthesize a polypeptide molecule comprising a series of
D-amino acids.
[0174] "Polypeptoids" are a class of non-natural, sequence-specific
polymers representing an alternative derivative of a peptide
backbone. Structurally, they differ from polypeptides in that their
sidechains are pendant groups of the amide nitrogen rather than the
.alpha.-carbon (see, e.g., Simon et al., 1992, Proc. Natl. Acad.
Sci. U.S.A. 89:9367-9371 and Zuckermann et al., 1992, J. Am. Chem.
Soc. 114:10646-10647). "Retropeptoids" are believed to have a
higher probability of bioactivity when protein binding is required,
as the relative positioning of sidechains and carbonyls "line up"
more closely with peptides (Kruijtzer, J. A, 1995 Tetrahedron
Letters 36:6969-72). N-Substitution prevents proteolysis of the
peptoid backbone (see, e.g., Miller et al., 1995, Drug Dev Res
35:20-32), giving enhanced biostability. Since polypeptoids are not
proteolyzed, they are not strongly immunogenic (Borman, 1998, C
& E News 76:56-57).
[0175] In another variation, one can wish to construct a molecule
that adopts a more "rigid" conformation; one means of accomplishing
this would be to add methyl or other groups to the a carbon atom of
the amino acids.
[0176] As noted above, other groups besides a CH.sub.3 group can be
added to the a carbon atom, that is, surfactant molecules of the
present invention are not limited to those incorporating a CH.sub.3
at the .alpha. carbon alone. For example, any of the side chains
and molecules described above can be substituted for the indicated
CH.sub.3 group at the .alpha. carbon component.
[0177] As used herein, "analogs" and "derivatives" of polypeptides
and amino acid residues are intended to encompass metabolites and
catabolites of amino acids, as well as molecules which include
linkages, backbones, side-chains or side-groups which differ from
those ordinarily found in what are termed "naturally-occurring"
L-form amino acids. (The terms "analog" and "derivative" can also
conveniently be used interchangeably herein.). Thus, D-amino acids,
molecules which mimic amino acids and amino acids with "designed"
side chains (i.e., that can substitute for one or more amino acids
in a molecule having surfactant activity) are also encompassed by
the terms "analogs" and "derivatives" herein.
[0178] A wide assortment of useful surfactant molecules, including
amino acids having one or more extended or substituted R or R'
groups, is also contemplated by the present invention.
[0179] Again, one of skill in the art should appreciate that one
can make a variety of modifications to individual amino acids, to
the linkages, and/or to the chain itself, which modifications will
produce molecules falling within the scope of the present
invention, as long as the resulting molecule possesses surfactant
activity as described herein.
[0180] F. Treatment Regimes
[0181] The invention provides pharmaceutical compositions
comprising a pulmonary surfactant for the treatment of a
respiratory disease, e.g., ARDS and RDS, formulated together with a
pharmaceutically acceptable carrier. Some compositions include a
combination of multiple (e.g., two or more) surfactants of the
invention.
[0182] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of a disease or condition (i.e., a respiratory
disease or disorder) in an amount sufficient to eliminate or reduce
the risk, lessen the severity, or delay the outset of the disease,
including biochemical, histological and/or behavioral symptoms of
the disease, its complications and intermediate pathological
phenotypes presenting during development of the disease. In
therapeutic applications, compositions or medicants are
administered to a patient suspected of, or already suffering from
such a disease in an amount sufficient to cure, or at least
partially arrest, the symptoms of the disease (biochemical and/or
histological), including its complications and intermediate
pathological phenotypes in development of the disease. An amount
adequate to accomplish therapeutic or prophylactic treatment is
defined as a therapeutically- or prophylactically-effective dose.
In both prophylactic and therapeutic regimes, agents are usually
administered in several dosages until a sufficient response has
been achieved. Typically, the response is monitored and repeated
dosages are given if the response starts to wane.
[0183] G. Effective Dosages
[0184] Effective doses of a pulmonary surfactant for the treatment
of disease, e.g., respiratory disease or disorder such as ARDS and
RDS, as described herein, vary depending upon many different
factors, including means of administration, target site,
physiological state of the patient, whether the patient is human or
an animal, other medications administered, and whether treatment is
prophylactic or therapeutic. Usually, the patient is a human but
nonhuman mammals can also be treated.
[0185] Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from days to several days to several months, or until a cure is
effected or a diminution of the disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of a patient or subject. Persons of
ordinary skill can easily determine optimum dosages, dosing
methodologies and repetition rates. Optimum dosages can vary
depending on the relative potency of individual surfactants and, in
the case of concomitant administration, the relative potency of
known drugs or other surfactants used in the treatment of disease.
Optimum dosages can generally be estimated based on EC.sub.50s
found to be effective in in vitro and in vivo animal models.
[0186] In general, dosage is from 0.01 .mu.g to 100 g per kg of
body weight and can be given once or more daily, weekly, monthly or
yearly, or even once every 2 to 20 years. The dosage and frequency
of administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic regime.
An optimal dosing schedule is used to deliver a therapeutically
effective amount of the nucleic acid being administered via a
particular mode of administration.
[0187] The term "therapeutically effective amount" as used herein
refers to a characteristic of an amount of a therapeutic compound,
or a characteristic of amounts of combined therapeutic compounds in
combination therapy. The amount or combined amounts achieve the
goal of preventing, avoiding, reducing or eliminating the
respiratory disease or condition. Although individual needs can
vary, determination of optimal ranges for effective amounts of
formulations is within the skill of the art. Human doses can be
extrapolated from animal studies (Remington's Pharmaceutical
Sciences, 20.sup.th ed., Gennaro, ed., Mack Publishing Co., Easton,
Pa., 2000). Generally the dosage required to provide an effective
amount of a formulation, which can be adjusted by one skilled in
the art, will vary depending on the age, health, physical
condition, weight, type and extend of the disease or disorder of
the recipient, frequency of treatment, the nature of concurrent
therapy (if any) and the nature and scope of the desired effect(s).
For additional guidance regarding formulation, dose and
administration regimen, see Berkow et al., 1997, The Merck Manual
Of Medical Information, Home, ed., Merck Research Laboratories,
Whitehouse Station, N.J.; Goodman et al., 1996, Goodman &
Gilman's the Pharmacological Basis of Therapeutics, 9.sup.th ed.
McGraw-Hill Health Professions Division, New York; Ebadi, 1998, CRC
Desk Reference of Clinical Pharmacology, CRC Press, Boca Raton,
Fla.; Katzung, 2001, Basic & Clinical Pharmacology, 8.sup.th
ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New
York; Speight et al., 1997, AverV's Drug Treatment: A Guide to the
Properties, Choice, Therapeutic Use and Economic Value of Drugs in
Disease Management, 4.sup.th ed. Adis International,
Auckland/Philadelphia, Pa.
[0188] For diagnostic applications, a detectable amount of a
composition of the invention is administered to a subject. A
"detectable amount," as used herein to refer to a diagnostic
composition, refers to a dose of such a composition that the
presence of the composition can be determined in vivo following
pulmonary administration.
[0189] Subjects that can be treated by the methods of the present
invention include those suffering from a respiratory disease or
disorder, and those at risk for developing a respiratory disease or
disorder. At-risk individuals include, but are not limited to,
individuals with a family history of respiratory disease or
disorder, individuals who have previously been treated for
respiratory diseases, and individuals presenting any other clinical
indicia suggesting that they have an increased likelihood of
developing the respiratory disease or disorder. Alternatively
stated, an at-risk individual is any individual who is believed to
be at a higher risk than the general population for developing a
respiratory disease or disorder. The term "prophylactically
effective amount" is meant to refer to an amount of a formulation
which produces an effect observed as the prevention of the onset or
recurrence of a respiratory disease or disorder. Prophylactically
effective amounts of a formulation are typically determined by the
effect they have compared to the effect observe when a second
formulation lacking the active agent is administered to a similarly
situated individual.
[0190] The compositions of the present invention can be
administered either before or during pulmonary crises. Further,
they can also be administered prior to single-lung, double-lung, or
heart-lung transplant. In addition, it can be desirable to give the
active compound to the subject over a long period as an adjunct to,
e.g., the standard therapies for respiratory diseases and
disorders.
[0191] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the surfactant or surfactants are
administered in maintenance doses, ranging from 0.1 .mu.g to 20 g
per kg of body weight, once or more daily, weekly, monthly or
yearly, or even once every 2 to 20 years. For example, in the case
of an individual known or suspected of being prone to a respiratory
disease or condition, prophylactic effects may be achieved by
administration of preventative doses, ranging from 0.1 .mu.g to 20
g per kg of body weight, once or more daily, weekly, monthly or
yearly, or even once every 2 to 20 years.
[0192] The compositions of the present invention can include
sterile aqueous solutions which can also include sterile aqueous
solutions which can also contain buffers, diluents and other
suitable additives. The pharmaceutical formulations, which can
conveniently be presented in unit dosage form, can be prepared
according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both. The formulations can be presented
in unit-dose or multi-dose containers, for example sealed ampoules
and vials, and can be stored in a frozen or freeze-dried
(lyophilized) condition requiring only the addition of sterile
liquid carrier immediately prior to use.
[0193] When used as a pharmaceutical treatment, the compositions of
the present invention can be administered either alone or
optionally in conjunction with other compounds or compositions that
are used in the treatment of respiratory diseases or disorders. For
example, if a subject is being treated for a respiratory disorder
caused by a bacterial infection, then a composition of the present
invention may be administered in conjunction with another compound
or treatment used to treat the bacterial infection, such as an
antibiotic. Examples of such compounds, referred to herein as
"supplemental compounds," or "supplemental compositions," include,
but are not limited to, antibiotics, anti-cytokines, anti-asthma
drugs, antiphospholipases (e.g., inhibitors of phospholipase),
vasodilators (e.g., adenosine, .beta.-adrenergic agonists or
antagonists, .beta.-adrenergic blockers, a-adrenergic blockers,
diuretics, smooth muscle vasodilators, nitrates, and
angiotensin-converting enzyme inhibitors), and compounds found to
be useful in the treatment of cystic fibrosis, such as
pyrazinoylguanidine sodium channel blockers (e.g., amiloride,
benzamil, phenamil).
[0194] The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
[0195] H. Therapeutic Methods
[0196] The present application also discloses a variety of
therapeutic methods that are useful in conjunction with various
novel compounds and compositions disclosed herein. While the use of
KL.sub.4-surfactant is described herein as exemplary, it should be
understood that the other compounds and compositions disclosed
herein, as well as compounds and compositions having surfactant
activity and known to those of skill in the art, are also useful
according to the described methods.
[0197] The surfactant compositions of the present invention, and
therapeutic methods for pulmonary administration of the
compositions to a subject, are useful for the treatment of a
disease or disorder of the lung, such as an infection, an
immunodeficiency syndrome, an inflammatory disease, an autoimmune
disease, a neoplasm, or cancer. In particular, the present
invention provides improved methods for formulating therapeutic
proteins for pulmonary delivery.
[0198] The present invention also discloses preferred methods of
treating respiratory diseases in patients of any age, including
neonates and adults. One such method comprises administering to a
patient in need of such treatment a therapeutically effective
amount of a surfactant composition, preferably, a lipid-based
surfactant composition, prepared from a polypeptide (or other
surfactant molecule) of the present invention and a
pharmaceutically acceptable phospholipid, wherein the polypeptide
is combined with the phospholipid in an amount sufficient to
increase the surfactant activity of the composition above that of
the phospholipid. The present invention also discloses a method of
treating respiratory diseases, for example ARDS and RDS, wherein
the polypeptide is constituted by about 10-60 amino acid residues
and alternating groupings of charged amino acid residues and
uncharged amino acid residues as represented by the formula
[(Charged).sub.a (Uncharged).sub.b].sub.C(Charged).sub.d, wherein a
has an average value of about 1 to about 5; b has an average value
of about 3 to about 20; c is 1 to 10; and d is 0 to 3. In various
preferred embodiments, such a polypeptide, when admixed with a
pharmaceutically acceptable phospholipid, forms a pulmonary
surfactant having a surfactant activity greater than the surfactant
activity of the phospholipid alone.
[0199] As disclosed herein and as further described in the Examples
set forth below, a variety of methods for administering the
surfactant compounds and compositions of the present invention are
available and are well known by one of skill in the art. Depending
on the needs of any individual needing treatment, e.g., an infant
or adult with respiratory distress syndrome, different treatment
methods can be appropriate.
[0200] Thus, in instances in which an infant suffers from a
respiratory disease, such as aspirated meconium, particular
treatment modalities can be recommended. In one such therapeutic
method, lavage of the patient's lungs with a surfactant composition
of the present invention is performed. A single lavage with
surfactant can be all that is required; alternatively, multiple
surfactant lavages can be appropriate. Moreover, a saline lavage
followed by one or more surfactant lavages can be an appropriate
treatment, albeit that it will be shown below that dilute
surfactant lavage tends to produce better results than a
combination of saline and surfactant lavage.
[0201] Lavage procedures using surfactant are performed essentially
as follows. KL.sub.4-surfactant or another surfactant (e.g., one of
the present invention) is preferably administered using tools
typically used in saline lavage procedures, which include various
flexible tube-like apparatus such as endotracheal tubes, cannulae
and catheters. Thus, for example, an endotracheal tube apparatus
which includes a cannula that can be inserted through the tube,
e.g., for suctioning purposes, is appropriate for use according to
the disclosed methods. Preferably, any apparatus appropriately used
to safely and efficaciously deliver and remove lavage fluids to and
from the lung, respectively, is contemplated for use herein.
[0202] Exemplary devices for pulmonary lavage are ventilator
devices equipped for bronchoalveolar lavage (BAL), which must
include a means for applying a positive end-expiratory pressure
(PEEP) to the lung, a means for instilling liquids into the lung
and a means for removing pulmonary fluids from the lung using
negative pressure suction.
[0203] Representative devices are described in U.S. Pat. Nos.
4,895,719, 5,207,220, 5,299,566 and 5,309,903, the disclosures of
which are hereby incorporated by reference in their entirety for
all purposes.
[0204] As shown herein, particularly advantageous results were
obtained by practicing a method of pulmonary lavage using dilute
surfactant that produced sustained recovery of arterial oxygen
(PaO.sub.2), normal lung compliance and diminished inflammation
following pulmonary injury by meconium aspiration or by partial
loss of intrinsic surfactant, such as is demonstrated herein in the
model using instillation of bacterial LPS. These methods can be
useful for use in treating any of a variety of pulmonary conditions
in which there is respiratory distress, particularly ARDS and
RDS.
[0205] Conditions in which respiratory stress can be present
include, but are not limited to, meconium aspiration in newborn
infants, pulmonary inflammation, and pulmonary infection.
Respiratory distress can be associated with a variety of
conditions, including sepsis, pulmonary trauma, accumulation of
pulmonary exudate, pancreatitis, aspiration of gastric contents,
heated gas inhalation, smoke or noxious gas inhalation, acute
hypoxemia, fetal circulation, congenital diaphramatic hernia,
pneumonia, inflammation arising from infection or multiple
transfusions, and the like.
[0206] As shown herein, the present dilute surfactant lavage
methods remove mediators of inflammation and simultaneously
preserve and/or restore pulmonary function, thereby providing
effective therapy.
[0207] The application of the pulmonary lavage provides several
beneficial features. The washing effect of the lavage removes
debris, dead cells, loose inflammatory cells and fluids, and the
like, cleaning the alveoli of occluding fluid and materials, and
removing typically 30 to 95% of the pulmonary and lavage fluids,
together with any undesirable materials, such as meconium or
inflammatory exudates. The dilute surfactant treats the alveolar
membranes, improving the compliance of the tissue. The application
of specified amounts of ventilator air pressure in the form of
positive end-expiratory pressure (PEEP) before, during and after
lavage with surfactant expands the lungs to maximize contact in the
wash and treatment phase and thereby improve the dynamics of the
lavage process, and in particular improves the oxygen tension and
gas exchange in the patient during a process that can precariously
burden oxygen exchange in the alveoli. Finally, the use of short
intervals of tracheo-bronchial suction to remove the pulmonary
(lavage) fluids are carefully administered in a manner that does
not allow the arterial oxygen saturation to be reduced below
acceptable and safe levels.
[0208] The pulmonary lavage method can be practiced on any mammal,
and is particularly suited for humans, including adults, juveniles
and infants, both newborn infants and babies experiencing
respiratory distress or suffering from a respiratory disease or
disorder.
[0209] The method for pulmonary lavage of a mammal comprises
applying vapor phase (gas) positive end-expiratory pressure (PEEP)
with a ventilator means to a lung, lung section or lobe of the
mammal. Thereafter, a lavage composition containing dilute
surfactant in a pharmaceutically acceptable aqueous medium is
instilled into the lung or lung section of the mammal. Afterwards,
some or all pulmonary fluid, including the lavage composition,
present in the lung section is removed by applying short intervals
of tracheo-bronchial suction using negative pressure.
[0210] The PEEP is typically administered at a pressure range of 4
to 20 centimeters (cm) water, although the pressure can vary
depending on the patient and the pulmonary condition. For adults,
juveniles and infants other than newborns, in which the lungs have
toughened, the range is preferably from 6 to 12 cm water, and more
preferably about 8-10 cm water. For newborn infants in which the
lung sacs are more delicate and more fragile to applied pressure,
the PEEP can range from about 4 to 15 cm water, preferably about 6
to 9 cm water, and more preferably about 8 cm water.
[0211] The administration of gas PEEP is typically applied to the
lung prior to instilling dilute surfactant lavage, typically for up
to about 30 minutes prior, more preferably about 5 to 30 minutes,
in order to stabilize the blood oxygen prior to the procedure. In
addition, PEEP is preferably applied continuously throughout the
procedure during both the instilling and removing steps. It is to
be understood that the combined effect on pressure of applying
continuous PEEP and a short interval of suction will result in a
brief, transient, drop in net pressure, with a rapid return to the
maintained PEEP level when the suction interval is terminated. In
addition, PEEP can be applied for a time period after the lavage
fluid removal step in order to maintain oxygen tension on the
alveoli following the procedure. Preferably, PEEP is maintained for
up to about 24 hours after the removing step, preferably up to
about 12 hours, and more preferably about 0.5 to 6 hours.
[0212] It is also contemplated that the applied gas can contain
supplemental oxygen, typically from about 21 to 100% oxygen,
preferably about 50 to 100% oxygen.
[0213] The suction phase of the method to remove the lavage and
pulmonary fluids is administered in short intervals, i.e, 1 to 120
seconds. A typical suctioning interval is less than 30 seconds,
preferably less than 20 seconds, and more preferably for about 5 to
20 seconds. A preferred interval is from 2 to 120 seconds, and
preferably 5 to 20 seconds. The suction time period is short in
order to minimize decreases in and saturated arterial oxygen
(SaO.sub.2) that can accompany the suction phase of the lavage
procedure.
[0214] In one permutation of the suction procedure where there is
more than one suction step required to remove the pulmonary fluid,
it is desirable to pause between short suction intervals rather
than to follow one suction interval immediately with another in
order to provide the opportunity for the PaO.sub.2 level to
recover. A typical pause period is from about 30 seconds to fifteen
minutes, preferably about 1-5 minutes.
[0215] The suction applied to remove pulmonary fluids is a negative
pressure of from about 10 to 150 millimeters (mm) mercury (Hg),
preferably about 20 to 120 mm Hg, and more preferably about 60 to
100 mm Hg. A suction catheter or similar suction means is present
in the ventilator device, typically as a cannula extending through
the ventilator tube of the apparatus and into the bronchus. The
cannula tip is typically guided into a segmental bronchus with the
aid of the fiber optic observing means, and the instilling and
removing are provided through the cannula tip.
[0216] In practicing the dilute lavage method, it is understood
that the lavage can be administered repeatedly. Thus, the
instilling and removing steps are repeated sequentially while
applying PEEP as described herein. Typically, instilling and
removing (lavage wash cycles) can be repeated in sequence from 2 to
5 times, although additional repetitions can be conducted if
warranted. In addition, the content of the dilute surfactant can be
varied over the course of the repeated lavage washes. For example,
it is contemplated that a first series of from 1 to 3 wash cycles
are conducted using dilute surfactant at a concentration of about
0.1 to 10 mg per ml lavage composition, and a second series of from
1 to 5 wash cycles are conducted using dilute surfactant at a
concentration of about 10 to 50 mg per ml.
[0217] Depending on the position of the endotracheal tube apparatus
in lung, the lavage composition will bathe a lung lobe, a lung
segment or an entire side of lung, this being determined by the
position in the bronchial tube where the apparatus terminates.
Thus, it is understood that the term "lung" connotes alternatively
that a lung lobe, a lung segment, a lung half containing two or
three lobes, or a whole lung is being referred to in the context of
the method, but adjusted for volume based on weight of the
patient.
[0218] In instilling a lavage composition, it is also understood
that the process can be conducted by a variety of methods, such as
by cannula, by bronchoscope, by endotracheal tube and the like. In
a preferred method, the instilling is typically monitored visually
by a means for observing the lung at the apparatus tube terminus,
typically by use of a fiber optic bronchoscope and illuminating
means for visual display of the bronchial tube and distal lung
lobe(s). Thus, although estimates of the appropriate lavage
composition volume are stated, it is understood that in practice,
the instilled volume can depend on the judgment of the practitioner
during the instilling process, aided by the observing means.
[0219] The pulmonary lavage process is typically conducted on as
many lungs, both left and right, and involved lung lobes, as needed
depending on the extent of the condition of the lungs. Typically
the procedure is conducted sequentially on 30 to 100% of the
bronchial segments of the left and right lungs.
[0220] The bronchoscope or endotracheal tube can be fitted with a
fixed or expanding collar designed to fit the inner diameter of a
bronchial passage, and thereby secure a fit that can withstand the
pressure ranges for practicing the method. This feature is
particularly desirable insofar as it allows a section of the lung
to be lavaged while the remaining lung portions can respire,
thereby minimizing the trauma of the procedure to gas exchange in
the patient.
[0221] KL.sub.4-surfactant solution or other novel surfactant
solutions according to the present invention can be administered
via lavage in a formulation appropriate for this procedure. While a
formulation of surfactant comprising about 10-20 ml/kg is useful in
the treatment of respiratory diseases such as ARDS and RDS,
formulations for lavage therapy tend to be more dilute, to
facilitate efficient delivery and removal via endotracheal
tube.
[0222] Thus, a "dilute surfactant" when used in the context of a
lavage composition indicates that the lavage composition contains
one or more substances which provide surfactant activity to the
composition as defined herein in an amount sufficient to provide
the surfactant activity but present in an amount such that the
composition has a liquid viscosity amenable to lavage.
[0223] Thus, a surfactant-containing composition useful for
administration to a subject (or patient) via lavage is preferably
diluted to a concentration of about up to about 100 mg/ml so long
as the viscosity is such that the composition is amenable to
suction removal in less than 30 seconds following instillation.
Dosages in the range of about 0.1-100 mg/ml are typically
contemplated for use herein. In addition, the administration of
higher dosages can be appropriate in various instances, e.g., when
the subject is not fully responsive to lower dosages, or where the
formulation lowers viscosity while allowing for increased
concentrations of surfactant activity-containing substances.
[0224] In general, quantities of surfactant of about 4 to 60 ml per
kg of the subject's (or patient's) body weight are given during
each administration, typically divided equally between right lung
and left lung or divided among lung sections. Depending on the
needs of the individual patient, which can readily be determined by
the physician or other individual of skill in the relevant art who
is administering the treatment, greater or lesser quantities of
surfactant can be delivered during each administration. Quantities
comprising about 8-30 ml/kg are preferred, with quantities
comprising about 10-25 ml/kg being somewhat more preferred. Thus,
lavage composition is typically instilled in a volume of about 4 to
60 ml per kilogram (kg), preferably about 8 to 30 ml per kg, and
more preferably about 16-25 ml per kg.
[0225] In describing the amount of surfactant present in a lavage
composition, the weight refers to an amount measured as
phospholipid phosphate per total volume of lavage composition,
unless otherwise specified.
[0226] The amount of surfactant administered via lavage can also be
described "per lung" in a particular patient. Thus, an effective
amount of surfactant for lavage purposes can comprise about
200-1000 ml/lung for a 70 kg adult, preferably about 400 ml/lung,
and about 30-60 ml/lung for a 3 kg infant, preferably about 50
ml/lung. As before, depending on the maturity and size of the
individual receiving treatment, the amount of surfactant
administered, as well as the dosage, can be adjusted as
appropriate.
[0227] A subject can receive one or more lavages, depending on the
severity of the individual's condition and depending on the
response of the subject to the first lavage. The dosages and
amounts of surfactant administered can likewise vary in subsequent
lavages. For example, if a subject receives a typical dose during
the first lavage, subsequent lavages can be administered using the
same dosage, a lesser dosage, or a higher dosage, depending on the
needs and response of the subject.
[0228] Similarly, the amounts of surfactant administered during
each lavage can vary, or can remain constant, depending on the
needs of the individual patient. In appropriate circumstances, the
first or subsequent lavage can be followed by a lavage
administering a higher dose, e.g., up to 10-50 mg/ml. For example,
a subject can receive 1-3 lavages with surfactant at a
concentration of 0.1-10 mg/ml followed by 1-5 lavages with
surfactant at a concentration of 10-50 mg/ml.
[0229] As noted previously, the dosage to be administered varies
with the size and maturity of the subject, as well as with the
severity of the subject's condition. Those of skill in the relevant
art will be readily able to determine these factors and to adjust
the dosage administered via lavage, as taught herein.
[0230] Bolus administration of surfactant can also be appropriate.
Thus, for example, a bolus of 10-300 mg/kg surfactant at 15-100
mg/ml can be administered prior to or subsequent to one or more
lavage treatments.
[0231] The type of treatment, dosage and amount utilized will
understandably vary depending on the nature and seriousness of an
individual subject's condition. Thus, for example, if a subject is
an infant suffering from meconium aspiration, a treatment regimen
comprising one or more surfactant lavages will likely be
administered. Bolus administration of surfactant can follow the
lavage(s) as well.
[0232] As noted previously, one aspect of the present invention was
the removal of meconium or inflammatory exudate from the airways
via lavage with dilute surfactant, in order to improve pulmonary
function and inhibit the inflammatory reaction that usually
develops in response to the presence of meconium or other injurious
substances in the respiratory pathway. Although many of the
examples focus on the use of one preferred embodiment, e.g., a
synthetic peptide-containing exogenous surfactant, it is expressly
to be understood that other surfactant-containing lavage
compositions can be used according to the disclosed methods.
Exemplary formulations for, and methods of using surfactants are
also disclosed in the present specification.
[0233] The compositions of the present invention can be
administered directly into the lungs of the subject by any suitable
means, but preferably by intratracheal instillation, and more
preferably bolus intratracheal instillation. An alternative
preferred method of administration is via inhalation of respirable
aerosol particles comprising the composition. Pulmonary lavage is
another alternative route of administration as discussed above.
Accordingly, the composition can be administered, as appropriate to
the dosage form, by endotracheal tube, by bronchoscope, by cannula,
by aerosol administration, or by nebulization of a suspension or
dust of the composition into a gas to be inspired. Compositions of
the present invention can also be delivered to the lung in an
aerosolized form using the pulmonary drug delivery systems set
forth in U.S. Pat. No. 5,874,064 and U.S. Pat. No. 5,934,273, the
disclosures of which are herein incorporated by reference in their
entireties for all purposes.
[0234] In one embodiment of the invention, the composition of the
invention can be administered by administering an aerosol of
respirable particles comprised of the composition, which the
subject inhales. The composition can be aerosolized in a variety of
forms, such as, but not limited to, dry powder inhalants, metered
dose inhalants, bolus or continuously delivered liquid solutions or
dispersions. The respirable particles can be liquid or solid. Solid
or liquid particulate forms of the compositions prepared for
practicing the present invention should include particles of
respirable size: that is, particles of a size sufficiently small to
pass through the mouth and larynx upon inhalation and into the
bronchi and alveoli of the lungs. In general, particles ranging
from about 0.5 to 5 microns in size are within the respirable
range. In a preferred embodiment, the microparticles have a
diameter between 0.5 microns and 5 microns mass median aerodynamic
diameter and viscosity less than 10 cp (measured at room
temperature) at a cholesterol concentration from about 8 mole % to
about 15 mole %, in a pharmaceutically acceptable carrier for
administration to the lungs. In another preferred embodiment, the
microparticles have a diameter between 0.5 microns and 5 microns
and viscosity less than 30 cP at a cholesterol concentration from
about 8 mole % to about 15 mole %, in a pharmaceutically acceptable
carrier for administration to the lungs. As described herein, the
viscosity less than 30 cP can be defined as the fluid viscosity at
25.degree. C. using an instrumented cone and plate viscometer
operating at a rate of approximately 160 sec.sup.-1. A step-flow
procedure can be employed that involves increasing the spin rate
from about 0 to 200 sec.sup.-1 allowing the viscosity to reach
equilibrium after each of 10 steps and then repeating the process
while decreasing the spin-rate.
[0235] Particles of non-respirable size that are included in the
aerosol tend to be deposited in the throat and swallowed, and the
quantity of non-respirable particles in the aerosol is preferably
minimized. The particulate pharmaceutical composition can
optionally be combined with a carrier to aid in dispersion or
transport. A suitable carrier such as a sugar (i.e., lactose,
sucrose, trehalose, and mannitol) can be blended with the active
compound or compounds in any suitable ratio (e.g., a 1 to 1 ratio
by weight).
[0236] Aerosols of liquid particles comprising the compositions can
be produced by any suitable means, such as with a pressure-driven
aerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat.
No. 4,501,729. Nebulizers are commercially available devices that
transform solutions or dispersions of the active compound (i.e.,
KL.sub.4 surfactant) into a therapeutic aerosol mist by means of
acceleration of compressed gas, typically air or oxygen, through a
narrow enturi orifice, by means of ultrasonic agitation, by means
of electrohydrodynamic, by liquid vaporization and condensation or
by agitation through vibrating membranes. Suitable formulations for
use in nebulizers consist of the active ingredient in a liquid
carrier, the active ingredient comprising up to 40% w/w of the
formulation, but preferably less than 20% w/w. The carrier medium
is typically water-based (and most preferably sterile, pyrogen-free
water) or a dilute aqueous alcoholic solution, preferably made
isotonic but can be hypertonic or hypotonic relative to body fluids
by the addition of, for example, sodium chloride. Optional
additives include preservatives if the formulation is not made
sterile, for example, methyl hydroxybenzoate, antioxidants,
flavoring agents, volatile oils, buffering agents, and
surfactants.
[0237] Aerosols of solid particles comprising the composition can
likewise be produced with any solid particulate medicament aerosol
generator. Aerosol generators for administering solid particulate
medicaments to a subject produce particles that are respirable, as
explained above, and generate a volume of aerosol containing a
predetermined metered dose of a medicament at a rate suitable for
human administration. One illustrative type of solid particulate
aerosol generator is an insufflator. Suitable formulations for
administration by insufflation include finely comminuted powders
that can be delivered by means of an insufflator or taken into the
nasal cavity in the manner of a snuff. In the insufflator, the
powder (e.g., a metered dose thereof effective to carry out the
treatments described herein) is contained in capsules or
cartridges, typically made of gelatin, plastic or aluminum foil,
which are either pierced or opened in situ and the powder delivered
by air drawn through the device upon inhalation or by means of a
manually-operated pump. The powder employed in the insufflator
consists either solely of the active ingredient or of a powder
blend comprising the active ingredient, a suitable powder diluent,
such as lactose, and an optional surfactant. The active ingredient
typically comprises from 0.1 to 100 w/w of the formulation.
[0238] A second type of illustrative aerosol generator comprises a
metered dose inhaler (MDI). MDIs are pressurized aerosol
dispensers, typically containing a suspension or solution
formulation of the active ingredient in a liquefied propellant.
During use, these devices discharge the formulation through a valve
adapted to deliver a metered volume, typically from 10 to 200 to
produce a fine particle spray containing the active ingredient.
Suitable propellants include certain chlorofluorocarbon or
hydrofluorocarbon compounds, for example, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane and mixtures
thereof. The formulation can additionally contain one or more
co-solvents, for example, ethanol, surfactants, such as oleic acid
or sorbitan trioleate, antioxidants and suitable flavoring
agents.
[0239] Any propellant can be used in carrying out the present
invention, including both chlorofluorocarbon-containing propellants
and non-chlorofluorocarbon-containing propellants. Thus,
fluorocarbon aerosol propellants that can be employed in carrying
out the present invention including fluorocarbon propellants in
which all hydrogens are replaced with fluorine, chlorofluorocarbon
propellants in which all hydrogens are replaced with chlorine and
at least one fluorine, hydrogen-containing fluorocarbon
propellants, and hydrogen-containing chlorofluorocarbon propellants
and mixtures thereof. Particularly preferred are hydrofluoroalkanes
such as 1,1,1,2-tetrafluoroethane and heptafluoropropane. A
stabilizer such as a fluoropolymer can optionally be included in
formulations of fluorocarbon propellants, such as described in U.S.
Pat. No. 5,376,359.
[0240] The aerosol, whether formed from solid or liquid particles,
can be produced by the aerosol generator at a rate of from about
0.1 to 1 mL per minute. Aerosols containing greater amounts of
medicament can be administered more rapidly. Typically, each
aerosol can be delivered to the patient for a period from about 30
seconds to about 20 minutes, with a delivery period of about five
to ten minutes being preferred.
[0241] Reperfusion is also associated with harmful effects of
neutrophil activation and tissue infiltration. The nature of the
neutrophil-mediated injury is not fully characterized but is in
part due to the production of superoxide anion (O.sub.2) and/or
related oxidative products. This sequence of events (activation of
white blood cells, release of toxic mediators, and resultant
pathophysiology in the host) is common to many inflammatory
diseases.
[0242] The present invention also provides compositions and methods
for treating conditions associated with oxidative injury. For
example, aerosol compositions comprising the compositions of the
present invention together with drugs or other active agents known
to reduce cell and/or tissue damage due to oxidative injury, and to
inhibit oxidant production by leukocytes are contemplated. Tissue
at risk of oxidative injury can include blood-perfused tissue and
inflamed tissue.
[0243] Inflammatory diseases and disorders that can be treated
using the disclosed compositions and methods include but are not
limited to acute lung injury, acute respiratory distress syndrome,
arthritis, asthma, bronchitis, cystic fibrosis, reperfusion injury
artery occlusion, stroke, ultraviolet light induced injury, and/or
vasculitis. The inflammation can be symptomatic of a separate
disease or condition, such as autoimmune disease and
transplantation. Inflammatory diseases and disorders also include
those conditions characterized by leukocyte dysfunction. The
inflammation can be acute, chronic, or temporary inflammation. See
e.g., Weissmann et al., 1982, Ann N Y Acad Sci 389:11-24, Goldstein
et al., 1982, Ann N Y Acad Sci 389:368-79, Janoff, 1985, Annu Rev
Med 36:207-16, Hart & Fritzler, 1989, J Rheumatol 16:1184-91,
Doring, 1994, Am J Respir Crit Care Med 150:S114-7, Demling, 1995,
Annu Rev Med 46:193-202, Hansen, 1995, Circulation 91:1872-85,
Dakik & Nasrallah, 2001, Heart Dis 3:362-4, Kehl et al., 1996,
Intensive Care Med 22:968-71, and Munkvad, 1993, Dan Med Bull
40:383-408.
[0244] Reduced tissue inflammation can be assayed by detecting
proteins induced by inflammation, such as cytokines, monokines,
receptors, and proteases. For example, histamine can be measured
using a fluorescent assay described by Shore et al., 1959, J
Pharmacol Exp Ther 127:182-186. Nitric oxide can be measured using
a chemiluminescent assay described by Hybertson, 1994, Anal Lett
127:3081-3093.
[0245] Reduced inflammation can also be assessed by measuring a
reduction in oxidant production, including oxidant production by
neutrophils, macrophages, monocytes eosinophils, mast cells and/or
basophils. Representative methods for assaying the production of
oxidants by inflammatory cells are described in the examples.
Neutrophil function can also be assayed using techniques known in
the art, for example, as described by Bell et al., 1990, Br Heart J
63:82-7, Riesenberg et al., 1995, Br Heart J 73:14-9, Zivkovic et
al., 1995, J Pharmacol Exp Ther 272:300-9.
[0246] An aerosol composition of the invention can also comprise a
detectable label. Preferably, the detectable label can be detected
in vivo, for example by using any one of techniques including but
not limited to magnetic resonance imaging, scintigraphic imaging,
ultrasound, or fluorescence. Thus, representative detectable labels
include fluorophores, epitopes, radioactive labels, and contrast
agents.
[0247] In one embodiment of the invention, the detectable label is
a protein, e.g., a fluorescent protein. Alternatively, the
detectable label is conjugated to a protein to be administered. For
example, a composition of the invention can comprise a diagnostic
protein which is conjugated or otherwise bound to a detectable
label. Representative detectable labels, labeling methods, and
imaging systems suitable for pulmonary imaging and diagnosis are
described, for example, in Desai, 2002, Clin Radiol 57:8-17,
McLoud, 2002, Clin Chest Med 23:123-36, and McWilliams et al.,
2002, Oncogene 21:6949-59.
[0248] In addition to being useful in methods of treating lung
disorders (i.e., where the composition is a pharmaceutical
composition), the compositions and methods of the present invention
can be useful in enhancing pulmonary or airway function in subjects
generally. In this embodiment, the subject is not necessarily
suffering from a respiratory disease or disorder, but rather can be
a subject who is generally physically healthy, but desires
improvement in pulmonary function (i.e., easier breathing).
Subjects can desire the administration of the compositions of the
present invention in this context, for example, because of
incidental exposure to cigarette smoke (primary or second-hand),
environmental toxins or air pollution or smog; because of a
decrease in pulmonary function due to normal aging processes;
because of the desire for improved cardiovascular fitness, and the
like. In this sense, the compositions of the present invention are
administered not as a prescribed pharmaceutical (i.e., a drug), but
rather as a general health-improving tonic. By "enhance" is meant
that pulmonary function (e.g., respiration) occurs at an improved
level, as compared to pulmonary function occurring with the lack of
administration of the compositions of the present invention.
[0249] Pulmonary administration of a surfactant composition of the
present invention can be combined with other techniques for
pulmonary delivery, for example carbon dioxide enhancement of
inhalation therapy (see, e.g., U.S. Pat. No. 6,440,393) and
bronchodilation (see e.g., U.S. Pat. No. 5,674,860 and U.S.
Published Patent Application No. 20020151597). A treatment regimen
can also comprise pulmonary delivery with other delivery routes
(e.g., oral and intravascular delivery).
[0250] I. Toxicity
[0251] Preferably, a therapeutically effective dose of the
surfactant compositions described herein will provide therapeutic
benefit without causing substantial toxicity.
[0252] Toxicity of the proteins described herein can be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., by determining the LD.sub.50 (the dose
lethal to 50% of the population) or the LD.sub.100 (the dose lethal
to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. The data obtained from
these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the proteins described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1)
[0253] J. Kits
[0254] A further subject of the invention is a commercial product
consisting of a customary secondary packaging, a primary packaging
comprising a pharmaceutical preparation and, if desired, a pack
insert, the pharmaceutical preparation being suitable for the
prophylaxis or treatment of chronic respiratory diseases or
disorders in patients and reference being made on the secondary
packaging or on the pack insert of the commercial product to the
suitability of the pharmaceutical preparation for the prophylaxis
or treatment of respiratory diseases in patients, and the
pharmaceutical preparation being a pulmonary surfactant formulation
or preparation. The secondary packaging, the primary packaging
comprising the pharmaceutical preparation and the pack insert
otherwise correspond to what the person skilled in the art would
regard as standard for pharmaceutical preparations of this type.
Suitable primary packaging is, for example, ampoules or bottles of
suitable materials such as transparent polyethylene or glass or
alternatively suitable means of administration such as are
customarily employed for the administration of active compounds
into the lungs. By way of example, mention can be made of means of
administration for the atomization of an active compound solution
or suspension or for the atomization of active compound powder.
Preferably, the primary packaging is a glass bottle which can be
sealed, for example, by a commercially available rubber stopper or
a septum. A suitable secondary packaging which may be mentioned by
way of example is a folding box.
[0255] The kits of the invention can also include an inhalation
apparatus, surfactant dry aerosol particle formulation and a
detection system. An inhalation apparatus, as used herein, is any
device for administering a dry aerosol. This type of equipment is
well known in the art and has been described in detail, such as
that description found in Remington's Pharmaceutical Sciences,
20.sup.th ed., 2000. Inhalation devices are described, for example,
in U.S. Pat. No. 6,116,237.
[0256] The following examples are intended to illustrate, but not
limit, the present invention.
EXEMPLARY EMBODIMENTS
Example 1
Development of Novel Lung Surfactant Formulation Containing DPPG
and Cholesterol
[0257] Formulation of Lung Surfactant:
[0258] Lab-scale formulations were prepared at 60 mg/ml (TPL) with
the cholesterol content variations described below (Table 3). Each
formulation was prepared in n=1-5 (see lot numbers and names in
Table 3) and individually characterized. The appropriate amounts
(Table 3) of DPPC, POPG/DPPG, PA/cholesterol and KL.sub.4 by weight
were sequentially added to a round bottom flask containing
appropriate volumes of ethanol, which was heated to 45-50.degree.
C. Each of the components was added with 1-2 minutes application of
bath sonication. Finally, after each of components was added, the
flask was bath sonicated for 5 more minutes. The flask was attached
to a rotary evaporator and the ethanol was evaporated at 55.degree.
C. The speed of the rotary evaporator was .about.50 rpm and a
vacuum of <10 mbar was applied. When there was about 15 mls of
ethanol remaining in the flask, the speed of the rotary evaporator
was increased to 60 rpm in order to form a frothy film. On
formation of a persistent film, the flask was placed in a vacuum
desiccator for a period of 20 hrs in order to remove all the
residual ethanol. The following day the formulations were hydrated
with 20 mM Tris-Acetate buffer at a pH of 7.5 pre heated to
.about.25.degree. C. The resulting aqueous dispersion was
bath-sonicated at 45-50.degree. C. with intermittent swirling of
the flask over 30 mins. This resulted in a fine white dense
dispersion. 5M NaCl was added under constant ultrasonication and
shaking of the flask drop wise at 45-50.degree. C. in order to
obtain a final concentration of 130 mM of NaCl in the buffer. The
ultrasonication was continued for 5 mins after complete addition of
NaCl. This yielded a very fine homogeneous dense white dispersion.
Table 3 summarizes the samples used for testing.
TABLE-US-00004 TABLE 3 Composition of formulations Formulation
Cholesterol Formulation Lot compositoin mol (%) A0170-10-0
DPPC/DPPG 0 A0170-5-01 3/1 1 A0154-22-01 2 A0170-5-02 2
A0170-14-2-1 2 A0170-14-2-2 2 A0170-14-2-3 2 A0154-22-02 5
A0170-5-05 5 A0154-22-03 8 A0170-5-08 8 A0170-15-8-1 8 A0170-15-8-2
8 A0170-15-8-3 8 A0154-22-04 10 A0170-5-10 10 A0170-14-10-1 10
A0170-14-10-2 10 A0170-14-10-3 10 A0170-5-12 12 A0170-5-15 15
A0170-10-20 20 A0170-10-25 25 A0170-15-30-1 30 A0170-15-30-2 30
A0170-12-0 DPPC/POPG 0 A0170-12-2 3/1 2 A0170-12-5 5 A0170-12-8 8
A0170-12-8 8 A0170-12-10 10
[0259] The physico-chemical characteristics of the formulations
were evaluated using analytical methods for viscosity, surface
activity, zeta potential, and aerosol output rate.
[0260] Rheometry for Apparent Viscosity.
[0261] The apparent viscosity was measured using a Rheometer
(AR1000, TA Instruments, Newcastle, Del.). An aliquot (.about.400
.mu.l) was analyzed using a step flow program as follows:
[0262] Conditioning step: Allow temperature to reach 25.degree.
C.
[0263] Step Flow 1: Linear ramp as 1/sec, 0 to 200 sec with 15 data
point collection
[0264] Step Flow 2: Linear ramp as 1/sec, 200 to 0 sec with 15 data
point collection
[0265] The viscosity value at the shear rate 157.2 sec.sup.-1 from
both the steps was recorded and the average of these two values is
reported here.
[0266] Pulsating Bubble Surfactometry (PBS) for Surface
Activity.
[0267] The surface activity of the formulations was measured using
a Pulsating Bubble Surfactometer (PBS, Electronetics Corporation,
Amherst, N.Y.) at an oscillation frequency of p cycles/min, 5 mins
read time at 37.degree. C. The minimum surface tension values were
determined for the samples at 3 mg/ml concentrations prior to
aerosolization. The samples were diluted to 3 mg/ml TPL in 20 mM
Tris-Ac buffer with 130 mM NaCl. The samples were aerosolized prior
to the PBS measurement.
[0268] Aerosol Output Rate Measurements.
[0269] An Aeroneb Pro nebulizer was used to aerosolize the samples.
This nebulizer uses vibrating mesh technology (with an aperture
size of 4.8 microns) to generate the aerosol with a frequency of
128 KHz. All output rates were calculated through gravimetric
analysis, and are averaged over a two-minute period of operation.
Two sets of measurements were made with each sample. Operation at
elevated temperatures was achieved using a heating tape which was
tightly wrapped around the outside of the nebulizer. The heating
tape was operated at 10% on-time, and the equipment was pre-heated
7 minutes before aerosolization. To avoid material losses to
evaporation during pre-heating, the rubber filler plug for the
nebulizer reservoir was closed until operation began. Aerosol
collection was accomplished using a centrifuge tube that was placed
at the output end of the nebulizer and submerged in an ice
bath.
[0270] The surface activity of each formulation lot was evaluated
by recording the minimum surface tension values of pre-aerosolized
formulation samples. These samples were measured using PBS at a
concentration of 3 mg/ml, as per the internal release assay.
[0271] The pre-aerosolization minimum surface tensions measured at
3 mg/ml by PBS are shown in FIG. 1. FIG. 1 shows that all DPPG
formulations exhibit minimum surface tensions less than 10 mN/m
and, moreover, these values are essentially indistinguishable from
each other in this assay.
[0272] FIG. 2 shows the comparison between the surface tension of
DPPG formulations and POPG formulations containing cholesterol up
to 10 mol % but lacking palmitic acid. FIG. 2 shows that all DPPG
formulations exhibit minimum surface tensions of less than 3 mN/m.
In contrast, formulations containing POPG and cholesterol all
failed to demonstrate minimum surface tension values of less than
10 mN/m.
[0273] The apparent viscosities of the DPPG formulation are plotted
in FIG. 3 for both days 1 and 4 post hydration. FIG. 3 shows that
all DPPG-cholesterol formulations up to 25 mol % cholesterol showed
very low viscosity (less than 6 cp) one day after hydration with
the exception of 30 mol % cholesterol formulations having higher
viscosity (160 cp). 4 days after hydration, the viscosity of the
formulations containing 0-10 mole % and 20-30 mole % cholesterol
increased significantly. The viscosity of 0-10 mole % cholesterol
formulations decreased substantially as the content of cholesterol
was increased ranging from about 10-200 cp. Formulations containing
12-20 mole % cholesterol show no increase in viscosity and remained
lower then 5 cp, while the viscosity of formulations in the range
of 25-30 mole % cholesterol started to increase again up to 160
cp.
[0274] The results of the particle size analyses performed on the
liquid-dispersion formulations are shown in FIG. 4. The apparent
viscosities of the formulations, 4 days post hydration, trend in a
similar direction with particle size, with higher apparent
viscosities observed for formulations having greater particle
size.
[0275] Aerosol output rates were measured at room temperature and
elevated temperature (40-45.degree. C. The aerosol output rates for
the 60 mg/ml formulations are shown in FIGS. 5 (mg-TPL/min). Values
presented represent averages for each formulation type, with
various individual lots produced (n=1-5) and tested for each
formulation type and two or more measurements made for each lot.
FIG. 5 shows that the output rates at RT of the test formulations
are strongly influenced by the cholesterol content. The
aerosolization rate increased with increasing cholesterol content
up to 15 mol % and then decreases again with increasing cholesterol
content up to 30 mol %. This trend was observed for formulations
tested at 60 mg/ml concentration. The aerosolization rates at ET
were about the same level (20-25 TPL mg/min) up to 20 mol % content
of cholesterol and then decreased with increasing cholesterol
content. The capability of these formulations to be aerosolized at
RT with an output rate close to 20 mg/min has potential benefit
over current KL.sub.4-formulations. The increased content of
cholesterol in DPPG formulations lacking palmitic acid were active
pre-aerosolization exhibiting < the 10 mN/m minimum surface
tension values when tested at 3 mg/ml TPL. All cholesterol
formulations containing POPG exhibited poor minimum surface tension
values. All cholesterol formulations prepared at 60 mg/ml TPL
exhibited higher aerosol output rates than Surfaxin.RTM. tested at
30 mg/mL.
Example 2
Development of Novel Lung Surfactant Formulation Containing DPPG
and Cholesterol-II
[0276] Formulation of Lung Surfactant.
[0277] Lab-scale formulations were prepared at 60 mg/ml (TPL)
according to DOP-017, with the appropriate changes for cholesterol
content variations described below (Table 4). Each formulation was
prepared in formulation was prepared in triplicate (see lot numbers
and names in Table 4) and individually characterized.
TABLE-US-00005 TABLE 4 Formulation Lot Numbers, Names, and
Cholesterol Target Concentrations Cholesterol Formulation Lot
Concentration Name Number mol (%) mg/mL 2Ch1 A0170-14-2Ch1 2 0.32
2Ch2 A0170-14-2Ch2 2 0.32 2Ch3 A0170-14-2Ch3 2 0.32 8Ch1
A0170-15-8Ch1 8 1.26 8Ch2 A0170-15-8Ch2 8 1.26 8Ch3 A0170-15-8Ch3 8
1.26 10Ch1 A0170-14-10Ch1 10 1.58 10Ch2 A0170-14-10Ch2 10 1.58
10Ch3 A0170-14-10Ch3 10 1.58 12Ch1 A0174-7-12Ch1 12 1.89 12Ch2
A0174-7-12Ch2 12 1.89 12Ch3 A0174-7-12Ch3 12 1.89 15Ch1
A0174-7-15Ch1 15 2.36 15Ch2 A0174-7-15Ch2 15 2.36 15Ch3
A0174-7-15Ch3 15 2.36
[0278] Analytical assays and procedures that were used to
characterize the formulations are summarized in Table 5. Each
formulation was independently assayed.
[0279] The PBS activity measurements were made using a
dilution-until-failure approach. That is, each formulation was
diluted to 10 mg/ml TPL and analyzed by PBS. If the steady-state
minimum surface tension did not meet an arbitrary failure criterium
of, >10 mN/m, then the formulation was diluted 2.times. and the
assay was repeated. This process was repeated until the formulation
lot was sufficiently dilute to fail the >10 mN/m criterium.
Triplicate measurements for each formulation were made at the
lowest dilutions (i.e., the concentration at which the failure
criterion was met for the average of the triplicate measurements).
PBS activity was measured singularly at the 3 mg/ml formulation
concentration.
TABLE-US-00006 TABLE 5 Summary of Assays Performed Assay 1. Surface
activity by PBS 2. Viscosity 3. Particle size (dispersion) 4. DPPC
content by HPLC 5. DPPG content by HPLC 6. Cholesterol content by
HPLC 7. KL4 content by HPLC
[0280] The surface activity of each formulation lot was evaluated
by two means: 1) minimum surface tensions of formulation samples
were measured using PBS at a concentration of 3 mg/ml, and 2) the
failure concentration was measured on formulation samples. The
second assay was designed to identify potential differences in
activity of the formulation lots.
[0281] The minimum surface tensions measured at 3 mg/ml by PBS are
shown in Table 6 (i.e., average (+/-standard deviation) of the
three lots of each formulation). Table 6 shows that all
formulations exhibit surface tension values of less than 10 mN/m
when measured 7 or 14 days post hydration, and, moreover, these
values are essentially indistinguishable from each other and from
Surfaxin.RTM. measured in this assay at 10 mg/mL.
TABLE-US-00007 TABLE 6 Surface activities of formulations measured
at 3 mg/ml TPL Formulation Minimum Surface Tension* (mN/m) Name Day
1 Day 7 Day 14 2Ch 5.6 .+-. 4.8 0.0 .+-. 0.0 0.0 .+-. 0.0 8Ch 4.2
.+-. 2.6 0.0 .+-. 0.0 1.1 .+-. .7 10Ch 7.2 .+-. 6.0 1.3 .+-. 1.4
2.8 .+-. 2.6 12Ch 13.6 .+-. 16.8 0.9 .+-. 1.3 0.0 .+-. 0.0 15Ch
15.8 .+-. 17.0 3.2 .+-. 2.8 0.8 .+-. 1.3 Surfaxin .RTM. 0.0 .+-.
0.0 *Average of measurements for the three lots of each
formulation
[0282] The "failure" concentrations for the individual formulation
lots are listed in Table 7.
[0283] The failure concentration of Surfaxin.RTM. was 1.25 mg/ml.
In general, the failure concentrations for the three lots of a
given formulation type (i.e., 2Ch1, 2Ch2, and 2Ch3) were either all
the same value or all within one dilution, as shown in Table 4,
demonstrating reasonable consistency between the triplicate
formulation lots. All formulations also demonstrate similar surface
activity.
TABLE-US-00008 TABLE 7 Failure concentrations* of individual
formulation lots measured by PBS after 2 weeks storage Formulation
Failure Concentration Name (mg/mL) 2Ch1 0.625 2Ch2 0.625 2Ch3 0.625
8Ch1 1.25 8Ch2 1.25 8Ch3 1.25 10Ch1 1.25 10Ch2 0.625 10Ch3 0.625
12Ch1 0.625 12Ch2 0.625 12Ch3 0.3125 15Ch1 1.25 15Ch2 0.625 15Ch3
0.625 *Failure concentration is defined as the formulation
concentration at which the average of triplicate PBS minimum
surface tension measurements is greater than 10 mN/m.
[0284] The apparent viscosities of the formulation are plotted in
FIG. 6. FIG. 6 shows that all cholesterol formulations up to 15 mol
% cholesterol showed very low viscosity (less than 6 cp) one day
after hydration. However, during the first week post preparation,
formulations containing up to 10 mol % (1.575 mg/mL) cholesterol
showed an increase in viscosity that leveled off after 1 week.
Nevertheless, formulations with 12 and 15 mol % cholesterol showed
no increase in viscosity during the two weeks tested-period, and
the viscosity remained below 4 cp.
[0285] The results of the particle size analyses performed on the
liquid-dispersion formulations are shown in FIG. 7. No significant
trends were observed--with the exception of the larger apparent
particle sizes for the 2 mol % cholesterol formulation lots at this
60 mg/ml concentration, which also exhibit the highest viscosity
after storage. Note that the measured median particle size for
Surfaxin.RTM. using this method was 11.0 .mu.m.
[0286] The results of the lipid and KL.sub.4 HPLC assays on the 60
mg/ml formulations are shown in Table 8 Note that the target DPPC,
POPG, and KL.sub.4 concentrations for all of the formulations were
45, 15, and 1.60 mg/ml, respectively. The target concentrations for
cholesterol are shown in Table 1. Table 8 shows that the measured
lipid and peptide concentrations are in good agreement with the
target values.
TABLE-US-00009 TABLE 8 DPPC, DPPG, Cholesterol and KL.sub.4
concentrations of the formulations Concentration (mg/ml) Name DPPC
DPPG Cholesterol KL.sub.4 2Ch Ave: 44.2 14.6 ND 1.6 Stdev: 0.81
0.06 0.08 8Ch Ave: 46.3 15.2 1.25 1.6 Stdev: 2.40 0.27 0.02 0.06
10Ch Ave: 45.2 15.1 1.55 1.6 Stdev: 2.15 0.35 0.04 0.06 12Ch Ave:
45.4 15.6 1.86 1.6 Stdev: 1.42 0.12 0.03 0.01 15Ch Ave: 45.4 15.5
2.36 1.6 Stdev: 0.06 0.07 0.03 0.02
[0287] The stability results of the formulations in terms of
apparent viscosity and surface activity after incubation at
25.degree. C. and 60% relative humidity for 30 days are plotted in
FIG. 8. FIG. 8 shows that in the period of 30 days, at 25.degree.
C. and 60% relative humidity there was no change in the initial
viscosity of the formulations which remained below 6 cp, the
formulations were still active in terms of surface activity (below
1 mN/m at 3 mg/mL) and there was no degradation of the formulations
components as detected by HPLC.
Example 3
In Vivo Activity of a Cholesterol-Containing Composition
[0288] The change in the volume of the respiratory system is
measured by placing an animal, in this case a pre-term rabbit, in a
plethysmograph, and measuring the change in volume of gas into and
out of the plethysmograph with a flow meter, or pneumotachograph.
The stiffness of the respiratory system is calculated by dividing
the tidal volume change by the airway pressure change during a
mechanical breath. The parameter for this stiffness is the
compliance of the respiratory system (Crs).
[0289] In surfactant-deficient fetal-rabbits treated with
surfactant, Crs is expected to increase during mechanical
ventilation, and reach a plateau in approximately 10-30 minutes.
This increase is due to the spreading of the surfactant and the
establishment of a surface-active lining of the alveoli. In
surfactant-deficient animals, there can be a small increase in the
Crs with mechanical ventilation due to the release of minimal
stores of surfactant, but their lungs remain stiff because there is
insufficient surfactant to prevent alveolar collapse with each
exhalation. The expected course of Crs is shown in FIG. 9 for the
mean Crs of six fetal rabbit pups treated with a 30 mg/ml DPPC,
DPPG (3:1) and cholesterol (10 mole %) formulation and six control
animals treated with vehicle (all at 27 days gestation) during 30
minutes of mechanical ventilation. The raw data is an average of 20
seconds worth of compliance data which is acquired every 2 minutes.
Compliance values rise over time. The values for compliance are
corrected for the weight of the pups. It can be seen that the
cholesterol-containing formulation exhibits a positive trend toward
improving compliance relative to a vehicle control.
Example 4
Development of Novel Lung Surfactant Formulations Containing No
Palmitic Acid
[0290] Formulation Details.
[0291] All of the formulations prepared comprised of DPPC, POPG and
KL.sub.4 at ratios of 3 to 1 to 0.11 by weight respectively. The
active components were dissolved in a 60% t-butanol (TBA) mixture
(remaining 40% H.sub.2O) such that the final total phospholipid
concentration in the pre-lyo solvent system was 30 mg-TPL/ml. The
TBA solution containing the actives was aliquoted into 20 mL serum
vials at a fill volume of 9.1+/-0.1 mL and lyophilized using a
developmental scale lyophilizer.
[0292] Reconstitution of Samples.
[0293] The lyophilized cakes were reconstituted by hand shaking
using 20 mM Tris-Ac buffer at pH 7.6 containing 130 mM NaCl. The
volume of buffer used for reconstitution was 4.3+/-0.1 ml resulting
in a final concentration of 60 mgTPL/ml for the reconstituted
samples. The samples were evaluated for apparent viscosity and in
vitro surface activity upon reconstitution.
[0294] Apparent Viscosity Measurements.
[0295] The apparent viscosities of the surfactant formulations were
measured at a temperature of 25.degree. C. using a TA AR1000
Rheometer (TA Instruments, New Castle, Del.) fitted with a 40
mm/1.degree. acrylic cone. Approximately 350 .mu.L of undiluted
surfactant was placed on the rheometer and allowed to thermally
equilibrate at 25.degree. C. A step flow procedure was utilized to
analyze the samples with a linear increase in the shear rate with
time (0 to 200 sec.sup.-1) followed by linear decrease in shear
rate (200 to 0 sec.sup.-1). During each ramp up and ramp down
process, 15 points were collected resulting in approximately 6
minutes of total run time. The viscosities measured at a shear rate
of 157 sec.sup.-1 during the ramp up and ramp down were averaged
and reported as the apparent viscosity for each sample.
[0296] In Vitro Activity Measurements Using the PBS.
[0297] The surface activities of reconstituted formulations were
measured at 3 mg-TPL/ml using a Pulsating Bubble Surfactometer
(PBS, Electronetics Corp, Seminole, Fla.). The samples were diluted
in 20 mM Tris-Ac buffer at pH 7.6 containing 130 mM NaCl. Upon
dilution, the samples were vortexed for 10 seconds and heated in a
37.degree. C. water bath for 10 minutes. After removal from the
water bath, the samples were vortexed for 10 seconds. Approximately
50 .mu.L of sample was then analyzed on the PBS at 37.degree. C. A
bubble was formed by the user and allowed to equilibrate for 1
minute. The bubble was then pulsated at an oscillation frequency of
20 cycles/min for 6 minutes, during which time the bubble cycled
from a minimum radius of 0.4 mm to a maximum radius of 0.55 mm. The
pressure transducer in the PBS instrument measures a pressure,
which is used to calculate the surface tension using the Laplace
equation. The surface tension after 100 cycles was determined and
reported as the minimum surface tension of the sample.
[0298] Results.
[0299] The apparent viscosities and in vitro activities for the
formulations tested are represented in Table 9. The data presented
in the table shows that the minimum surface tension of all the
formulations were <15 mN/m.
TABLE-US-00010 TABLE 9 Apparent Viscosities and in vitro activities
of formulations studied Viscosity Min ST Sample # 60 mg/ml @ 3
mg/ml ID Component Name Samples (cp) (mN/m) Control Palmitic Acid
15 82 +/- 27 0 2-MeP Methyl Palmitate 10 16 +/- 2 0 +/- 0 2-EtP
Ethyl Palmitate 1 13 9 2-IpP Isopropyl Palmitate 1 19 13 2-ChP
Cholesteryl Palmitate 1 39 0 2-PaP Palmitoyl Palmitate 1 157 10
2-NaP Sodium Palmitate 1 229 0 2-KP Potassium Palmitate 1 162 0
2-TPT Tripalmitin 1 91 0 2-0PaCh Cholesterol 1 57 13 4C Cetyl
Alcohol 10 45 +/- 7 2 +/- 5
Example 5
Development of Novel Lung Surfactant Formulations Containing No
POPG (Palmitoyloleoyl Phosphatidylglycerol)
[0300] Formulation Details.
[0301] All of the formulations prepared comprised of DPPC, PA and
KL.sub.4 at ratios of 3 to 0.54 to 0.11 by weight respectively. The
active components were dissolved in a 60% t-butanol (TBA) mixture
(remaining 40% H.sub.2O) such that the final total phospholipid
concentration in the pre-lyo solvent system was 30 mg-TPL/ml. The
TBA solution containing the actives was aliquoted into 20 mL serum
vials at a fill volume of 9.1+/-0.1 mL and lyophilized using a
developmental scale lyophilizer.
[0302] Reconstitution of Samples.
[0303] The lyophilized cakes were reconstituted by hand shaking
using 20 mM Tris-Ac buffer at pH 7.6 containing 130 mM NaCl. The
volume of buffer used for reconstitution was 4.3+/-0.1 ml resulting
in a final concentration of 60 mgTPL/ml for the reconstituted
samples. The samples were evaluated for apparent viscosity and in
vitro surface activity upon reconstitution.
[0304] Apparent Viscosity Measurements.
[0305] As described above in Example 4.
[0306] In vitro activity measurements using the PBS.
[0307] As described above in Example 4.
[0308] Results.
[0309] The apparent viscosities and in vitro activities for the
formulations tested are represented in Table 10. The data presented
in the table shows that the minimum surface tension of all the
formulations were <15 mN/m. Additionally, the replacement of
POPG in the formulation with other lipids (both neutral and
charged) resulted in reduced apparent viscosities of the
formulation.
TABLE-US-00011 TABLE 10 Apparent Viscosities and in vitro
activities of formulations studied Viscosity Min ST Sample # 60
mg/ml @ 3 mg/ml ID Component Name Samples (cp) (mN/m) Control POPG
15 82 +/- 27 0 3-DSPC Di-stearoyl phosphatidylcholine 7 33 +/- 5 2
+/- 2 3-DSPE Di-stearoyl phosphatidylethanolamine 1 41.9 0 3-POPC
Palmitoyl Oleoyl Phosphatidylcholine 7 19 +/- 5 1.2 3-POPE
Palmitoyl Oleoyl Phosphatidylethanolamine 1 54.2 0 3-PPoPC
Palmitoyl Palmito-oeloyl Phosphatidylcholine 4 17 +/- 10 0 3-DMPC
Di-myristoyl phosphatidylcholine 7 27 +/- 5 3 +/- 4 3-DMPE
Di-myristoyl phosphatidylethanolamine 1 62 1 3-POPS Palmitoyl
Oleoyl Phosphatidylserine 1 76 0 3-DMPG Di-myristoyl
phosphatidylglycerol 1 21.3 0 3-DPPG Di-palmitoyl
phosphatidylglycerol 1 43.5 0 3-DPPE Di-palmitoyl
phosphatidylethanolamine 1 64.5 2.5
Example 6
Development of Novel Lung Surfactant Formulations Containing No
POPG (Palmitoyloleoyl Phosphatidylglycerol) and No Palmitic
Acid
[0310] Formulation Details.
[0311] All of the formulations prepared comprised of DPPC and
KL.sub.4 at ratios of 3 to 0.11 by weight respectively. The active
components were dissolved in a 60% t-butanol (TBA) mixture
(remaining 40% H.sub.2O) such that the final total phospholipid
concentration in the pre-lyo solvent system was 30 mg-TPL/ml. The
TBA solution containing the actives was aliquoted into 20 mL serum
vials at a fill volume of 9.1+/-0.1 mL and lyophilized using a
developmental scale lyophilizer.
[0312] Reconstitution of Samples.
[0313] The lyophilized cakes were reconstituted by hand shaking
using 20 mM Tris-Ac buffer at pH 7.6 containing 130 mM NaCl. The
volume of buffer used for reconstitution was 4.3+/-0.1 ml resulting
in a final concentration of 60 mgTPL/ml for the reconstituted
samples. The samples were evaluated for apparent viscosity and in
vitro surface activity upon reconstitution.
[0314] Apparent Viscosity Measurement.
[0315] As described above in Example 4.
[0316] In vitro activity measurements using the PBS.
[0317] As described above in Example 4.
[0318] Results.
[0319] The apparent viscosities and in vitro activities for the
formulations tested are represented in Table 11. The data presented
in the table shows that the minimum surface tension of all the
formulations were <15 mN/m. Additionally, the replacement of
POPG and palmitic acid in the formulations with alternate
lipids/esters resulted in lower apparent viscosities of the
samples.
TABLE-US-00012 TABLE 11 Apparent Viscosities and in vitro
activities of formulations studied Re- place- ment Com- Replacement
ponent Viscosity Min ST Sample Component for # 60 mg/ml @ 3 mg/ml
ID for PA POPG Samples (cp) (mN/m) Control Palmitic POPG 15 82 +/-
27 0 Acid 5-CH-2 Cholesterol DPPG 1 33.2 0.4 5-CH-5 Cholesterol
DPPG 1 33.6 5.5 5-CH-8 Cholesterol DPPG 1 39.3 0.8 5-CH-10
Cholesterol DPPG 4 51 +/- 12 0.3 +/- 0.6 5-CH-12 Cholesterol DPPG 1
36.1 0 5-CH-15 Cholesterol DPPG 1 42.6 3.8 5-VE-1 Vitamin E DPPG 1
22.4 0 5-VE-5 Vitamin E DPPG 1 34.5 0 5-VE-10 Vitamin E DPPG 1 26.7
0 5-VE-15 Vitamin E DPPG 1 32.7 0 5-VE-20 Vitamin E DPPG 1 24.6 0
3-POPC-2 Methyl POPC 3 16 +/- 2 5 +/- 9 Palmitate 3-POPC-3 Cetyl
POPC 3 12 +/- 0.8 8 +/- 4 Alcohol 3-DMPC-2 Methyl DMPC 3 37 +/- 1.9
0 +/- 0 Palmitate 3-DMPC-3 Cetyl DMPC 3 20 +/- 3.8 5 +/- 7.9
Alcohol 3-DSPC-2 Methyl DSPC 3 51 +/- 3.2 1 +/- 2.4 Palmitate
3-DSPC-3 Cetyl DSPC 3 40 +/- 4 1 +/- 0.6 Alcohol
Example 7
Improved Thermal Stability of a Formulation Containing No Palmitic
Acid and No POPG
[0320] Formulation Details.
[0321] Two formulations were prepared. One formulation comprised of
DPPC/POPG/Palmitic Acid/KL.sub.4 at ratios of 3/1/0.54/0.11 by
weight respectively, was prepared at a total phospholipid
concentration of 30 mg-TPL/ml. The second formulation comprised of
DPPC/DPPG/Cholesterol/KL.sub.4 at ratios of 3/1/1.9/0.11 by weight
and prepared at a total phospholipid concentration of 60 mg-TPL/ml.
The samples were prepared by the rotary evaporation process as
described in Example 4.
[0322] Stability Study.
[0323] The samples prepared above were aliquoted into 10 ml vials
at a fill volume of 8 mls. The formulations were stored under three
different conditions as below and tested over time at 0, 7, 14, 30,
60, 90 and 180 days.
[0324] 1. Storage at 5.degree. C..+-.3.degree. C., ambient RH
[0325] 2. Storage at 25.degree. C..+-.3.degree. C., 60% RH
[0326] 3. Storage at 40.degree. C..+-.3.degree. C., 75% RH
[0327] Thermal stability, which is equivalent to accelerated
(thermal) storage stability testing, was designed to determine
enhanced storage stability.
[0328] The analytical testing included measurements of apparent
viscosity, in vitro surface activity, KL.sub.4 concentration using
RP-HPLC and aerosol output rate.
[0329] Apparent Viscosity Measurements.
[0330] The apparent viscosity measurements were performed as
described in Example 4.
[0331] In Vitro Activity Measurements Using the PBS.
[0332] The surface activities of reconstituted formulations were
measured using dilution curves using a Pulsating Bubble
Surfactometer (PBS, Electronetics Corp, Seminole, Fla.). The
samples were intitally diluted to 10 mg-TPL/ml followed by doubling
dilutions until the minimum surface tension at 100 cycles was
>10 mN/m (this concentration is referred to as the failure
concentration for the sample). The surface tension at this dilution
and one prior dilution were then measured two more times. The key
readouts for the samples included the failure concentration and the
minimum surface tension at 10 mg-TPL/ml. The measurement details
for the PBS are described in Example 4.
[0333] Aerosol Output Rate Measurements.
[0334] An Aeroneb Pro nebulizer was used to aerosolize the samples.
This nebulizer uses vibrating mesh technology (with an aperture
size of 4.8 microns) to generate the aerosol with a frequency of
128 KHz. All output rates were calculated through gravimetric
analysis, and are averaged over a two-minute period of operation.
Two sets of measurements were made with each sample. Operation at
elevated temperatures was achieved using a heating tape which was
tightly wrapped around the outside of the nebulizer. The heating
tape was operated at 10% on-time, and the equipment was pre-heated
7 minutes before aerosolization. To avoid material losses to
evaporation during pre-heating, the rubber filler plug for the
nebulizer reservoir was closed until operation began.
[0335] Measurement of KL.sub.4 Concentration Using RP-HPLC.
[0336] Approximately 250 mg of the sample was weighed in a 15 ml
eppendorf tubes and 1 ml of TFA was added to the sample. Upon
dissolution of the sample in TFA, 10 ml of a 1:1 solution of
H.sub.2O:ACN was added. The mixture was vortexed and then
centrifuged at 3000 rpm for 20 min in order to pellet the lipids. A
250-ul aliquot of the supernatant was loaded onto the column.
KL.sub.4 analysis was carried out on a HP1100 (Agilent
Technologies, Palo Alto, Calif.). A Zorbax-SB, C-18, 5.mu.,
4.6.times.250 mm (Agilent Technologies, Palo Alto, Calif.) was used
for the analysis. The eluent system comprised of a gradient of
H.sub.2O and ACN with TFA as a modifier (details provided below) at
a flow rate of 1 mL/min. The column was maintained at 60.degree. C.
with UV signal detection at 214 nm. The running buffers were 60%
H.sub.2O, 40% ACN, 0.1% TFA (Solvent A) and 90% ACN, 10% H.sub.2O,
0.1% TFA (Solvent B). The eluent system was 0% B over 10 min,
0-100% B in 10 mins, 100% B for 10 mins; 100-0% B in 1 min and 0% B
for 4 min.
[0337] Results
[0338] Apparent Viscosity.
[0339] The apparent viscosity of the formulations is depicted in
FIG. 10. The data indicates that the apparent viscosity for the
cholesterol (CH) containing formulation does not change at
25.degree. C. up to 180 days of storage. An increase in viscosity
is observed for the cholesterol containing formulation at 5.degree.
C.
[0340] In Vitro Surface Activity.
[0341] The in vitro surface activity data for the samples is
presented in Table 12. The data indicates that the failure
concentrations of both control (9P) and CH formulations noisy. The
failure concentrations for the cholesterol formulations are greater
than the palmitic acid containing formulations at higher storage
temperatures.
TABLE-US-00013 TABLE 12 In vitro surface activity data for
formulations placed on thermal stability Time Failure Conc (mg/ml)
Time Min ST (mN/m) (days) 5 C. 25 C. 40 C. (days) 5 C. 25 C. 40 C.
A0174-040 (9P) Min ST @ 10 mg/ml (9P) 0 5 0 0.00 7 1.25 0.625 1.3 7
0.00 3.8 0.0 14 1.25 1.25 0.625 14 0.0 3.6 5.1 30 0.625 0.3125 2.5
30 0.0 0.4 3.9 60 0.625 <0.625 0.625 60 0 1.2 0 90 <0.625
<0.625 <0.625 90 0 0.4 0.4 180 0.3125 <1.25 <1.25 180 0
0 0 A0174-038 (12% CH) Min ST @ 10 mg/ml (12% CH) 0 10 0 7.7 7 2.5
10 5 7 7.7 14.4 2.7 14 5 5 10 14 0.8 2.7 10.6 30 0.63 1.25 1.25 30
0.0 0.8 1.6 60 1.25 1.25 1.25 60 0 0 0 90 0.625 1.25 2.5 90 0 0.8 0
180 0.625 1.25 10 180 0.4 0 16.6
[0342] Aerosol Output Rate Measurements.
[0343] The aerosol output rate measurements are depicted in FIG.
11. The data shows that the aerosol output rates for the
cholesterol containing formulations are greater than those for the
palmitic acid containing formulations at all storage temperatures
and times.
[0344] Change in the KL.sub.4 Concentration of the Formulations
Over Time.
[0345] The change in the KL.sub.4 concentrations for the samples
containing cholestereol and palmitic acid stored at 5.degree. C.
and 25.degree. C. are depicted in FIGS. 12 and 13 respectively. The
data presented in the figures shows much improved thermal stability
for the KL.sub.4 in the cholesterol containing formulation when
compared with the palmitic acid containing formulation. At
5.degree. C., the % loss of KL.sub.4 for the cholesterol containing
formulation is 7.5% as compared to 25% for the palmitic acid
formulation (a threefold improvement in the thermal stability for
the cholesterol containing formulation). Similarly, FIG. 13 shows
that the loss in KL.sub.4 is 69% for the palmitic acid formulation
as compared to 10% for the cholesterol containing formulation.
[0346] Although the foregoing invention has been described in
detail by way of example for purposes of clarity of understanding,
it will be apparent to the artisan that certain changes and
modifications are comprehended by the disclosure and can be
practiced without undue experimentation within the scope of the
appended claims, which are presented by way of illustration not
limitation.
[0347] All publications and patent documents cited above are hereby
incorporated by reference in their entirety for all purposes to the
same extent as if each were so individually denoted.
[0348] Each recited range includes all combinations and
sub-combinations of ranges, as well as specific numerals contained
therein.
Sequence CWU 1
1
15121PRTArtificial SequenceSynthetic Construct 1Lys Leu Leu Leu Leu
Lys Leu Leu Leu Leu Lys Leu Leu Leu Leu Lys1 5 10 15Leu Leu Leu Leu
Lys 20221PRTArtificial SequenceSynthetic Construct 2Lys Leu Leu Leu
Leu Leu Leu Leu Leu Lys Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Lys
Leu Leu 20321PRTArtificial SequenceSynthetic Construct 3Lys Lys Leu
Leu Leu Leu Leu Leu Leu Lys Lys Leu Leu Leu Leu Leu1 5 10 15Leu Leu
Lys Lys Leu 20421PRTArtificial SequenceSynthetic Construct 4Asp Leu
Leu Leu Leu Asp Leu Leu Leu Leu Asp Leu Leu Leu Leu Asp1 5 10 15Leu
Leu Leu Leu Asp 20521PRTArtificial SequenceSynthetic Construct 5Arg
Leu Leu Leu Leu Arg Leu Leu Leu Leu Arg Leu Leu Leu Leu Arg1 5 10
15Leu Leu Leu Leu Arg 20621PRTArtificial SequenceSynthetic
Construct 6Arg Leu Leu Leu Leu Leu Leu Leu Leu Arg Leu Leu Leu Leu
Leu Leu1 5 10 15Leu Leu Arg Leu Leu 20721PRTArtificial
SequenceSynthetic Construct 7Arg Arg Leu Leu Leu Leu Leu Leu Leu
Arg Arg Leu Leu Leu Leu Leu1 5 10 15Leu Leu Arg Arg Leu
20819PRTArtificial SequenceSynthetic Construct 8Arg Leu Leu Leu Leu
Cys Leu Leu Leu Arg Leu Leu Leu Leu Cys Leu1 5 10 15Leu Leu
Arg921PRTArtificial SequenceSynthetic Construct 9Arg Leu Leu Leu
Leu Cys Leu Leu Leu Arg Leu Leu Leu Leu Cys Leu1 5 10 15Leu Leu Arg
Leu Leu 201028PRTArtificial SequenceSynthetic Construct 10Arg Leu
Leu Leu Leu Cys Leu Leu Leu Arg Leu Leu Leu Leu Cys Leu1 5 10 15Leu
Leu Arg Leu Leu Leu Leu Cys Leu Leu Leu Arg 20 251121PRTArtificial
SequenceSynthetic Construct 11His Leu Leu Leu Leu His Leu Leu Leu
Leu His Leu Leu Leu Leu His1 5 10 15Leu Leu Leu Leu His
201220PRTArtificial SequenceSynthetic Construct 12Arg Leu Leu Leu
Leu Cys Leu Leu Leu Arg Leu Leu Leu Leu Leu Cys1 5 10 15Leu Leu Leu
Arg 201322PRTArtificial SequenceSynthetic Construct 13Arg Leu Leu
Leu Leu Leu Cys Leu Leu Leu Arg Leu Leu Leu Leu Cys1 5 10 15Leu Leu
Leu Arg Leu Leu 201449PRTArtificial SequenceSynthetic Construct
14Arg Leu Leu Leu Leu Cys Leu Leu Leu Arg Leu Leu Leu Leu Cys Leu1
5 10 15Leu Leu Arg Leu Leu Leu Leu Cys Leu Leu Leu Arg Asp Leu Leu
Leu 20 25 30Asp Leu Leu Leu Asp Leu Leu Leu Asp Leu Leu Leu Asp Leu
Leu Leu 35 40 45Asp159PRTArtificial SequenceSynthetic Construct
15Leu Leu Glu Lys Leu Leu Gln Trp Lys1 5
* * * * *