U.S. patent application number 16/418265 was filed with the patent office on 2019-10-10 for composition comprising a peptide and an inhibitor of viral neuraminidase.
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, Hendrik Fischer, Rudolf Lucas.
Application Number | 20190309021 16/418265 |
Document ID | / |
Family ID | 45001564 |
Filed Date | 2019-10-10 |
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United States Patent
Application |
20190309021 |
Kind Code |
A1 |
Fischer; Bernhard ; et
al. |
October 10, 2019 |
Composition Comprising a Peptide and an Inhibitor of Viral
Neuraminidase
Abstract
Described is a composition comprising a peptide which consists
of 7-17 adjacent amino acids and comprises the hexamer
TX.sub.1EX.sub.2X.sub.3E, where X.sub.1, X.sub.2, and X.sub.3 can
be any natural or non-natural amino acid, and the peptide is
cyclized and does not exhibit TNF receptor binding activity, and an
inhibitor of viral neuraminidase.
Inventors: |
Fischer; Bernhard; (Vienna,
AT) ; Lucas; Rudolf; (Aartselaar, BE) ;
Fischer; Hendrik; (Vienna, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Apeptico Forschung UND Entwicklung GMBH |
Vienna |
|
AT |
|
|
Assignee: |
Apeptico Forschung UND Entwicklung
GMBH
Vienna
AT
|
Family ID: |
45001564 |
Appl. No.: |
16/418265 |
Filed: |
May 21, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13885705 |
Jun 20, 2013 |
10344055 |
|
|
PCT/AT11/00462 |
Nov 15, 2011 |
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16418265 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 43/00 20180101; A61K 38/191 20130101; A61P 31/16 20180101;
C07K 7/64 20130101; A61K 2300/00 20130101; A61K 38/191 20130101;
A61P 11/00 20180101 |
International
Class: |
C07K 7/64 20060101
C07K007/64; A61K 38/19 20060101 A61K038/19; A61K 45/06 20060101
A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 18, 2010 |
AT |
A 1908/2010 |
Claims
1.-16. (canceled)
17. A composition, comprising: a peptide, selected from the group
consisting of CGQRETPEGAEAKPWYC (SEQ ID NO: 1), QRETPEGAEAKPWY (SEQ
ID NO: 5), PKDTPEGAEALKPWY (SEQ ID NO: 6) and CGPKDTPEGAELKPWYC
(SEQ ID NO: 7), wherein the peptide has no TNF receptor binding
activity and is cyclized, and an inhibitor of viral neuraminidase,
wherein the inhibitor of viral neuraminidase is a sialic acid
analog.
18. The composition of claim 17, wherein the peptide comprises the
amino acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1) and is
cyclized via the C-residues.
19. The composition of claim 18, wherein the peptide is cyclized
via a disulfide bridge between said C-residues.
20. The composition of claim 17, further comprising a
pharmaceutically acceptable carrier.
21. The composition of claim 20, wherein the pharmaceutically
acceptable carrier is selected from the group consisting of water,
sodium chloride, sodium phosphate, sodium acetate, sodium
carbonate, citrate, glycine, glycylglycine, histidine, lysine,
arginine, TRIS, sodium citrate, Ringer's solution, dextrose,
mannite, trehalose, saccharose, sorbite, fructose, maltose, lactose
or dextrane, Hank's solution, fixed oils, ethyl oleate, stabilizing
agents, pharmaceutically acceptable proteins, polysaccharides,
polylactic acids, polyglycolic acids, polymeric amino acids and
amino acid copolymers.
22. The composition of claim 17, wherein the composition comprises,
independently of each other, the peptide in an amount of 1 .mu.g to
10 g, and the inhibitor of viral neuraminidase in an amount of 1
.mu.g to 10 g.
23. The composition of claim 17, wherein the composition is in a
liquid form and comprises, independently of each other, the peptide
in an amount of 1 .mu.g to 10 g, and the inhibitor of viral
neuraminidase in an amount of 1 .mu.g to 10 g, and is provided in a
volume of 0.5 to 10 ml.
24. The composition of claim 17, wherein the composition is further
defined as a nebulizable powder formulation or as a nebulizable
liquid formulation.
25. A set, comprising: a first container comprising a peptide,
selected from the group consisting of TABLE-US-00006 (SEQ ID NO: 1)
CGQRETPEGAEAKPWYC, (SEQ ID NO: 5) QRETPEGAEAKPWY, (SEQ ID NO: 6)
PKDTPEGAEALKPWY and (SEQ ID NO: 7) CGPKDTPEGAELKPWYC,
wherein the peptide has no TNF receptor binding activity and is
cyclized, and a second container comprising an inhibitor of viral
neuraminidase, wherein the inhibitor of viral neuraminidase is a
sialic acid analog.
26. The set of claim 25, wherein the peptide comprises the amino
acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1) and is cyclized via
the C-residues.
27. A method of treating one or more symptoms of influenza
comprising the steps of: obtaining a peptide, selected from the
group consisting of CGQRETPEGAEAKPWYC (SEQ ID NO: 1),
QRETPEGAEAKPWY (SEQ ID NO: 5), PKDTPEGAEALKPWY (SEQ ID NO: 6) and
CGPKDTPEGAELKPWYC (SEQ ID NO: 7), wherein the peptide has no TNF
receptor binding activity and is cyclized, and an inhibitor of
viral neuraminidase; and treating one or more symptoms of
influenza, wherein the inhibitor of viral neuraminidase treats one
or more symptoms of influenza and the peptide treats pulmonary
inflammation associated with influenza; wherein the inhibitor of
viral neuraminidase is a sialic acid analog.
28. The composition of claim 17, wherein the inhibitor of viral
neuraminidase is selected from the group consisting of Laninamivir,
its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate,
Oseltamivir carboxylate, Oseltamivir, including racemic, optically
pure, salt-free, salt, enantiomeric and diastereomeric forms
thereof.
29. The set of claim 25, wherein the inhibitor of viral
neuraminidase is selected from the group consisting of Laninamivir,
its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate,
Oseltamivir carboxylate, Oseltamivir, including racemic, optically
pure, salt-free, salt, enantiomeric and diastereomeric forms
thereof.
30. The set of claim 26, wherein the inhibitor of viral
neuraminidase is selected from the group consisting of Laninamivir,
its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate,
Oseltamivir carboxylate, Oseltamivir, including racemic, optically
pure, salt-free, salt, enantiomeric and diastereomeric forms
thereof.
31. The method of claim 27, wherein the inhibitor of viral
neuraminidase is selected from the group consisting of Laninamivir,
its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate,
Oseltamivir carboxylate, Oseltamivir, including racemic, optically
pure, salt-free, salt, enantiomeric and diastereomeric forms
thereof.
Description
[0001] The present invention relates to pharmaceutical compositions
for the treatment of influenza.
[0002] In humans, influenza is a serious disease of the respiratory
tract and of the whole organism, which is caused by influenza
viruses. The influenza viruses belong to the family of
orthomyxoviruses, which are characterized by a segmented RNA genome
in a negative strand orientation. The types relevant to humans are
the influenza A and B viruses, of which subtype A in particular is
known as a causative agent of highly feverish diseases of the
respiratory tract. Besides the veterinary importance, all influenza
viruses have a zoonitic potential, i.e. there is a possibility of
transmission from a chicken or pig to a human being.
[0003] Influenza emerges periodically as a pandemic which in most
cases has its origine in South East Asia and China and spreads
worldwide from there. Pandemics of influenza viruses are associated
with a high number of deaths not only with elderly persons but also
with juveniles. According to the WHO, the annual seasonal influenza
results in a worldwide rate of 3 to 5 million cases of a severe
disease, with a death rate of 250,000 to 500,000. The most frequent
cause of death is an influenza pneumonia with resulting lung
failure, but cardiovascular damages such as myocarditis (myocardial
inflammation) or pericarditis (inflammation of the pericardial sac)
may occur as well. Further rather frequent causes of death can be
an inflammation of the brain or the meninges (meningoencephalitis)
or the damage of other organ systems (in particular the
kidneys).
[0004] Normally, the incubation period is 4 to 5 days, but it may
be shorter. The disease starts out with a sudden onset of
headaches, shivering, chills and cough. This is followed by a high
fever of up to 41.degree. C., muscles pains, loss of appetite and a
general sense of weakness. This phase lasts for about 3 days,
following which the fever declines and is in most cases sunk back
to normal values from the sixth day on, which means the virus is
eliminated out of the body. The cough may last for several
weeks.
[0005] A severe, life-threatening influenza may occur if a primary
viral interstitial (in many cases hemorrhagic) pulmonary
inflammation is developing following the above described symptoms.
In addition to weakened persons, it also occurs in approximately
25% of healthy or not predamaged persons and may last up to 2
weeks. Such a pulmonary inflammation may be detected by measuring
an increase in the lung weight.
[0006] Pneumonias may also develop secundarily by bacterial
superinfections (among others by Streptococcus pneumoniae,
Staphylococcus aureus and Hemophilus influenzae). Further factors
contributing to these complications are, among others, other
pulmonary diseases (e.g. asthma), immunodeficiency, age (infants
and elderly persons), diabetes, lung injuries, smoking. Therefore,
persons having these complications are the primary target group for
a vaccination.
[0007] Influenza viruses reach into the organism by droplet
infection and infect by binding the HA protein to terminal
neuraminic acid residues on the epithelial cells of the oral, nasal
and pharyngeal mucosa. From there, they spread to the lower
respiratory tract. Destructions of cells can be observed in
ciliated epithelia and in mucigenous skin layers of all areas of
the respiratory tract. If a primary, interstitial pulmonary
inflammation is developing, the virus is transferred to the cells
of the lung parenchyme. Strong swellings of the alveolar walls can
be seen, the epithelium of which often becomes entirely ablated by
the cell destruction. Such a swelling of the lung tissue can be
detected by measuring an increase in the lung weight.
[0008] There are both, prophylactic and therapeutic treatments, and
vaccines against both, influenza A and B infections, are available.
These consist of killed viruses which were grown in chicken eggs
and/or cell culture. The protection provided by the vaccine has
reached its full effectiveness approximately two weeks after the
vaccination. However, due to the high variability of the influenza
viruses, the vaccines must be adapted to the currently circulating
virus subtypes or variants of subtypes annually.
[0009] In addition, inhibitors of viral neuraminidase (Zanamivir,
Oseltamivir) are being applied which prevent the release of newly
replicated virus particles from the host cell. They are preferably
used shortly after an occurred and detected influenza virus
infection in order to constrain the spread of the virus in an early
infection phase (WO 2003/026567 A2).
[0010] However, the inhibitors of viral neuraminidase merely
interfere with the proliferation of viruses but cannot inactivate
viruses that are already present in the body. Neuraminidase
inhibitors may contribute to shortening the duration of disease
only minimally (on the average by one day in humans).
[0011] It is a goal of the present invention to significantly
increase the therapeutic effect of such inhibitors of virus
proliferation. The present invention aims at providing improved
pharmaceutical compositions for the treatment of infections with
influenza viruses.
[0012] Accordingly, the present invention relates to a composition
comprising [0013] a peptide, which consists of 7-17 adjacent amino
acids and comprises 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, and wherein the peptide has no TNF receptor binding
activity and is cyclized, and [0014] an inhibitor of viral
neuraminidase, in particular for the application of preventing and
treating influenza.
[0015] It was now found according to the invention that the effect
of neuraminidase inhibitors can surprisingly be improved by using a
peptide, as defined above, in combination with a neuraminidase
inhibitor for the treatment of influenza infections. The
prophylactic use of the compound according to the invention is
thereby indicated, too. The present invention has proved to be
particularly efficient in the treatment of pulmonary inflammations
which are induced by the influenza viruses.
[0016] The peptides to be used according to the invention are
known, for example, from European Patent EP 1 264 599 B1 (or from
US 2007/299003 A, WO 94/18325 A1 or WO 2008/148545 A1) and have
been used in the prior art for treating fluid accumulations
(pulmonary edema) and in particular for reabsorbing these fluid
accumulations. Surprisingly, these peptides are also appropriate
for influencing the fluid flow in the inverse direction via the
endothelium of the capillaries into the epithelium of the lungs and
can thus be employed to prevent and treat hyperpermeability of
epithelial cells and endothelial cells as well (WO 2010/099556
A).
[0017] These peptides, which are known per se and which are used
together with the neuraminidase inhibitor according to the
invention, do not exhibit any TNF receptor binding activity (Hribar
et al., Eur. J. Immunol. 1999; Elia et al., AJRCCM 2003; see also:
Example part below) and are cyclized. Preferred variants of these
peptides consist of 7-17 adjacent amino acids and contain the
hexamer TPEGAE (SEQ ID NO: 4).
[0018] While WO 2010/099556 A1 and EP 1 264 599 A1 are targeted to
the treatment of certain pathological symptoms in the lung and not
to the prevention and treatment of a disease by a certain pathogen,
the application according to the present invention, however, is a
complete different one.
[0019] As has been shown in the course of the studies concerning
the present invention, the peptides according to the claims (a.o.
according to WO 2010/099556 A1) support the effect of the
neuraminidase inhibitors in the combination preparation of the
invention in a synergistical way. The application according to the
present invention is thus targeted directly to an infection by
influenza viruses and not to general symptoms, which may have been
induced by several different pathogens (as is the case for example
in WO 2010/099556 A1 or in EP 1 264 599 A1).
[0020] From the point of view of a person skilled in this art it is
clear, however, that neuraminidase inhibitors can merely inhibit
the proliferation of influenza viruses but not thereby reduce
already existing viruses; it is, however, entirely surprising and
not shown by the prior art that the combination of neuraminidase
inhibitors and the peptides according to the invention yields a
substantial improvement in the control of influenza. The prior art
does not bestow any suggestions of this combination to a person
skilled in the art, on the contrary:
[0021] In WO 2003/026567 A2 it is described that the neuraminidase
inhibitors used for the treatment of influenza are also appropriate
for preventing any influenza associated bacterial infections. Based
on this document, a person skilled in the art can not at all
perceive any reasons why to add further additives to the
neuraminidase inhibitors, which can improve the antiviral
efficiency or the effectiveness of an influenza treatment. In a
particularly preferred embodiment of the present invention, the
composition according to the invention is comprised of a cyclized
peptide, consisting in a sequence of consecutive amino acids,
selected from the group, consisting in
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.
[0022] Preferably, the peptide in the composition is comprised of
the amino acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1) and is
cyclized via the C-residues. Thus, this particularly preferred
peptide has the following amino acid sequence (SEQ ID NO: 1) (NH2)
Cys-Gly-Gln-Arg-Glu-Thr-Pro-Glu-Gly-Ala-Glu-Ala-Lys-Pro-Trp-Tyr-Cys
(COOH). This peptide is also referred to as "AP301".
[0023] Thereby, the cyclization of the peptides according to the
invention can be achieved, for example, by a direct cyclization via
a disulfide bridge between the two C-residues at the N- and
C-terminus or by coupling the peptide via both cysteins to a
carrier substance. In the peptides according to the invention, the
cystein residues are preferably provided at the beginning and the
end of the molecule. Other functional groups, which can achieve a
cyclization of the peptide, may be employed as well; hereby, for
example, an acidic group can accomplish an amide or ester ring
closure with an amine or alcohol (for example, the amino acids
asparaginic acid and glutaminic acid can be cyclized preferably
intramolecularly with serine, threonine, tyrosine, asparagine,
glutamine or lysine). The cyclization of the peptide is preferably
effected by a disulfide bridge between the C-residues of the
peptide (if present).
[0024] Thus, additional preferred peptides according to the
invention are for example
TABLE-US-00002 (SEQ ID NO: 8) CGQKETPEGAEAKPWYC, (SEQ ID NO: 9)
CGQRETPEGAEARPWYC, (SEQ ID NO: 10) CGQRETPEGAEAKPC, (SEQ ID NO: 11)
CQRETPEGAEAKPWYC or (SEQ ID NO: 12) CGQRETPEGAEAKFWYC.
[0025] However, the cyclization can also be accomplished by binding
the peptide to carrier substances. As the carrier substances for a
cyclization of this kind, all customary pharmaceutically applicable
substances are eligible, which are able to covalently bind to the
SH groups of the cysteins, for example (or to other naturally
present or artificially introduced chemically reactive groups of
the peptides); hereby, the customary carrier proteins such as
keyhole limpet hemocyanine (KLH), tetanus toxin etc. are
particularly preferred. Adjacent bifunctional residues on the
carrier can also be envisaged (e.g. an acid group next to an amine
or alcohol group). In this context, it is important to understand
that the term "cyclization" comprises both, the intramolecular ring
closure and the bonding to a carrier (from which the bound peptide
projects out (by the N- and C-terminus of the peptide being bound
to the carrier)); hereby, a peptide cyclized like this is showing
the cyclic spatial structure and is stabilized appropriately.
[0026] Inhibitors of viral neuraminidase are known and have already
proved themselves in the prevention and treatment of influenza.
Such viral neuraminidase inhibitors (such as e.g. Zanamivir,
Oseltamivir, Laninamivir or Peramivir) prevent the release of newly
replicated virus particles from the host cell. In particular, the
spread of viruses shortly after an occurred and detected infection
with influenza viruses can be constrained by them in the early
phase of infection.
[0027] These neuraminidase inhibitors, however, merely interfere
with the proliferation of viruses but cannot inactivate viruses
that are already present in the body. Thus, in many cases, it is
merely the duration of the disease which can be shortened minimally
(on the average by one day in adults). In front of all, however,
the symptoms of influenza, in particular of a pneumonia, which in
most cases lead to particular complications, cannot be combated or
prevented efficiently enough as a rule.
[0028] Neuraminidase is an essential enzyme for the reproduction of
the influenza virus, and it has been described as "molecular
scissor" which is responsible for the release of the viruses.
[0029] Neuraminidase inhibitors are comprised of analogues of
sialic acid, which represent a new class of the second generation
of antiviral agents which show efficacy against influenza A and B
viruses. Neuraminidase inhibitors according to the present
invention can be all compounds suggested for it hitherto, as
summarized for example in US 2008/0063722 A1 (as well as for
preferred pharmaceutical formulations of such substances). These
substances can inhibit at least one enzymatic activity of the
neuraminidase protein of a virulent strain of the type A or type B
influenza virion. Such substances can be used for both, the
prophylaxis and treatment of influenza; however, in combination
with the above defined peptides according to the invention, this
effect is significantly improved.
[0030] Examples of neuraminidase inhibitors, which may be employed
in the present composition, are described for example in U.S. Pat.
Nos. 5,453,533, 5,763,483, 5,952,375, 5,958,973, 5,512,596,
5,886,213, 5,602,277, 6,410,594, 5,360,817, 5,866,601, 6,340,702,
6,451,766, 6,455,571, 6,593,314, 6,509,359, 6,518,305 and
6,831,096. Substances, which are already being employed with humans
(or are at least undergoing clinical trial) and are therefore
particularly preferred, are for example CS-8958 (RI 18958; US
2008/0063722 A1), Zanamivir (GG167, RELENZA.RTM.), Peramivir
(RWJ-270201, BCX-1812), Oseltamivir phosphate (Ro64-0796, GS4104),
Oseltamivir carboxylate (Ro64-0802, GS4071), or Oseltamivir
(GS4104, TAMIFLU.RTM.). Of course, all effective chemical forms of
the neuraminidase inhibitors are comprised according to the
invention, that is salts, racemic, optically pure and/or salt-free
forms (also in the form of enantiomers or diastereomers, for
example).
[0031] Preferably, the inhibitor of viral neuraminidase is
Zanamivir or Oseltamivir; these substances are preferred because
they have already been employed particularly successfully in the
treatment of humans.
[0032] Preferably, the composition according to the invention
comprises a pharmaceutically acceptable carrier and is prepared in
the form of a pharmaceutical composition which is suitable for
administration to humans.
[0033] The expression "a pharmaceutical composition" refers to any
composition, which is comprised of a neuraminidase inhibitor and a
peptide as defined above (of course to appropriate (i.e. not
negatively interfering with each other) mixtures of such
substances, too), which prevents, improves or cures the conditions
described herein. The expression "a pharmaceutical composition"
refers in particular to a composition, which is comprised of a
neuraminidase inhibitor and a peptide, such as described above, and
a pharmaceutically acceptable carrier or excipient (both
expressions may be used interchangeably). Suitable examples of
carriers or excipients, which are known to someone of skill in the
art, are water, isotonic solution, sodium phosphate, sodium
acetate, sodium carbonate, citrate, glycine, glycylglycine,
histidine, lysine, arginine, TRIS and sodium citrate or mixtures
thereof. Of course, Ringer solution, dextrose solution or solutions
of non-reducible sugars can be employed as well; accordingly,
mannite, trehalose, saccharose, sorbite, fructose, maltose, lactose
or dextrane, Hank solution, fixed oils, ethyl oleate, 5% dextrose
in isotonic solution, substances, which improve the isotonicity and
the chemical stability, buffers and stabilizing agents are also
suitable as such carriers. Other suitable carriers include any such
which do not induce by themselves the production of antibodies
which will be harmful to the individual, the composition is to be
administered to, such as proteins, polysaccharides, polylactic
acids, polyglycolic acids, polymeric amino acids and amino acid
copolymers. In formulating the composition according to the
invention, the relevant guidelines (e.g. the (European or US)
pharmacopoeia) have of course to be complied with. Thereby, the
peptide to be provided in the composition according to the
invention can also be cyclized to these carriers by direct covalent
binding.
[0034] The pharmaceutical composition according to the invention
can be administered (as a drug) by any appropriate method known to
a person skilled in the art, and in particular it is preferable to
administer the peptide to be used according to the invention or the
composition according to the invention into the lung. Although
influenza does play a certain role with animals, too, and the
composition according to the invention can of course be employed in
the prevention and prophylaxis of animals as well, the focus of the
present invention is on preventing and treating humans, i.e. of
persons who have already become infected with influenza or who are
at risk of getting infected with this virus (especially at
influenza epidemics or pandemics). The preferred administration
route is inhalation (via aerosols), followed by an intravenous
administration, instillation, oral administration or a combination
thereof. In the inhalatory, parenteral or oral administration, the
drug of this invention is formulated in unit dosage form as a
solution, suspension or emulsion, which is combined with the above
defined pharmaceutically acceptable excipients. In indivival cases,
the dosage and administration form can of course depend on the
respective individual.
[0035] Therefore, the effective amount required in each case is
administered to the individual that needs the administration. The
"effective amount" is to be understood as an amount effective
enough to provide the intended therapeutic or prophylactic effect,
for example to prevent a further spread of the disease or to
effectively treat it. In most cases, an average patient is taken as
a basis for this; however, the actual effective amounts of the
components in the composition can be formulated in a way that is
taking the administration form and the age, weight, condition of
the patient as well as the degree and progress of the disease into
consideration (e.g. by way of an adequate, conventional
pharmacological protocol).
[0036] Preferably, the pharmaceutically acceptable carrier in the
composition according to the invention is selected from water
(particularly preferred: water for injection), sodium chloride,
sodium phosphate, sodium acetate, sodium carbonate, citrate,
glycine, glycylglycine, histidine, lysine, arginine, TRIS, sodium
citrate, Ringer solution, dextrose, mannite, trehalose, saccharose,
sorbite, fructose, maltose, lactose or dextrane, Hank solution,
fixed oils, ethyl oleate, substances, which improve the isotonicity
and the chemical stability, stabilizing agents, pharmaceutically
acceptable proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids and amino acid
copolymers.
[0037] The composition according to the invention may as well be
provided by arranging the two active components, namely the
neuraminidase inhibitor and the peptide, as described above, in a
spatially separated form, that is in form of a set, which is
comprised of at least a neuraminidase inhibitor and a peptide (each
provided in a separate container). Accordingly, the present
invention relates to a set as well, which is comprised of at least
a neuraminidase inhibitor and a peptide (each provided in a
separate container). The separate administration (spatially, but
also temporarily) of the peptide and the inhibitor, which is made
possible by this, is mainly preferred in those cases in which
different administration routes of the two active components
according to the invention are desired for a respective patient.
For example, Oseltamivir is given orally in most cases, whereas the
peptide to be used according to the invention is mainly given
inhalatively. In many cases, however, a simultanous administration
is desired; Zanamivir, for example, is administered inhalatively as
well. Of course it may be indicated to provide an oral inhibitor
systemically by way of inhalation. In some cases, the peptide to be
used according to the invention can be mixed with the neuraminidase
inhibitor only with difficulty, for example if the inhibitor is
administered i.v. or orally and the peptide inhalatively. In many
cases, however, the inhibitor and the peptide can be administered
inhalatively in admixture, whereby the inhibitor directly reaches
into the lung and possibly through the lung into the blood
(influenza viruses are present in the lung tissue first).
[0038] In such a set according to the invention, comprising the
neuraminidase and peptide components separately, the features
described herein and the preferred embodiments for the mixed
composition can of course be provided in all kinds of combinations
perceivable to a person skilled in the art.
[0039] The drug according to the invention may for example be
administered by giving the peptide of the present invention in a
dosage of between 1 .mu.g/kg and 10 mg/kg, more preferred between
10 .mu.g/kg and 5 mg/kg, and most preferred between 0.1 and 2
mg/kg. Preferably, it is given as a bolus dose. A continuous
inhalation or infusion or a repetitive dosing regime may be
employed as well.
[0040] Particularly preferred compositions according to the
invention contain, independently of each other, [0041] the peptide
in an amount of 1 .mu.g to 10 g, preferably of 10 .mu.g to 1 g, in
particular of 1 mg to 100 mg, and [0042] the inhibitor of viral
neuraminidase in an amount of 1 .mu.g to 10 g, preferably of 100
.mu.g to 1 g, in particular of 1 mg to 200 mg.
[0043] Particularly preferred compositions according to the
invention in a liquid form contain, independently of each other,
[0044] the peptide in an amount of 1 .mu.g to 10 g, preferably of
10 .mu.g bis 1 g, in particular of 1 mg to 100 mg, and [0045] the
inhibitor of viral neuraminidase in an amount of 1 .mu.g to 10 g,
preferably of 100 .mu.g to 1 g, in particular of 1 mg to 200 mg,
and are provided in a volume of 0.5 to 10 ml, in particular in a
volume of 1 to 5 ml.
[0046] The composition according to the invention can preferably be
administered in a dry form via a powder inhaler. Examples of such
powder inhalers, which may be employed in the present invention,
are described in U.S. Pat. Nos. 4,995,385 and 4,069,819; already
established products are SPINHALER.RTM., ROTAHALER.RTM.,
FLOWCAPS.RTM., INHALATOR.RTM., DISKHALER.RTM. and
AEROLIZER.RTM..
[0047] The composition according to the invention can preferably be
administered as an aerosol by means of a fluid nebulizer as well.
Examples of such fluid nebulizers are established products such as
Aeroneb.RTM. and Pari.RTM..
[0048] According to a preferred embodiment, the composition
according to the invention is characterized by a peptide and/or an
inhibitor of viral neuraminidase being provided in a nebulizable
powder formulation or in a nebulizable fluid formulation.
[0049] The composition according to the invention is preferably
used for the treatment and prophylaxis of infections with the
influenza virus type A and type B, in particular type A. In
principle, however, the composition is suitable to prevent or treat
an infectious disease by any possible strain of the influenza
virus, which may cause this disease in an animal or in a human
being. The databases relevant for information about the various
influenza types are well-known to persons skilled in this art; in
particular, many isolated strains of type A are described or even
sequenced.
[0050] The invention will be explained in more detail by way of the
following examples and drawings, to which it is not limited, of
course.
[0051] It is shown in
[0052] FIG. 1: the relative lung weight of C57BL/6 mice on days 3,
5, 7 and 9 following an infection with influenza A or (as a
control) without infection;
[0053] FIG. 2: the relative lung weight of C57BL/6 mice on days 5,
7 and 9 following an infection with influenza A and treatment with
Oseltamivir, treatment with the composition according to the
invention, and without treatment (with PBS, for a control);
[0054] FIG. 3: the relative lung weight of C57BL/6 mice on days 7
and 9 following an infection with influenza A and treatment with
Zanamivir, treatment with the composition according to the
invention, and without treatment (with PBS, for a control).
EXAMPLES
[0055] The present examples are provided to show in an approved
experimental mouse model that the goal of the present invention has
been achieved by administering to mice, which had been infected
with influenza virus, a neuraminidase inhibitor or a combination of
the neuraminidase inhibitor and the synthetic peptide AP301 (SEQ ID
NO: 1).
Example 1
[0056] Infection with influenza virus causes the development of a
pulmonary inflammation.
[0057] Laboratory mice (strain C57BL/6, 8 weeks old) were infected
per-nasal with the influenza strain A (PR8/34) at a dose of 150
PFU. On days 3, 5, 7 and 9 following the infection, the lungs were
taken out of 8 mice, respectively, and the relative lung weight was
determined as a measure of the pulmonary inflammation.
[0058] The examination showed that with increasing length of time
following the mice's infection with the influenza virus the lung
weight strongly increased compared to control lungs. The results
are shown graphically in FIG. 1.
Example 2
[0059] Treatment of the pulmonary inflammation by administering the
neuraminidase inhibitor or administering a combination of
neuraminidase inhibitor and peptide AP301.
[0060] Laboratory mice (strain C57BL/6, 8 weeks old) were infected
per-nasal with the influenza strain A (PR8/34) at a dose of 150
PFU. Subsequently, each test animal received an oral administration
of 10 mg/kg Oseltamivir (neuraminidase inhibitor) and an
intratracheal administration of 10 .mu.g/test animal of peptide
AP301. The treatment was repeated on test days 2 and 4.
[0061] On days 5, 7 and 9 following the infection, the lungs were
taken out of 30 mice, respectively, and the relative lung weight
was determined as a measure of the pulmonary inflammation. The
results are shown graphically in FIG. 2.
[0062] The examination showed that the neuraminidase inhibitor
exhibited just a moderate effect on reducing the pulmonary
inflammation, as measured by the lung weight. If, however, peptide
AP301 was administered to the mice infected by the influenza virus
in addition to the neuraminidase inhibitor, the pulmonary
inflammation was essentially more reduced.
Example 3
[0063] Ex vivo assessment of proinflammatory properties of the
AP301 peptide in human whole blood.
[0064] An ex vivo safety pharmacological study concerning the AP301
peptide in human whole blood was performed to assess whether the
AP301 peptide results in the release of the proinflammatory marker
interleukin-6 (IL-6) from fresh human whole blood (i.e. whether APN
301 exhibits TNF-specific inflammatory activity (i.e. TNF receptor
binding activity)). In this study, fresh human whole blood has been
used, as it represents an approved predictive model system for the
assessment of inflammatory response in vivo.
[0065] Summary of Methodology
[0066] It was the goal of this study to determine the
proinflammatory signalling capacity of the peptide AP301. Whole
blood cultures were used and the secretion of interleukin-6 (IL-6),
a very sensitive marker for proinflammatory stimulation, was
quantified by ELISA.
[0067] Test System
TABLE-US-00003 Test system 25 ml of freshly taken heparinized blood
from 5 healthy volunteers (HV) was used in the tests. Test item
Identification: AP301 peptide (dose: 1 ng/ml to 10 .mu.g/ml; single
administration in solution) Description: White powder, purity
96%
[0068] Whole Blood Cultures
[0069] Whole blood (WB) cultures are performed by pipetting 1 ml WB
into wells of 24-well-plates. In each experiment, unstimulated and
control-stimulated cultures were included.
[0070] If possible, the substances and stimulants to be
investigated were always added in an identical volume to each well
of a given experiment, which is not greater than 10% of the total
volume contained in a well. Unstimulated controls received PBS.
Volume adjustments and dilutions for different treatments were also
done with PBS.
[0071] The content of each well was mixed and the plates incubated
at 37.degree. C. and 5% CO.sub.2 for 24 hours. After incubation the
content of each well was transferred to a fresh 1.5 ml microtube
and centrifuged at 8,000-9,000.times.g for 15 minutes. The
supernatant of each sample was transferred individually to two 1.5
ml microtubes and kept at -20.degree. C. until use.
[0072] Detection of Interleukin-6
[0073] Interleukin-6 was quantified by a specific ELISA (Human IL-6
ELISA Set, BD Biosciences, Cat. No. 555220) employing an anti-human
IL-6 antibody as capture antibody, a biotinylated anti-human IL-6
detection antibody, avidin-horseradish peroxidase conjugate as
enzyme reagent and recombinant IL-6 as standard. Absorbance
measurement was performed at 450 nm using the Packard Fusion
Reader.
[0074] Data Analysis
[0075] The results for each plate were stored and evaluated using
the FusionDataAnalysis Software.
[0076] Summary of Study Results
[0077] It was the goal of this study to determine the
proinflammatory signalling capacity of the AP301 peptide. Whole
blood cultures were used and the secretion of IL-6, a very
sensitive marker for proinflammatory stimulation, was quantified by
ELISA.
[0078] Whole blood samples of five healthy volunteers were either
left unstimulated (negative control), stimulated with high and low
doses of LPS (positive controls), or incubated with peptide with
nine semi-logarithmic dilutions ranging from 10 .mu.g/ml to 1
ng/ml. The results are shown in the following table:
TABLE-US-00004 TABLE Release of interleukin-6 from fresh human
whole blood on addition of peptide AP301 and LPS AP301 peptide
Positive control (LPS) Concentration concentration of IL-6 (pg/ml,
n = 5) 0 (negative control) less than 0.5 less than 0.5 10 mg/ml
less than 0.5 195.640 1 mg/ml less than 0.5 108.370 3 ng/ml less
than 0.5 34.867 1 ng/ml less than 0.5 not determined
[0079] The results clearly reveal that the AP301 peptide did not
induce any detectable level of IL-6 secretion at any of the
concentrations tested. The positive controls (LPS) resulted in a
strong induction of IL-6 secretion.
[0080] Discussion
[0081] The experiments have been performed to assess whether the
AP301 peptide mediates the induction of a proinflammatory cascade.
Readout parameter was the induced secretion of IL-6 in whole blood
cultures from five healthy donors. The results clear showed that
the AP301 peptide did not induce any detectable level of IL-6 in
any donor's cultures. Therefore, it is demonstrated that the AP301
peptide did not induce a proinflammatory response in the chosen ex
vivo model and, thus, does not exhibit any TNF receptor binding
activity.
Example 4
[0082] Treatment of pulmonary inflammation by administering
neuraminidase inhibitor (Zanamivir) or administering a combination
of neuraminidase inhibitor (Zanamivir) and peptide AP301.
[0083] Laboratory mice (strain C57BL/6, 8 weeks old) were infected
per-nasal with the influenza strain A (PR8/34) at a dose of 150
PFU. Subsequently, each test animal received a nasal administration
of 1.5 mg/kg Zanamivir (neuraminidase inhibitor) and an
intratracheal administration of 50 .mu.g/test animal of peptide
AP301. The treatment was repeated on test days 2 and 4.
[0084] On days 7 and 9 following the infection, the lungs were
taken out of 20 mice, respectively, and the relative lung weight
was determined as a measure of the pulmonary inflammation. The
results are shown graphically in FIG. 3.
[0085] The examination showed that the neuraminidase inhibitor
(Zanamivir) exhibited just a moderate effect on reducing the
pulmonary inflammation, as measured by the lung weight. If,
however, peptide AP301 was administered to the mice infected by the
influenza virus in addition to the neuraminidase inhibitor, the
pulmonary inflammation was essentially more reduced.
SUMMARY
[0086] The peptides according to the invention exhibit a
synergistic effect in the treatment of influenza infections in
combination with a neuraminidase inhibitor.
[0087] In WO 2010/099556 A1, the treatment of various pulmonary
symptoms is documented, as mentioned, which is targeted to the
hyperpermeability of epithelial cells and endothelial cells in such
lung diseases. According to WO 2010/099556 A1 it was shown that the
claimed peptides are excellently suited for preventing and treating
these symptoms. Although, according to WO 2010/099556 A1,
hyperpermeability of epithelial cells and endothelial cells can
also be treated in cases of infections with influenza viruses (in
the context of a pneumonia developing with this infection), the
present invention is of course not suggested by this. The
possibility of generally treating an influenza with a combination
preparation, comprising this peptide, is an entirely new and
inventive teaching as to the applicability of the peptide for
treating a--facultatively--emerging symptom of influenza.
[0088] The basic difference can also be derived from a view into
the detailled experiments according to WO 2010/099556 A1: In the
examples of WO 2010/099556 A1, it is experimentally demonstrated
that the peptides in the lung tissue: [0089] i) influence the
content of reactive oxygen, [0090] ii) the effect of the bacterial
gram positive toxines "listeriolysine" and "pneumolysine" also
influences hyperpermeability, a.o. by regulating the level of
phosphorylated myosin light chain, infiltration of leucocytes,
activated protein kinase C, [0091] iii) influence the body weight
after an influenza infection, [0092] iv) influence the body
temperature after an influenza infection, [0093] v) influence the
survival rate of the test animals after an influenza infection.
[0094] Throughout the whole WO 2010/099556 A1, however, there are
no experimental hints as to an infection of test animals with the
influenza virus leading to an alteration of the relative lung
weight and as to halting and treating such a process by the
administration of the peptide. Only now, by the present invention,
it is shown that an infection of the lungs of test animals with the
influenza virus leads to a substantial increase in the relative
lung weight.
[0095] The lung constitutes one of the most important organs of
all. An increase in the relative lung weight is connected to a
damage of the lung function which cannot be compensated for by any
other organ. It is a property of a healthy lung to contain as many
air-filled spaces (alveoles) as possible. At an increase of the
relative lung weight, it must strongly be assumed that the share of
air-filled alveoles will decline severely which will restrict the
function of the lung. Therefore, the relative lung weight
constitutes an essential factor for the treatment of influenza. The
results shown in the experimental part of the present application
thus demonstrate impressively the synergistic effect of the
combination preparation according to the present invention, based
on an extremely critical and relevant parameter.
[0096] It has hitherto been known from neuraminidase inhibitors
that these may suppress the proliferation of the influenza virus.
Neuraminidase inhibitors do not cause a reduction of living
influenza viruses. Hitherto, it could not be shown in the state of
the art that influenza virus leads to an increase of the relative
lung weight. Only with the present invention has it surprisingly
been shown that the administration of a neuraminidase inhibitor
after an influenza infection reduces the relative weight gain of
the lung weight. Additionally, by the present invention could it be
shown for the first time that a simultaneous treatment of test
animals, which were infected with the influenza virus, with a
neuraminidase inhibitor and a peptide according to WO 2010/099556
A1 in combination results in a significant and unforeseeable
synergistic effect on the relative lung weight. While neuraminidase
inhibitors inhibit the proliferation of influenza virus, without
reducing the already living viruses, the synergistic combination of
a neuraminidase inhibitor and a peptide according to the invention
obviously leads to a significant improvement of the influenza
treatment. None of the drugs (neuraminidase inhibitor and the
peptide according to the invention) administered individually does
by itself lead to the effect shown by the present invention.
[0097] Therefore, the present invention could not be rendered
obvious in any way by the results disclosed in WO 2010/099556
A1.
[0098] Summary of Sequences:
TABLE-US-00005 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 CGQKETPEGAEAKPWYC 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 Tyr1 5 10
15Cys220PRTArtificial Sequencesynthetic peptide 2Lys Ser Pro Gly
Gly Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys1 5 10 15Pro Trp Tyr
Glu 20317PRTArtificial Sequencesynthetic peptide 3Cys Gly Gln Arg
Glu Ala Pro Ala Gly Ala Ala Ala Lys Pro Trp Tyr1 5 10
15Cys46PRTArtificial Sequencesynthetic peptide 4Thr Pro Glu Gly Ala
Glu1 5514PRTArtificial Sequencesynthetic peptide 5Gln Arg Glu Thr
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr1 5 10614PRTArtificial
Sequencesynthetic peptide 6Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu
Lys Pro Trp Tyr1 5 10717PRTArtificial Sequencesynthetic peptide
7Cys Gly Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp Tyr1 5
10 15Cys817PRTArtificial Sequencesynthetic peptide 8Cys Gly Gln Lys
Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr1 5 10
15Cys917PRTArtificial Sequencesynthetic peptide 9Cys Gly Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Arg Pro Trp Tyr1 5 10
15Cys1015PRTArtificial Sequencesynthetic peptide 10Cys Gly Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Cys1 5 10
151116PRTArtificial Sequencesynthetic peptide 11Cys Gln Arg Glu Thr
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Cys1 5 10
151217PRTArtificial Sequencesynthetic peptide 12Cys Gly Gln Arg Glu
Thr Pro Glu Gly Ala Glu Ala Lys Phe Trp Tyr1 5 10 15Cys
* * * * *