U.S. patent application number 14/201119 was filed with the patent office on 2014-12-11 for method for preventing and treating hyperpermeability.
This patent application is currently assigned to Apeptico Forschung UND Entwicklung GMBH. The applicant listed for this patent is Apeptico Forschung UND Entwicklung GMBH. Invention is credited to Bernhard Fischer, Rudolf Lucas.
Application Number | 20140364358 14/201119 |
Document ID | / |
Family ID | 42115985 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140364358 |
Kind Code |
A1 |
Fischer; Bernhard ; et
al. |
December 11, 2014 |
Method for Preventing and Treating Hyperpermeability
Abstract
A peptide is described, which consists of 7-17 adjacent amino
acids and comprises the hexamer TXEXXE, wherein X, X and X can be
any natural or non-natural amino acid, wherein the peptide has no
TNF receptor binding activity and is cyclized, for the prevention
and treatment of hyperpermeability of epithelial cells and
endothelial cells.
Inventors: |
Fischer; Bernhard; (Vienna,
AT) ; Lucas; Rudolf; (Aartselaar, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Apeptico Forschung UND Entwicklung GMBH |
Vienna |
|
AT |
|
|
Assignee: |
Apeptico Forschung UND Entwicklung
GMBH
Vienna
AT
|
Family ID: |
42115985 |
Appl. No.: |
14/201119 |
Filed: |
March 7, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13254273 |
Sep 1, 2011 |
|
|
|
PCT/AT2010/000056 |
Mar 5, 2010 |
|
|
|
14201119 |
|
|
|
|
Current U.S.
Class: |
514/1.5 ;
514/1.6; 514/2.4; 514/3.7; 530/317 |
Current CPC
Class: |
A61K 38/191 20130101;
A61P 11/00 20180101; A61P 31/12 20180101; C07K 7/64 20130101; A61P
43/00 20180101; A61K 38/12 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/1.5 ;
514/1.6; 514/2.4; 514/3.7; 530/317 |
International
Class: |
C07K 7/64 20060101
C07K007/64 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 2009 |
AT |
359/2009 |
Claims
1. A method for the prevention of oedema by reduction of
hyperpermeability, based on injuries of the endothelium and
epithelium layers, comprising the step of: administering a peptide
consisting the amino acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1)
and is cyclized via the C residues in a patient suffering from
pneumonia, acute lung injury, acute respiratory distress syndrome
(ADRS) or bacterial or viral lung disease.
2.-3. (canceled)
4. The method according to claim 1, wherein the bacterial or viral
lung diseases are selected from Listeria monocytogenes,
Streptococcus pneumoniae, SARS viruses, RSV or influenza
viruses.
5. (canceled)
6. The method according to claim 1, wherein the peptide is cyclized
via a disulfide bridge between said C residues.
7. The method according to claim 1, wherein the cells of the
endothelial layers are protected against hyperpermeability
triggered by reactive oxygen molecules.
8. The method according to claim 1, wherein wherein the cells of
the endothelial layers are protected against hyperpermeability
triggered by bacterial toxins.
9. The method according to claim 1, wherein the phosphorylation of
the myosin light chain is inhibited.
10. The method according to claim 1, wherein the peptide is used
for inhibiting the activation of protein kinase C.
11. The method according to claim 1, wherein the peptide is used
for increasing the expression of the epithelial sodium channel.
12. The method according to claim 1, wherein the peptide is used
for treating hyperpermeability triggered by reactive oxygen
molecules, microbial toxins, or pulmonary virus infections.
13. The method according to claim 1, wherein the peptide is
contained in a pharmaceutical composition comprising the peptide
and a pharmaceutical carrier.
14. A method for the prevention of oedema by reduction of
hyperpermeability, based on injuries of the endothelium and
epithelium layers, comprising the administration of a peptide
consisting the amino acid sequence 7-17 adjacent amino acids and
comprising the hexamer TX.sub.1EX.sub.2X.sub.3E, wherein X.sub.1,
X.sub.2 and X.sub.3 can be any natural or non-natural amino acid,
wherein the peptide has no TNF receptor binding activity and is
cyclized, in a patient suffering from pneumonia, acute lung injury,
acute respiratory distress syndrome (ADRS) or bacterial or viral
lung disease.
15. The method according to claim 14, wherein peptide comprises the
hexamer TPEGAE (SEQ. ID. No. 4).
16. The method according to claim 14, wherein peptide comprises
peptide is a cyclized peptide consisting of a sequence of
consecutive amino acids selected from the group consisting of
TABLE-US-00008 (SEQ. ID. No. 5) QRETPEGAEAKPWY; (SEQ. ID. No. 6)
PKDTPEGAELKPWY; (SEQ. ID. No. 7) CGQRETPEGAEAKPWYC; and (SEQ. ID.
No. 8) CGPKDTPEGAELKPWYC;
and fragments of at least 7 amino acids thereof, which fragments
include the hexamer TPEGAE.
17. The method according to claim 14, wherein the bacterial or
viral lung diseases are selected from Listeria monocytogenes,
Streptococcus pneumoniae, SARS viruses, RSV or influenza
viruses.
18. The method according to claim 14, wherein the peptide is
cyclized via a disulfide bridge between said C residues.
19. The method according to claim 14, wherein the cells of the
endothelial layers are protected against hyperpermeability
triggered by reactive oxygen molecules.
20. The method according to claim 14, wherein wherein the cells of
the endothelial layers are protected against hyperpermeability
triggered by bacterial toxins.
21. The method according to claim 14, wherein the phosphorylation
of the myosin light chain is inhibited.
22. The method according to claim 14, wherein the peptide is used
for inhibiting the activation of protein kinase C.
23. The method according to claim 14, wherein the peptide is used
for increasing the expression of the epithelial sodium channel.
24. The method according to claim 14, wherein the peptide is used
for treating hyperpermeability triggered by reactive oxygen
molecules, microbial toxins, or pulmonary virus infections.
25. The method according to claim 14, wherein the peptide is
contained in a pharmaceutical composition comprising the peptide
and a pharmaceutical carrier.
Description
PRIORITY CLAIM AND CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 13/254,273, filed Sep. 1, 2011,
which is a U.S. National Phase application of PCT Patent
Application No. PCT/AT2010/000056, filed Mar. 5, 2010.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods for preventing and
treating hyperpermeability in endothelial cells and epithelial
cells.
[0003] Endothelial cells and epithelial cells have decisive
functions in all tissues and organs of the human and animal
body.
[0004] The endothelium consists of a thin layer of endothelial
cells. The layer of endothelial cells forms, among others, the
inner surface of the blood vessels, like veins and capillaries, and
the barrier between the blood and the outer wall of the blood
vessels. Endothelial cells line the entire blood system, from the
large blood vessels up to the smallest capillaries. Epithelial
cells form single- or multi-layer cell layers, which cover all
inner and outer body surfaces of the human and animal organs.
Epithelial cells are in close proximity to each other and are rich
in cell contacts. For epithelial cells, a distinction can be made
into an outer, apical side facing towards the outside or the lumen,
and a basal side. Furthermore, epithelial cells have an adhesion
complex (junctional complex), consisting of zonula occludens (tight
junction), zonula adhaerens (adhaerens junction) and desmosome
(macula adhaerens), which on the one hand represents a
physicochemical barrier and on the other hand interconnects
adjacent epithelial cells.
[0005] For the physiological function of all animal and human
organs and organelles, the intactness, in particular of the
restricting cells and cell layers, is extremely important. If, for
example, there is an injury of the endothelial cells or an injury
of the endothelium of the blood vessels, respectively, liquid can
escape from the blood vessels and result in massive disturbances in
the vitality of the entire organism.
[0006] If, for example, there is an injury of the epithelial cells
or an injury of the epithelium of organs, liquid can escape from
the organs or liquid can penetrate, respectively, and thus
seriously damage the functionality of the organs.
[0007] An injury of the endothelium and the epithelium may cause a
so-called hyperpermeability, i.e. an uncontrolled passage of liquid
from blood vessels into vital organs and tissues.
[0008] Beside mechanical causes, an infection or the impact of
toxins can result in hyperpermeability. Microbial toxins are
pore-forming molecules binding to cholesterol, which are released
by gram-positive bacteria. Due to the effect of toxins, first pores
are formed in cell membranes, and then macro-pores. Thus, cell
layers become permeable for liquid and substances contained
therein.
[0009] Known toxins are, among others, listeriolysin from Listeria
monocytogenes or also pneumolysin from Streptococcus pneumoniae.
These toxins can result in the formation of reactive oxygen
molecules in the cells. The reactive oxygen molecules caused by
toxins then result in damages to endothelium and epithelium due to
the fact, among others, that the barrier function of the cells is
damaged.
[0010] For retention of the barrier function of endothelial cell
layers and epithelial cell layers, the cells are interconnected via
protein fibers. Components of such protein fibers are e.g. the
myosin light chain. However, due to phosphorylation of the myosin
light chain, stresses are caused in the cells and the cell-cell
connections, and intercellular gaps are formed, whereby liquid can
penetrate and also leak in an uncontrolled manner.
[0011] A further component in the regulation of the barrier
function of the epithelial cells and endothelial cells is protein
kinase C. For protein kinases C, several isoenzymes are known, e.g.
protein kinase alpha and zeta. These protein kinase C isoenzymes
are activated by reactive oxygen molecules, hydrogen peroxide,
microbial toxins, like pneumolysin and listeriolysin, and
hydrophilic coronavirus proteins. Activated protein kinase C
additionally results in a reduction of the expression of the
epithelial sodium channel (ENaC), which is responsible for the
sodium and liquid transport in epithelial cells, and thus,
activated protein kinase C essentially contributes to the
development of hyperpermeability.
[0012] Further causes for the development of hyperpermeability in
the lungs are e.g. viruses, like influenza viruses, the severe
acute respiratory syndrome-associated coronavirus (SARS-CoV) or the
respiratory syncytial virus, which can result in hyperpermeability
of the endothelium and epithelium as well as in atypical pneumonia.
It is known that SARS-CoV proteins due to the activation of the
protein kinase C isoform result in a reduction of the size and
activity of the epithelial sodium channel, which promotes the
development of hyperpermeability. It is also known that for these
viral diseases of the lungs, the frequently used beta-2 adrenergic
agonists show no effect.
[0013] Thus, in total, it is known that microbial toxins result in
an increased level of reactive oxygen molecules in endothelial and
epithelial cells. This causes phosphorylation of the myosin light
chain, which again results in a disturbance of the cell-cell
interaction and in the development of hyperpermeability.
[0014] Microbial toxins, reactive oxygen molecules as well as viral
proteins result in an activation of protein kinase C isoenzymes.
The activation of protein kinase C then results in a decrease of
the expression of the epithelial sodium channel (ENaC) and the
inhibition of its activity. These mechanisms, too, result in the
development of hyperpermeability in the endothelium and
epithelium.
[0015] Hyperpermeability of lung tissues is an essential component
of various diseases of the lungs, e.g. acute lung injury, acute
respiratory distress syndrome (ARDS), pneumonia. Currently, there
is no standard therapy for treating hyperpermeability of the
endothelium and epithelium.
[0016] US 2003/0185791 A1, EP 2 009 023 A1, WO 2006/013183 A1, EP 1
264 559 A1 and Marquardt et al. (J. Pept. Sci. 13 (2007): 803-810)
disclose TNF-derived peptides for treating edemas.
SUMMARY OF THE INVENTION
[0017] The object of the present invention therefore is to provide
means and methods, by means of which diseases, for which the
prevention of hyperpermeability of epithelial cells and endothelial
cells plays an essential role in the treatment, in particular lung
diseases, like acute lung injuries, ARDS or viral lung diseases,
can be prevented or treated.
[0018] In particular, the invention is to provide a biologically
effective molecule for the prevention and treatment of
hyperpermeability of the endothelium and epithelium and for the
prevention and treatment of acute lung damage and the consequences
of pneumonia.
[0019] Accordingly, the present invention relates to a peptide,
which consists of 7-17 adjacent amino acids and comprises the
hexamer TXEXXE, wherein X, X and X can be any natural or
non-natural amino acid, wherein the peptide has no TNF receptor
binding activity and is cyclized, for the prevention and treatment
of hyperpermeability of epithelial cells and endothelial cells.
[0020] Preferably, the present invention relates to a peptide
consisting of 7-17 adjacent amino acids and comprising the hexamer
TPEGAE (SEQ ID No. 4), wherein the peptide has no TNF receptor
binding activity and is cyclized, for the prevention and treatment
of hyperpermeability of epithelial cells and endothelial cells.
[0021] One particularly preferred embodiment of the present
invention relates to a cyclized peptide consisting of a sequence of
consecutive amino acids selected from the group consisting of
TABLE-US-00001 (SEQ ID No. 5) QRETPEGAEAKPWY (SEQ ID No. 6)
PKDTPEGAELKPWY (SEQ ID No. 1) CGQRETPEGAEAKPWYC and (SEQ ID No. 7)
CGPKDTPEGAELKPWYC
and fragments of at least 7 amino acids thereof, which fragments
include the hexamer TPEGAE, for manufacturing of a drug for
preventing and treating hyperpermeability of epithelial cells and
endothelial cells.
[0022] The peptides according to the invention are preferably used
for preventing the outbreak of or for treating pneumonia, acute
lung injury, acute respiratory distress syndrome (ARDS) or
bacterial or viral lung diseases, in particular infections with
Listeria monocytogenes, Streptococcus pneumoniae, influenza
viruses, SARS or RSV. The cause of pneumonia, which can be treated
or prevented according to the invention, is independent of the
cause of pneumonia and independent of whether it is an acute or
chronic inflammation. Accordingly, according to the invention,
preferably pneumonias, which are caused by an infection with
bacteria, viruses, mycoplasmas, protozoa, worms or fungi, can be
treated, but also toxically (e.g. by inhalation of toxic
substances) or immunologically caused pneumonias or such ones
caused by radiation (e.g. X-Rays, radiation therapy in cancer
patients). Especially for pneumonias caused by inhalation of toxic
substances or radiation, the preventive aspect of the present
invention is particularly essential, however, also for bedridden
persons, in particular older people, or for immunocompromised
persons, like HIV patients or transplant patients. In particular,
according to the invention, the pneumonia can be fought or
prevented at a time, when no damages are recognizable on the X-ray
yet.
[0023] Pathogens of primary pneumonias are mostly pneumococci,
staphylococci, Haemophilus influenzae, mycoplasmas, chlamydia,
legionella (Legionella pneumophila) and viruses like the flu virus,
adenovirus and parainfluenza viruses. For secondary pneumonias, the
spectrum of pathogens is shifted to Herpes viruses (CMV, HSV),
fungi, Pneumocystis jirovecii, protozoa (toxoplasmosis) as well as
anaerobic bacteria. In particular pneumonias caused by these
pathogens are, according to the invention, particularly preferably
treatable or (in particular in respect of secondary pneumonias)
preventable, respectively.
[0024] The peptides according to the invention are for example
known from the European patent EP 1 264 599 B1 and were suggested
in the state of the art for the treatment of liquid accumulations
(lung edema) and in particular for the re-absorption of these
liquid accumulations, wherein the edema liquid is returned from the
alveoli of the lung tissue into the capillaries, i.e. pumped out of
the alveoli.
[0025] According to the invention, it was completely surprisingly
demonstrated, that these peptides also influence the opposite
liquid flow via the endothelium of the capillaries into the
epithelium of the lung, however, in a contrary manner: while for
the treatment of edemas, the transporting out of the liquid
requires open and fully active pumping mechanisms, according to the
invention, the passage of the liquid into the alveoli is stopped;
the influx is thus prevented in the first place. The activation of
edema resorption according to EP 1 264 599 B1 by the peptides
according to the invention therefore seems to be based on a
completely different mechanism--running in the opposite direction
and in a regulating manner--than the reduction of hyperpermeability
according to the invention, based on injuries of the endothelium
and epithelium layers, whereby edemas are even prevented by
avoiding the liquid transfer into the alveoli. Accordingly, with
the present invention, completely new and surprising indications
open up for the peptides according to the invention--beside the
edema treatment from EP 1 264 599 B1 (which is only indicated at a
later stage of the course of the disease).
[0026] Accordingly, the present invention is based on the
circumstance, which was also found within the course of the work
for the invention, that the peptides used according to the
invention, as defined in EP 1 264 599 B1, influence the effects of
toxins, reactive oxygen molecules, the activation of protein kinase
C, the phosphorylation of the myosin light chain, and the
expression of the epithelial sodium channel. This was not to be
expected based on the existing knowledge about these peptides.
[0027] A very particularly preferred peptide according to the
present invention consists of the amino acid sequence
CGQRETPEGAEAKPWYC and is cyclized via the C residues (at positions
1 and 17).
[0028] The cyclization of the peptides according to the invention
may either be achieved via a direct cyclization with a disulfide
bridge between the two C residues at the N and C terminus or by
coupling the peptide via both cysteines to a carrier substance. In
that, in the peptides according to the invention, the cysteine
residues are preferably provided at the beginning and at the end of
the molecule. Other functional groups achieving a cyclization of
the peptide can also be used, e.g. with an acid group resulting in
an amide or ester ring closure with an amine or alcohol (for that,
e.g. the amino acids aspartic acid and glutamic acid can be
preferably intramolecularly cyclized with serine, threonine,
tyrosine, asparagine, glutamine, or lysine). Therefore, further
preferred peptides according to the invention are, for example,
CGQKETPEGAEAKPWYC (SEQ ID No. 8), CGQRETPEGAEARPWYC (SEQ ID No. 9),
CGQRETPEGAEAKPC (SEQ ID No. 10), CQRETPEGAEAKPWYC (SEQ ID No. 11),
or CGQRETPEGAEAKFWYC (SEQ ID No. 12).
[0029] As carrier substances, any common pharmaceutically
acceptable substances can be used, which are able, e.g., to form a
covalent bond with the SH groups of the cysteines, wherein common
carrier proteins, like keyhole limpet hemocyanin (KLH), tetanus
toxin, etc. are particularly suited. Adjacent bifunctional residues
may also be provided at the carrier (e.g. acid group beside amine
or alcohol group). In this connection, it is important that
"cyclization" comprises the intramolecular ring closure as well as
the integration of a carrier (from which the bound peptide
protrudes (with the N and the C terminus of the peptide being bound
to the carrier)), wherein the peptide cyclized in such manner shows
the cyclic threedimensional structure and is respectively
stabilized.
[0030] The peptides according to the invention may preferably be
used for protecting epithelial cells and endothelial cells against
hyperpermeability caused by reactive oxygen molecules or by
bacterial toxins.
[0031] The peptides according to the invention may also be used for
inhibiting the phosphorylation of the myosin light chain, for
inhibiting the activation of protein kinase C or for increasing the
expression of the epithelial sodium channel.
[0032] In that, the peptides according to the invention can be used
for treating hyperpermeability caused by reactive oxygen molecules,
microbial toxins, gram-positive microorganisms or pulmonary virus
infections.
[0033] According to a further aspect, the present invention relates
to a pharmaceutical composition containing a peptide according to
the invention (or a mixture of various peptides according to the
invention) and a pharmaceutical carrier. According to the
invention, this pharmaceutical composition is used for preventing
and treating hyperpermeability, as described above, in particular
for preventing and treating pneumonia, acute lung injury, acute
respiratory distress syndrome (ARDS) or viral lung diseases, in
particular infections with Listeria monocytogenes, Streptococcus
pneumoniae, SARS, RSV or influenza viruses, in particular influenza
A viruses. The term "a pharmaceutical composition" refers to any
composition comprising a peptide as defined above, which prevents,
enhances or heals the conditions described herein. In particular,
the term "a pharmaceutical composition" refers to a composition
having a peptide as described above and a pharmaceutically
acceptable carrier or excipient (both terms may be used
interchangeably). Suitable carriers or excipients known to the
expert are saline solution, Ringer's solution, dextrose solution,
Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline
solution, substances improving isotonia and chemical stability,
buffers and preservative agents. Further suitable carriers include
any carrier, which does not induce the production of antibodies
itself, which are harmful for the individual receiving the
composition, like proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids and amino acid
copolymers. In that, the peptide according to the invention may
also be cyclized to these carriers via a direct covalent bond. This
pharmaceutical composition may (as a drug) be administered using
any suitable method known by the expert. The preferred
administration path is parenteral, in particular by inhalation
(with aerosols) or intravenous administration. For parenteral
administration, the drug of this invention is formulated in an
injectable unit dosage form, like a solution, suspension or
emulsion, in connection with the pharmaceutically acceptable
excipient defined above. Dosage and type of administration,
however, depend on the individual. In general, the drug is
administered such that the peptide of the present invention is
administered at a dose of between 1 .mu.g/kg and 10 .mu.g/kg, more
preferably between 10 .mu.g/kg and 5 mg/kg, most preferably between
0.1 and 2 mg/kg. Preferably, it is administered as a bolus dose. A
continuous infusion may be used as well. In this case, the drug may
be infused at a dose of between 5 and 20 .mu.g/kg/minute, more
preferably between 7 and 15 .mu.g/kg/minute.
[0034] According to the present invention, a particularly preferred
peptide according to the invention has the following amino acid
sequence: SEQ ID No. 1
(NH2)Cys-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-AlaGlu-Ala-Lys-Pro-Trp-Tyr-Cys(C-
OOH).
[0035] The determination of the concentration of reactive oxygen
molecules in cultivated endothelial cells of the lungs showed that
upon culture of the endothelial cells under a normal oxygen content
of 21% (normoxic gas mixture), there is only a low formation of
reactive oxygen molecules. With lack of oxygen (0.1% of oxygen,
hypoxic gas mixture), however, there is a 3-fold increased
formation of reactive oxygen molecules. If, however, a peptide
according to the invention, in particular peptide SEQ ID No. 1, is
added to endothelial cells cultivated under lack of oxygen (oxygen
content 0.1%, hypoxic gas mixture), surprisingly no reactive oxygen
molecules are formed by the endothelial cells.
[0036] Further examinations determined the electric resistance of
cell layers of human endothelial and epithelial cells by means of
electrical cell-substrate impedance analysis before, during and
after the addition of the microbial toxins pneumolysin and
listeriolysin. The examinations showed, that with an addition of
125 ng/ml and 250 ng/ml of listeriolysin to cultivated human
endothelial cells, the development of hyperpermeability is
initiated. This process was still enhanced by a toxin concentration
of 250 ng/ml of listeriolysin. The addition of 62.5 ng/ml of
pneumolysin to cultivated human endothelial cells also resulted in
the development of hyperpermeability. This process was still
enhanced by a toxin concentration of 125 ng/ml of pneumolysin.
Surprisingly, however, it was found, that with the addition of a
peptide according to the invention, in particular 50 .mu.g/ml of
peptide SEQ ID No. 1, the pneumolysin-induced as well as the
listeriolysin-induced hyperpermeability is inhibited.
[0037] Further examinations showed that hyperpermeability can also
be induced in human epithelial cells by microbial toxins. Thus, the
incubation of human epithelial cells with 1 .mu.g/ml of
listeriolysin results in clear hyperpermeability. Surprisingly,
however, it was found, that the hyperpermeability is inhibited with
the addition of a peptide according to the invention, in particular
50 .mu.g/ml of peptide SEQ ID No. 1.
[0038] Further examinations showed that an addition of 125 ng/ml of
the toxin listeriolysin to human endothelial lung cells results in
an increase in the content of phosphorylated myosin light chain.
This effect is still enhanced by a toxin concentration of 250 ng/ml
of listeriolysin. An addition of 62.5 ng/ml of the toxin
pneumolysin to human endothelial lung cells also resulted in an
increase in the relative content of phosphorylated myosin light
chain. This effect was still enhanced by a toxin concentration of
125 ng/ml of pneumolysin. Surprisingly, however, it was found, that
the addition of a peptide according to the invention, in particular
50 .mu.g/ml of peptide SEQ ID No. 1, inhibits the phosphorylation
of the myosin light chain caused by the toxins listeriolysin and
pneumolysin.
[0039] Further examinations demonstrated that with the
intratracheal application of toxins in mice, hyperpermeability of
the lungs of mice is triggered, which was verified by the fact that
Evans blue dye passes from the blood vessels into the lung tissue.
Surprisingly, however, it was found, that with the intratracheal
application of a peptide according to the invention, in particular
50 .mu.g of peptide SEQ ID No. 1, there is an inhibition of the
hyperpermeability caused by the toxin.
[0040] Further examinations showed that by triggering
hyperpermeability in the lungs of mice, triggered by intratracheal
application of toxin, e.g. 250 ng of pneumolysin, there is an
increased number of leukocytes in the bronchoalveolar liquid.
Surprisingly, however, it was found, that with the intratracheal
application of a peptide according to the invention, in particular
50 .mu.g of peptide SEQ ID No. 1, the toxinrelated development of
hyperpermeability is inhibited and clearly less leukocytes are
present in the bronchoalveolar liquid in the lungs of mice.
[0041] Further examinations demonstrated that bacterial toxins
result in a substantial increase in the content of activated
protein kinase C alpha in human endothelial cells of the lungs.
Surprisingly, however, it was found, that the addition of a peptide
according to the invention, in particular of peptide SEQ ID No. 1,
inhibits this toxin-mediated effect and thus results in an increase
in the expression of the epithelial sodium channel. Surprisingly,
it was also found, that an addition of the peptide according to the
invention, in particular of peptide SEQ ID No. 1, to human
epithelial cells results in a substantial increase in the
expression of the epithelial sodium channel (ENaC). The invention
will now be explained in more detail on the basis of the following
examples and figures, to which it shall not be limited.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] For a more complete understanding of the present invention,
and the advantages thereof, reference is now made to the following
descriptions taken in conjunction with the accompanying drawings,
in which:
[0043] FIG. 1A shows the HPLC chromatogram of the protein with the
amino acid sequence SEQ ID No. 1. Units: Y axis "Absorption in
mAU"; X axis "Time in minutes".
[0044] FIG. 1B shows the HPLC chromatogram of the protein with the
amino acid sequence SEQ ID No. 2. Units: Y axis "Absorption in
mAU"; X axis "Time in minutes".
[0045] FIG. 1C shows the HPLC chromatogram of the protein with the
amino acid sequence SEQ ID No. 3. Units: Y axis "Absorption in
mAU"; X axis "Time in minutes".
[0046] FIG. 2A shows the electron paramagnetic resonance (EPR)
spectra of endothelial cells, which were cultivated at either 21%
oxygen (normoxic gas mixture) or 0.1% oxygen (hypoxic gas mixture)
with and without the addition of peptide SEQ ID No. 1 or peptide
SEQ ID No. 3, respectively.
[0047] FIG. 2B shows the relative content of reactive oxygen
molecules (superoxide) in endothelial cells, which were cultivated
at either 21% oxygen (normoxic gas mixture) or 0.1% oxygen (hypoxic
gas mixture) with and without the addition of peptide SEQ ID No. 1,
or at 0.1% oxygen (hypoxic gas mixture) with and without the
addition of peptide SEQ ID No. 3.
[0048] FIG. 3A shows the course of the electric resistance of human
epithelial cells of the lungs without addition of the toxin
listeriolysin as well as following addition of 125 ng/ml of
listeriolysin (125 ng/ml of LLO) and following addition of 500
ng/ml of listeriolysin (500 ng/ml of LLO).
[0049] FIG. 3B shows the course of the electric resistance of human
epithelial cells of the lungs without addition of the toxin
pneumolysin as well as following addition of 62.5 ng/ml of
pneumolysin (62.5 ng/ml of PLY) and following addition of 250 ng/ml
of pneumolysin (250 ng/ml of PLY).
[0050] FIG. 3C shows the course of the electric resistance of human
epithelial cells of the lungs without addition of the toxin
pneumolysin/peptide SEQ ID No. 1 (control) as well as following
addition of 125 ng/ml of pneumolysin (125 ng/ml of PLY) as well as
following addition of 125 ng/ml of pneumolysin/50 .mu.g/ml of
peptide SEQ ID No. 1 (125 ng/ml of PLY/50 .mu.g/ml of peptide SEQ
ID No. 1).
[0051] FIG. 3D shows the course of the electric resistance of human
epithelial cells of the lungs without addition of the toxin
listeriolysin/peptide SEQ ID No. 1 (control) as well as following
addition of 500 ng/ml of listeriolysin (500 ng/ml of LLO) as well
as following addition of 500 ng/ml of listeriolysin/50 .mu.g/ml of
peptide SEQ ID No. 1 (500 ng/ml of LLO/50 .mu.g/ml of peptide SEQ
ID No. 1).
[0052] FIG. 3E shows the course of the electric resistance of human
epithelial cells of the lungs without addition of the toxin
listeriolysin/peptide SEQ ID No. 1 (control) as well as following
addition of 1 .mu.g/ml of listeriolysin (1 .mu.g/ml of LLO) as well
as following addition of 1 .mu.g/ml of listeriolysin/50 .mu.g/ml of
peptide SEQ ID No. 1 (1 .mu.g/ml of LLO/50 .mu.g/ml of peptide SEQ
ID No. 1).
[0053] FIG. 4A shows the relative content of phosphorylated myosin
light chain in human endothelial cells of the lungs depending on
the concentration of the toxin listeriolysin (125 ng/ml of LLO, 250
ng/ml of LLO, 500 ng/ml of LLO).
[0054] FIG. 4B shows the relative content of phosphorylated myosin
light chain in human endothelial cells of the lungs depending on
the concentration of the toxin pneumolysin (62.5 ng/ml of PLY, 125
ng/ml of PLY, 250 ng/ml of PLY).
[0055] FIG. 4C shows the relative content of phosphorylated myosin
light chain in human endothelial cells of the lungs depending on
the addition of 50 .mu.g/ml of peptide SEQ ID No. 1, 250 ng/ml of
the toxin listeriolysin (LLO), 50 .mu.g/ml of peptide SEQ ID No.
1/250 ng/ml of the toxin listeriolysin (LLO), 50 .mu.g/ml of
peptide SEQ ID No. 3/250 ng/ml of the toxin listeriolysin
(LLO).
[0056] FIG. 4D shows the relative content of phosphorylated myosin
light chain in human endothelial cells of the lungs depending on
the addition of 50 .mu.g/ml of peptide SEQ ID No. 1, 125 ng/ml of
the toxin pneumolysin (PLY), 50 .mu.g/ml of peptide SEQ ID No.
1/125 ng/ml of the toxin pneumolysin (PLY), 50 .mu.g/ml of peptide
SEQ ID No. 3/125 ng/ml of the toxin pneumolysin (PLY).
[0057] FIG. 5A shows the content of Evans blue dye in the lung
tissue of mice 5.5 hours following intratracheal administration of
the toxin pneumolysin with the doses 250 ng of pneumolysin per
mouse (250 ng of PLY) and 500 ng of pneumolysin per mouse (500 ng
of PLY).
[0058] FIG. 5B shows the content of Evans blue dye in the lung
tissue of mice 5.5 hours following intratracheal administration of
250 ng of the toxin pneumolysin per mouse as well as following
intratracheal administration of 250 ng of the toxin pneumolysin and
50 .mu.g of peptide SEQ ID No. 1 per mouse.
[0059] FIG. 5C shows the content of leukocytes in the
bronchoalveolar liquid in the lungs of mice 5.5 hours following
intratracheal administration of 250 ng of the toxin pneumolysin per
mouse as well as following intratracheal administration of 250 ng
of the toxin pneumolysin and 50 .mu.g of peptide SEQ ID No. 1 per
mouse.
[0060] FIG. 6 states the content of activated protein kinase C
alpha in relation to the overall content of protein kinase C alpha,
depending on the incubation of human endothelial lung cells with
250 ng/ml of the toxin pneumolysin (250 ng/ml of PLY) and the
mixture of 250 ng/ml of the toxin pneumolysin and 50 .mu.g/ml of
peptide SEQ ID No. 1 (250 ng/ml of PLY/50 .mu.g/ml of peptide SEQ
ID No. 1).
[0061] FIG. 7 shows the expression of the epithelial sodium channel
(ENaC) in human epithelial lung cells compared to cell culture
conditions without and following addition of 50 .mu.g/ml of peptide
SEQ ID No. 1 as well as following addition of 50 .mu.g/ml of
peptide SEQ ID No. 3. The content of mRNS for ENaC was determined
using "real-time PCR".
[0062] FIG. 8 shows the change in the body weight of the test
animals with viral pneumonia (group 1: negative control (PBS);
group 2: positive control (influenza A via nasal); group 3:
influenza A via nasal+10 .mu.g of peptide SEQ ID No. 1
intratracheal).
[0063] FIG. 9 shows the change in the body temperature of test
animals of these groups 1 to 3.
[0064] FIG. 10 shows the survival rate of test animals of these
groups 1 to 3.
DETAILED DESCRIPTION OF THE INVENTION
Examples
Example 1A
Synthesis of a Peptide with the Amino Acid Sequence SEQ ID No.
1
[0065] A peptide with the amino acid sequence SEQ ID No. 1 was
fully automatically synthesized using Fmoc solid phase synthesis
with the following steps:
TABLE-US-00002 Step Process Product 1 Coupling of the amino acids
Peptide bound to the solid phase 2 Split-off from the solid Peptide
in solution phase 3 Purification Purified peptide as TFA salt 4
Purification/salt exchange Purified peptide as acetate salt 5
Analytical examination Purified peptide
[0066] Subsequently, the peptide SEQ ID No. 1 was cyclized by
oxidative formation of a disulfide bridge between the side chains
of the amino acids cysteine (position 1) and cysteine (position
17).
[0067] Subsequently, the peptide was examined using reverse HPLC,
wherein the result as shown in FIG. 1A was obtained. The purity of
the peptide SEQ ID No. 1 was higher than 95%.
Example 1B
Synthesis of a Peptide with the Amino Acid Sequence SEQ ID No.
2
TABLE-US-00003 [0068] SEQ ID No. 2
(NH2)Lys-Ser-Pro-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-
Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Glu(COOH),
wherein an amide bond is formed between the amino group of the side
chain of lysine Lys (1) and the carboxyl group of the side chain of
glutamic acid Glu (19).
[0069] A peptide with the amino acid sequence SEQ ID No. 2 was
fully automatically synthesized using Fmoc solid phase synthesis
with the following steps:
TABLE-US-00004 Step Process Product 1 Coupling of the amino acids
Peptide bound to the solid phase 2 Split-off from the solid Peptide
in solution phase 3 Purification Purified peptide as TFA salt 4
Purification/salt exchange/ Purified peptide as acetate salt
oxidative cyclization 5 Analytical examination Purified peptide
[0070] The cyclization took place by the connection of the epsilon
amino group of lysine (position 1) with the gamma carboxyl group of
glutamic acid (position 19) forming an amide bond. This is
achieved, for example, by transferring the gamma carboxyl group of
the glutamine group into an active ester by means of
dicyclohexylcarbodiimide (DHC), which active ester subsequently
spontaneously reacts with the epsilon amino group of the lysine,
forming a ring closure in the peptide.
[0071] Subsequently, the peptide was examined using reverse HPLC,
wherein the result as shown in FIG. 1B was obtained. The purity of
the peptide SEQ ID No. 2 was higher than 95%.
Example 1C
Synthesis of a Peptide with the Amino Acid Sequence SEQ ID No.
3
TABLE-US-00005 [0072] SEQ ID No. 3
(NH2)Cys-Gly-Gln-Arg-Glu-Ala-Pro-Ala-Gly-Ala-Ala-
Ala-Lys-Pro-Trp-Tyr-Cys(COOH)
(NH2)Cys-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-
Ala-Lys-Pro-Trp-Tyr-Cys(COOH)
[0073] A peptide with the amino acid sequence SEQ ID No. 3 was
fully automatically synthesized using Fmoc solid phase synthesis
with the following steps:
TABLE-US-00006 Step Process Product 1 Coupling of the amino acids
Peptide bound to the solid phase 2 Split-off from the solid Peptide
in solution phase 3 Purification Purified peptide as TFA salt 4
Purification/salt exchange Purified peptide as acetate salt 5
Analytical examination Purified peptide
[0074] Subsequently, the peptide SEQ ID No. 3 was cyclized by
oxidative formation of a disulfide bridge between the side chains
of the amino acids cysteine (position 1) and cysteine (position
17).
[0075] Subsequently, the peptide was examined using reverse HPLC,
wherein the result as shown in FIG. 1C was obtained. The purity of
the peptide SEQ ID No. 3 was higher than 95%.
[0076] The difference between peptide SEQ ID No. 3 and peptide SEQ
ID No. 1 consists in the fact that the amino acids Thr (6), Glu (8)
and Glu (11) from peptide SEQ ID No. 1 are replaced by Ala (6), Ala
(8) and Ala (11) in peptide SEQ ID No. 3.
Example 2
Influence of the Peptide SEQ ID No. 1 on Reactive Oxygen
Molecules
Cell Culture of Endothelial Cells
[0077] The cell culture of endothelial cells took place with
addition and without addition of 50 .mu.g/ml of peptide SEQ ID No.
1 or with addition and without addition of 50 .mu.g/ml of peptide
SEQ ID No. 3, respectively.
[0078] For the generation of reactive oxygen molecules, arterial
endothelial cells were cultivated in an oxygen-deficient gas
mixture of 0.1% oxygen, 5% carbon monoxide and 94.9% nitrogen
(hypoxic gas mixture). In control experiments, the gas
concentrations were 21% oxygen, 5% carbon monoxide and 74% nitrogen
(normoxic gas mixture).
[0079] After 90 minutes under oxygen-deficient conditions, the
endothelial cells were cultivated with 21% oxygen for a further 30
minutes. Thereafter, 20 .mu.l of a solution consisting of 20 uM
1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine HCl
(CHM), 20 .mu.M DPBS, 25 .mu.M desferrioxamine and 5 .mu.M
diethyldithiocarbamate as well as 2 .mu.l of DMSO were added to the
cells.
Trypsinization of the Cells.
[0080] Following cell culture, the cells were individualized in a
manner common in the laboratory by adding a trypsin solution. The
endothelial cells were washed and suspended in 35 .mu.A of a
solution consisting of DPBS and 25 .mu.M desferrioxamine and 5
.mu.M diethyldithiocarbamate.
Measurement of the Electron Paramagnetic Resonance (EPR)
[0081] The determination of the electron paramagnetic resonance
(EPR), also called electron spin resonance, serves the
investigation of paramagnetic substances, e.g. for detection of
unpaired electrons in reactive oxygen molecules (radicals of the
oxygen).
[0082] For that, the previously treated cells were placed into 50
.mu.l capillaries and examined in a MiniScope MS200 ESR of the
company Magnettech (Berlin, Germany) at 40 mW microwaves, 3000 mG
modulation amplitude, 100 kHz modulation frequency.
[0083] As FIGS. 2A and 2B show, with a normal oxygen concentration
of 21% (normoxic gas mixture), there only is a low formation of
reactive oxygen molecules. Under oxygen deficiency (0.1% oxygen,
hypoxic gas mixture), there is a 3-fold higher formation of
reactive oxygen molecules. If, however, the peptide SEQ ID No. 1 is
added to endothelial cells cultivated under oxygen deficiency
(oxygen content 0.1%, hypoxic gas mixture), then no reactive oxygen
molecules are formed by the endothelial cells.
[0084] Contrary to peptide SEQ ID No. 1, an addition of peptide SEQ
ID No. 3 to endothelial cells cultivated under oxygen deficiency
(oxygen content 0.1%, hypoxic gas mixture), does not result in an
inhibition of the formation of reactive oxygen molecules by the
endothelial cells.
[0085] The difference between peptide SEQ ID No. 3 and peptide SEQ
ID No. 1 is that the amino acids Thr (6), Glu (8) and Glu (11) of
peptide SEQ ID No. 1 are replaced with Ala (6), Ala (8) and Ala
(11) in SEQ ID No. 3.
Example 3
Inhibition of Hyperpermeability in Endothelial Cells and Epithelial
Cells by the Peptide SEQ ID No. 1
Materials
[0086] Human epithelial cells of the lungs of type H441 were
acquired from the company ATTC.
[0087] Human endothelial cells of the lungs, isolated from
capillaries of the lungs, were acquired from the company Lonza.
[0088] The microbial toxins listeriolysin (LLO) and pneumolysin
(PLY) were acquired from the University of Giessen.
Cell Culture
[0089] Human endothelial cells of the lungs, isolated from
capillaries of the lungs, were cultivated in a manner common in the
laboratory.
[0090] Epithelial cells of the lungs of type H441 were cultivated
in a manner common in the laboratory in a commercial RPMI 1640
medium with the additives 2 mM L-glutamine, 1.5 g/l of sodium
carbonate, 4.5 g/l of glucose, 10 mM HEPES buffer pH 7.4, 10%
bovine serum. The ECIS experiments took place in serum-free
medium.
Hyperpermeability
[0091] In order to cause hyperpermeability, i.e. injuries of the
endothelial cells and epithelial cells, the human epithelial cells
of the lungs as well as the human endothelial cells of the lungs
were cultivated in a manner common in the laboratory up to the
formation of a continuous cell layer, and subsequently, the toxins
listeriolysin or pneumolysin, respectively, were added.
Determination of the Transendothelial Resistance
[0092] Before, during and after the addition of the microbial
toxins pneumolysin and listeriolysin to human endothelial cells,
the electric resistance of the cell layer (transendothelial
resistance) was determined by means of electrical cell-substrate
impedance analysis.
[0093] As FIG. 3A shows, the electric resistance decreases with an
addition of 125 ng/ml of listeriolysin to cultivated human
endothelial cells. Hyperpermeability is developed. This effect is
even more significant with a higher amount of 500 ng/ml of
listeriolysin.
[0094] As FIG. 3B shows, the electric resistance decreases with an
addition of 62.5 ng/ml of pneumolysin to cultivated human
endothelial cells. Hyperpermeability is developed. This effect is
even more significant with a higher amount of 250 ng/ml of
pneumolysin.
[0095] As FIG. 3C shows, the electric resistance decreases with an
addition of 125 ng/ml of pneumolysin to cultivated human
endothelial cells. Hyperpermeability is developed. However, the
hyperpermeability caused by the addition of the toxin pneumolysin
is inhibited by addition of 50 .mu.g/ml of peptide SEQ ID No.
1.
[0096] As FIG. 3D shows, the electric resistance decreases with an
addition of 500 ng/ml of listeriolysin to cultivated human
endothelial cells. Hyperpermeability is developed. However, the
hyperpermeability caused by the addition of the toxin listeriolysin
is inhibited by addition of 50 .mu.g/ml of peptide SEQ ID No.
1.
[0097] As FIG. 3E shows, the electric resistance decreases with an
addition of 1 .mu.g/ml of listeriolysin to cultivated human
epithelial cells. Hyperpermeability is developed. However, the
hyperpermeability caused by the addition of the toxin listeriolysin
is inhibited by addition of 50 .mu.g/ml of peptide SEQ ID No.
1.
Example 4
Inhibition of the Phosphorylation of the Myosin Light Chain by the
Peptide SEQ ID No. 1
Materials
[0098] Human endothelial cells of the lungs, isolated from
capillaries of the lungs, were acquired from the company Lonza.
[0099] The microbial toxins listeriolysin (LLO) and pneumolysin
(PLY) were acquired from the University of Giessen.
Cell Culture
[0100] Human endothelial cells of the lungs, isolated from
capillaries of the lungs, were cultivated in a manner common in the
laboratory.
Determination of Phosphorylation of the Myosin Light Chain
[0101] For the determination of phosphorylation of the myosin light
chain and the influence of the peptide SEQ ID No. 1 on the
phosphorylation, the previously cultivated human endothelial cells
of the lungs were washed with phosphate buffer pH 7.4, which
contained 1 mM orthovanadate. The cell contents was lysed by
incubation of the cells with a solution of 20 mM tris buffer (pH
7.4), 150 mM mol/l of NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
2.5 mM sodiumpyrophosphate, 1 mM betaglycerophosphate, 1 mM
sodiumvanadate, 1 .mu.g/ml of leupeptine, 1 mM
phenylmethylsulfonylfluoride. In addition, the cells were digested
with ultrasound. The cell lysate was centrifuged in order to obtain
the soluble components. The soluble cell lysate was subsequently
applied to denaturing sodium dodecyl sulfate polyacrylamide gel
electrophoresis in a manner common in the laboratory, and the
proteins were separated according to their masses. Thereafter, the
proteins were transferred onto nitrocellulose membranes. The
protein blots were treated with a solution of 0.1% Tween 20 and 5%
dry milk powder for 1 hour in a manner common in the laboratory.
Subsequently, the protein blots were incubated with antibodies
directed against either the myosin light chain or the
phosphorylated myosin light chain.
[0102] In order to make either the myosin light chain or the
phosphorylated myosin light chain visible, the antibodies were made
visible on diagnostic film using chemiluminescence in a manner
common in the laboratory. The signal strength was determined with
densitometry, and the ratio of myosin light chain to phosphorylated
myosin light chain was determined.
[0103] As FIG. 4A shows, an addition of 125 ng/ml of the toxin
listeriolysin to human endothelial lung cells results in an
increase in the relative content of phosphorylated myosin light
chain. This effect is still enhanced by a toxin concentration of
250 ng/ml of listeriolysin.
[0104] As FIG. 4B shows, an addition of 62.5 ng/ml of the toxin
pneumolysin to human endothelial lung cells results in an increase
in the relative content of phosphorylated myosin light chain. This
effect is still enhanced by a toxin concentration of 125 ng/ml of
pneumolysin.
[0105] As FIG. 4C shows, an addition of 125 ng/ml of the toxin
listeriolysin to human endothelial lung cells results in an
increase in the relative content of phosphorylated myosin light
chain. An addition of 50 .mu.g/ml of peptide SEQ ID No. 1 has no
influence on the content of phosphorylated myosin light chain. The
increase in the content of phosphorylated myosin light chain by 250
ng/ml of the toxin listeriolysin is inhibited by an addition of 50
.mu.g/ml of peptide SEQ ID No. 1. A peptide SEQ ID No. 3 has no
influence on the increase in the content of phosphorylated myosin
light chain mediated by the toxin listeriolysin.
[0106] As FIG. 4D shows, an addition of 125 ng/ml of the toxin
pneumolysin to human endothelial lung cells results in an increase
in the relative content of phosphorylated myosin light chain. An
addition of 50 .mu.g/ml of peptide SEQ ID No. 1 has no influence on
the content of phosphorylated myosin light chain. The increase in
the content of phosphorylated myosin light chain by 125 ng/ml of
the toxin pneumolysin is inhibited by an addition of 50 .mu.g/ml of
peptide SEQ ID No. 1. A peptide SEQ ID No. 3 has no influence on
the increase in the content of phosphorylated myosin light chain
mediated by the toxin pneumolysin.
[0107] The difference between peptide SEQ ID No. 3 and peptide SEQ
ID No. 1 is that the amino acids Thr (6), Glu (8) and Glu (11) of
peptide SEQ ID No. 1 are replaced with Ala (6), Ala (8) and Ala
(11) in SEQ ID No. 3.
Example 5
Influence of the Peptide SEQ ID No. 1 on Hyperpermeability and
Acute Lung Damage in an Animal Model
Induction of Hyperpermeability in Mice
[0108] Laboratory mice were intratrachealy treated with a mixture
of isoflurane/oxygen prior to preparation of the lungs, as well as
with 100 .mu.l per mouse of a mixture of ketamine/rompun (1.33:1).
Following anesthesia, a venous catheter was implanted into the
mice. For induction of hyperpermeability of the lungs, 25 .mu.l of
liquid were subsequently nebulized into the lungs with a fine
syringe. The liquid either contained 0.9% saline solution or 250 ng
of the toxin pneumolysin or 250 ng/ml of pneumolysin/50 .mu.g/ml of
peptide SEQ ID No. 1.
Visualization of Hyperpermeability by Evans Blue
[0109] 5.5 hours following administration of the toxin pneumolysin,
Evans blue dye, dissolved in 0.9% saline solution, was
intravenously applied to the mice at 100 mg/kg of mouse weight.
After 30 minutes, blood was withdrawn from the animals by means of
heart puncture. Subsequently, the lungs were removed, washed with 1
ml of EDTA phosphate buffer (pH 7.4), and quick-frozen in liquid
nitrogen. For determination of the Evans blue dye content in the
lung tissue, the lungs were then homogenized in cold phosphate
buffer (1 ml of buffer per 100 mg of lung tissue), incubated with
formalin solution for 18 hours, and subsequently centrifuged
(5,000.times.g, 30 minutes). In the liquid supernatant, the
absorptions were then determined photometrically at 620 nm and at
740 nm. The Evans blue dye content in the lung tissue was
determined on the basis of a reference curve for Evans blue dye
dissolved in formalin solution, deducting the content of hemoglobin
pigments. The discharge of Evans blue dye from the capillaries into
the lung tissue due to hyperpermeability induced by the toxin
pneumolysin was compared to the amount of dye in the blood
serum.
[0110] As FIG. 5A shows, an intratracheal application of the toxin
pneumolysin with doses of 250 ng and 500 ng per mouse results in
hyperpermeability, which is determined by the fact that blood with
the Evans blue dye passes from the lung capillaries into the lung
tissue and can be verified in the lung tissue.
[0111] As FIG. 5B shows, an intratracheal application of the toxin
pneumolysin with a dose of 250 ng per mouse results in
hyperpermeability, which is determined by the fact that blood with
the Evans blue dye passes from the lung capillaries into the lung
tissue and can be verified in the lung tissue. With the
intratracheal application of 50 .mu.g of peptide SEQ ID No. 1,
there is an inhibition of the toxin-mediated development of
hyperpermeability.
[0112] As FIG. 5C shows, an intratracheal application of the toxin
pneumolysin with a dose of 250 ng per mouse results in an increased
number of leukocytes in the bronchoalveolar liquid of the lungs in
mice due to the development of hyperpermeability. With the
intratracheal application of 50 .mu.g of peptide SEQ ID No. 1,
there is an inhibition of the toxin-mediated development of
hyperpermeability and a clear reduction in the number of leukocytes
in the bronchoalveolar liquid in the lungs of mice.
Example 6
Inhibition of the Activation of Protein Kinase C by the Peptide SEQ
ID No. 1
Materials
[0113] Human endothelial cells of the lungs, isolated from
capillaries of the lungs, were acquired from the company Lonza.
[0114] The microbial toxin pneumolysin (PLY) was acquired from the
University of Giessen.
Cell Culture
[0115] Human endothelial cells of the lungs, isolated from
capillaries of the lungs, were cultivated in a manner common in the
laboratory. During cell culture, the toxin pneumolysin was added
with a concentration of 250 ng/ml, or the toxin pneumolysin with a
concentration of 250 ng/ml and the peptide SEQ ID No. 1 with a
concentration of 50 .mu.g/ml.
Determination of the Content of Activated Protein Kinase C
Alpha
[0116] The content of activated protein kinase C alpha was
determined by ELISA measurement using an antibody directed against
the activated protein kinase C alpha (phospho-threonine 638 protein
kinase C alpha). Simultaneously, the overall content of protein
kinase C alpha was determined using a commercially available ELISA
assay.
[0117] As FIG. 6 shows, due to the effect of the toxin pneumolysin,
there is a strong increase in the content of activated protein
kinase C alpha compared to the overall concentration of protein
kinase C alpha. With the addition of peptide SEQ ID No. 1, there is
an inhibition of the activation of protein kinase C alpha
Example 7
Increase in the Expression of the Epithelial Sodium Channel (ENaC)
in Epithelial Cells by the Peptide SEQ ID No. 1
Materials
[0118] Human epithelial cells of the lungs of type H441 were
acquired from the company ATTC.
Cell Culture
[0119] Epithelial cells of the lungs of type H441 were cultivated
in a manner common in the laboratory in a commercial RPMI 1640
medium with the additives 2 mM L-glutamine, 1.5 g/l of sodium
carbonate, 4.5 g/l of glucose, 10 mM HEPES buffer pH 7.4, 10%
bovine serum.
Verification of the Expression of the Epithelial Sodium Channel
[0120] In the cultivated epithelial cells, the expression of the
sodium channel (ENaC) was determined by means of "real-time PCR".
These examination took place in cells without and with the addition
of 50 ug/ml of peptide SEQ ID No. 1, as well as following the
addition of 50 .mu.g/ml of peptide SEQ ID No. 3.
[0121] As examination 7 shows, with the addition of 50 ug/ml of
peptide SEQ ID No. 1 to epithelial cells of the lungs, there is a
triplication of the expression of the sodium channel ENaC.
[0122] With an addition of 50 .mu.g/ml of peptide SEQ ID No. 3,
there is no substantial increase in the expression of the sodium
channel ENaC.
[0123] The difference between peptide SEQ ID No. 3 and peptide SEQ
ID No. 1 consists in the fact that the amino acids Thr (6), Glu (8)
and Glu (11) from peptide SEQ ID No. 1 are replaced by Ala (6), Ala
(8) and Ala (11) in peptide SEQ ID No. 3.
Example 8
Effect of Peptide SEQ ID No. 1 on the Course of Disease in Mice
with Viral Lung Infection
[0124] The following animal study groups were examined in respect
of the effect of peptide SEQ ID No. 1 on a viral lung
infection:
[0125] Group 1. Negative control (PBS via nasal).
[0126] Group 2. Positive control (infection with approx. 2,000
units of influenza A virus via nasal).
[0127] Group 3. Test group (infection with approx. 2,000 units of
influenza A virus via nasal, as well as intratracheal
administration of 10 .mu.g of peptide SEQ ID No. 1).
[0128] In each group, 6 BALB/c mice were used.
[0129] The following treatment scheme was followed:
[0130] Day of treatment 0:
[0131] Group 1: Administration of PBS via nasal.
[0132] Group 2: Infection of the mice with influenza virus A via
nasal.
[0133] Group 3: Infection of the mice with influenza virus A via
nasal and administration of peptide SEQ ID No. 1.
[0134] Days of treatment 0, 2, 4, 6, 8:
[0135] Group 1: Intratracheal administration of PBS.
[0136] Group 2: Intratracheal administration of PBS.
[0137] Group 3: Intratracheal administration of peptide SEQ ID No.
1.
[0138] Days of treatment 0 to 10:
Daily Observation of Body Temperature, Body Weight and Survival
Rate of the Test Animals.
[0139] The examinations demonstrated that test animals with viral
lung infection (group 2) lost approx. 20% of their body weight
within 10 days.
[0140] Compared to that, the body weight of the test animals
reduced by only approx. 10%, when the peptide SEQ ID No. 1 was
administered (group 3).
[0141] The results are shown in FIG. 8.
[0142] The examinations furthermore demonstrated, that in the test
animals with viral lung infection (group 2), the body temperature
cooled down from 37.5.degree. C. to 33.degree. C. after 7 days.
Subsequently, the body temperature increased to 35.degree. C.
[0143] Compared to that, in the test animals with administration of
peptide SEQ ID No. 1 (group 3), it only reduced to 35.degree. C.
after 7 days. Subsequently, the body temperature increased to
37.degree. C. again.
[0144] The results are shown in FIG. 9.
[0145] The examinations furthermore demonstrated, that 10 days
after the viral lung infection, 2/3 of the test animals of group 2
had died.
[0146] Compared to that, the mortality of the test animals with
administration of peptide SEQ ID No. 1 (group 3) after 10 days was
only 1/3.
[0147] The results are shown in FIG. 10.
[0148] In total, the examinations of test animals with viral lung
infection show that the administration of peptide SEQ ID No. 1
reduces the decrease in body weight, reduces the lowering of the
body temperature and results in a clearly increased survival
rate.
Example 9
Application of Peptide SEQ ID No. 1 ("AP301") in a Lavage-Induced
Large Animal ARDS Model
[0149] Material & methods: With the consent of the competent
animal protection commission, lung damage was induced in two pigs
(25 kg) under general anesthesia by surfactant depletion (four-time
bronchoalveolar lavage, 30 ml/kg of body weight each).
Subsequently, 1 mg/kg of body weight AP301 (peptide SEQ ID No. 1)
was endotracheally applied. Animal 1 (1) received a deep tracheal
injection of the overall dose, while for animal 2 (2), nebulization
of the same dosage over 30 min was performed. Thereafter, there was
a five-hour ventilation period. The arterial oxygen partial
pressure (paO.sub.2) was recorded using an intra-aortic real-time
measuring probe (FOXY, Ocean Optics, USA) validated in advance.
Spirometry and hemodynamics were permanently registered as well as
measurements with the PiCCO technology performed at half-hour
intervals.
[0150] Results: During application of the drug, no undesired
hemodynamic effects were demonstrated. The ventilation settings
were constantly kept in the non-protective range (Pmax 40 mbar,
tidal volume.gtoreq.10 ml/kg of body weight, PEEP.ltoreq.10 mbar,
frequency 25-35/min) in order to avoid therapeutic effects. Both
animals showed continuous improvement of oxygenation limited to
about 1.5 hours with a paO.sub.2 increase by max. 162.8 mmHg (1) or
224.6 mmHg (2), respectively. With nebulization of AP301, this
occurred delayed compared to the deep tracheal application,
however, it was more pronounced. In parallel to the improvement of
gas exchange, a reduction of the extra-vascular lung water by
15.8-52.5% compared to the initial value could be registered
following surfactant depletion.
[0151] These results impressively show that the new pharmacological
effect approach for treatment of ARDS according to the invention
also proves to be efficient in the approved large animal model for
treatment of ARDS.
[0152] Summary of the Sequences
TABLE-US-00007 SEQ ID No. 1 CGQRETPEGAEAKPWYC SEQ ID No. 2
KSPGGQRETPEGAEAKPWYE SEQ ID No. 3 CGQREAPAGAAAKPWYC SEQ ID No. 4
TPEGAE SEQ ID No. 5 QRETPEGAEAKPWY SEQ ID No. 6 PKDTPEGAELKPWY SEQ
ID No. 7 CGPKDTPEGAELKPWYC SEQ ID No. 8 CGQRETPEGAEAKPWYC SEQ ID
No. 9 CGQRETPEGAEARPWYC SEQ ID No. 10 CGQRETPEGAEAKPC SEQ ID No. 11
CQRETPEGAEAKPWYC SEQ ID No. 12 CGQRETPEGAEAKFWYC
Sequence CWU 1
1
12117PRTArtificial sequenceSynthetic peptide. 1Cys Gly Gln Arg Glu
Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 1 5 10 15 Cys
220PRTArtificial sequenceSynthetic peptide. 2Lys Ser Pro Gly Gly
Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 1 5 10 15 Pro Trp Tyr
Glu 20 317PRTArtificial sequenceSynthetic peptide. 3Cys Gly Gln Arg
Glu Ala Pro Ala Gly Ala Ala Ala Lys Pro Trp Tyr 1 5 10 15 Cys
46PRTArtificial sequenceSynthetic peptide. 4Thr Pro Glu Gly Ala Glu
1 5 514PRTArtificial sequenceSynthetic peptide. 5Gln Arg Glu Thr
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 1 5 10 614PRTArtificial
sequenceSynthetic peptide. 6Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu
Lys Pro Trp Tyr 1 5 10 717PRTArtificial sequenceSyntehtic peptide.
7Cys Gly Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp Tyr 1
5 10 15 Cys 817PRTArtificial sequenceSynthetic peptide. 8Cys Gly
Gln Lys Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 1 5 10 15
Cys 917PRTArtificial sequenceSyntehtic peptide. 9Cys Gly Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Arg Pro Trp Tyr 1 5 10 15 Cys
1015PRTArtificial sequenceSyntehtic peptide. 10Cys Gly Gln Arg Glu
Thr Pro Glu Gly Ala Glu Ala Lys Pro Cys 1 5 10 15 1116PRTArtificial
sequenceSynthetic peptide. 11Cys Gln Arg Glu Thr Pro Glu Gly Ala
Glu Ala Lys Pro Trp Tyr Cys 1 5 10 15 1217PRTArtificial
sequenceSynthetic peptide. 12Cys Gly Gln Arg Glu Thr Pro Glu Gly
Ala Glu Ala Lys Phe Trp Tyr 1 5 10 15 Cys
* * * * *