U.S. patent application number 11/798610 was filed with the patent office on 2009-11-12 for methods and compounds for the treatment of mucus hypersecretion.
Invention is credited to John Chaddock, Keith Alan Foster, Conrad Padraig Quinn.
Application Number | 20090280066 11/798610 |
Document ID | / |
Family ID | 32071324 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280066 |
Kind Code |
A1 |
Quinn; Conrad Padraig ; et
al. |
November 12, 2009 |
Methods and compounds for the treatment of mucus hypersecretion
Abstract
A method of treating mucus hypersecretion, the causative factor
in chronic obstructive pulmonary disease (COPD), asthma and other
clinical conditions involving COPD, comprises administering a
compound that inhibits exocytosis in mucus secreting cells or
neurones that control or direct mucus secretion. Also described is
a compound, for use in the treatment of hypersecretion of mucus,
which inhibits mucus secretion by inhibiting mucus secretion by
mucus secreting cells, and/or inhibiting neurotransmitter release
from neuronal cells controlling or directing mucus secretion.
Inventors: |
Quinn; Conrad Padraig;
(Lilburn, GA) ; Foster; Keith Alan; (Salisbury,
GB) ; Chaddock; John; (Salisbury, GB) |
Correspondence
Address: |
MORRIS MANNING MARTIN LLP
3343 PEACHTREE ROAD, NE, 1600 ATLANTA FINANCIAL CENTER
ATLANTA
GA
30326
US
|
Family ID: |
32071324 |
Appl. No.: |
11/798610 |
Filed: |
May 15, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11518213 |
Sep 11, 2006 |
|
|
|
11798610 |
|
|
|
|
10633698 |
Aug 5, 2003 |
|
|
|
11518213 |
|
|
|
|
09763669 |
May 29, 2001 |
6632440 |
|
|
PCT/GB99/02806 |
Aug 25, 1999 |
|
|
|
10633698 |
|
|
|
|
Current U.S.
Class: |
424/45 ;
424/94.63; 435/219; 435/68.1; 536/23.2 |
Current CPC
Class: |
A61K 38/4886 20130101;
A61K 47/62 20170801; C07K 14/33 20130101; A61P 25/00 20180101; C07K
2319/33 20130101 |
Class at
Publication: |
424/45 ; 435/219;
536/23.2; 424/94.63; 435/68.1 |
International
Class: |
A61K 9/12 20060101
A61K009/12; C12N 9/50 20060101 C12N009/50; C07H 21/04 20060101
C07H021/04; A61K 38/48 20060101 A61K038/48; C12P 21/06 20060101
C12P021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 25, 1998 |
GB |
9818548.1 |
Claims
1-11. (canceled)
12. A single-chain fusion protein comprising: (a) a light chain
(L-chain) or L-chain fragment of a clostridial neurotoxin, which
L-chain or L-chain fragment includes the active proteolytic enzyme
domain of the L-chain; (b) a targeting domain comprising or
consisting of a growth factor; (c) a translocating domain that
translocates the L-chain or L-chain fragment into the target cell;
and (d) a site for cleavage by a proteolytic enzyme.
13. A fusion protein according to claim 12, wherein the growth
factor is an epidermal growth factor (EGF).
14. A fusion protein according to claim 12, wherein the cleavage
site has been introduced into the fusion protein.
15. A fusion protein according to claim 12, wherein the cleavage
site is selected from the group consisting of: IEGR; DDDDK;
EXXYXQSG; LEVLFQGP; LVPRGS; HY; and YH.
16. A fusion protein according to claim 12, wherein said cleavage
site is not present in a native clostridial neurotoxin.
17. A fusion protein according to claim 12, wherein said cleavage
site is located between the L-chain or L-chain fragment and the
translocating domain.
18. A fusion protein according to claim 12, wherein the cleavage
site is cleavable by a proteolytic enzyme selected from the group
consisting of: factor Xa; enterokinase; TEV protease; precission;
thrombin; and genenase.
19. A nucleic acid encoding a fusion protein according to claim
12.
20. A pharmaceutical composition for topical administration to a
patient suffering from mucus hypersecretion, comprising: (i) a
polypeptide in an amount effective to inhibit mucus hypersecretion,
wherein the polypeptide comprises: (a) a light chain (L-chain) or
L-chain fragment of a clostridial neurotoxin, which L-chain or
L-chain fragment includes the active proteolytic enzyme domain of
the L-chain; (b) a targeting domain comprising or consisting of a
growth factor; and (c) a translocating domain that translocates the
L-chain or L-chain fragment into the target cell; and (ii) a
formulation component selected from the group consisting of an
excipient, an adjuvant and a propellant.
21. A method of treating hypersecretion of mucus, comprising
administering to a patient in need thereof, a therapeutically
effective amount of a polypeptide comprising: (a) a light chain
(L-chain) or L-chain fragment of a clostridial neurotoxin, which
L-chain or L-chain fragment includes the active proteolytic enzyme
domain of the L-chain; (b) a targeting domain comprising or
consisting of a growth factor; and (c) a translocating domain that
translocates the L-chain or L-chain fragment into the target cell;
wherein following said administration, hypersecretion of mucus is
reduced.
22. A method of treating chronic obstructive pulmonary disease
(COPD), comprising administering to a patient in need thereof, a
therapeutically effective amount of a polypeptide comprising: (a) a
light chain (L-chain) or L-chain fragment of a clostridial
neurotoxin, which L-chain or L-chain fragment includes the active
proteolytic enzyme domain of the L-chain; (b) a targeting domain
comprising or consisting of a growth factor; and (c) a
translocating domain that translocates the L-chain or L-chain
fragment into the target cell; wherein following said
administration, hypersecretion of mucus is reduced.
23. A method of treating asthma, comprising administering to a
patient in need thereof, a therapeutically effective amount of a
polypeptide comprising: (a) a light chain (L-chain) or L-chain
fragment of a clostridial neurotoxin, which L-chain or L-chain
fragment includes the active proteolytic enzyme domain of the
L-chain; (b) a targeting domain comprising or consisting of a
growth factor; and (c) a translocating domain that translocates the
L-chain or L-chain fragment into the target cell; wherein following
said administration, hypersecretion of mucus is reduced.
24. A method for activating a single-chain fusion protein,
comprising: (i) providing a single-chain fusion protein comprising:
(a) a light chain (L-chain) or L-chain fragment of a clostridial
neurotoxin, which L-chain or L-chain fragment includes the active
proteolytic enzyme domain of the L-chain; (b) a targeting domain
comprising or consisting of a growth factor; (c) a translocating
domain that translocates the L-chain or L-chain fragment into the
target cell; and (d) a site for cleavage by a proteolytic enzyme;
(ii) contacting said single-chain fusion protein with a proteolytic
enzyme that cleaves said site for cleavage; and (iii) cleaving said
single-chain fusion protein at said site for cleavage, and thereby
providing a di-chain polypeptide wherein (a) and (c) are linked
together by a disulphide bond.
25. A method according to claim 24, wherein the proteolytic enzyme
is selected from the group consisting of: factor Xa; enterokinase;
TEV protease; precission; thrombin; and genenase.
26. A method according to claim 24, wherein the cleavage site is
selected from the group consisting of: IEGR; DDDK; EXXYXQSG;
LEVLFQGP; LVPRGS; HY; and YH.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to treatment of mucus
hypersecretion, to compositions therefor and manufacture of those
compositions. The present invention relates particularly, though
not exclusively, to the treatment of chronic bronchitis in chronic
obstructive pulmonary disease (COPD), asthma and other clinical
conditions involving COPD.
[0003] 2. Description of Related Art
[0004] Mucus is a thin film of protective viscoelastic liquid which
lines the airways. It is a 1-2% aqueous solution, in which the
major components are the glycoconjugates known as mucins. Mucus,
including the mucins, is secreted by mucus secretory cells, the
surface epithelial goblet cells of the large airways and the mucus
cells of the submucosal glands. Mucin release occurs by three
mechanisms: constitutive secretion, regulated secretion and
protease cell surface activity. Of these it is regulated secretion
that responds to external stimuli and is amenable to therapeutic
intervention in COPD and asthma. Regulated secretion involves
release from intracellular granules by docking and fusion of the
granules with the cell exterior to release their contents onto the
airway surface. Fusion of the granules can either be with the
plasma membrane of the epithelial cell or with the membrane of
other granules leading to release via multigranular complexes fused
at the cell surface. Regulated secretion of mucins is controlled by
humoral factors and by neural mechanisms. The neural mechanisms in
humans involve a minor contribution from the adrenergic,
sympathetic pathway and a major cholinergic, parasympathetic
component. Another important neural pathway regulating mucin
secretion, particularly the hypersecretion of pathological
conditions, is that of the Non-Adrenergic Non-Cholinergic (NANC)
pathway. The NANC component involves both an orthodromic pathway
involving neuropeptide and nonpeptide transmitters, and a local
sensory efferent pathway involving antidromic fibres from sensory C
fibres.
[0005] COPD is a common respiratory condition, being the fourth
most common cause of death in middle age in the Western world. COPD
comprises two related diseases, which usually occur together,
emphysema and chronic bronchitis. The pathological basis of chronic
bronchitis is mucus hypersecretion. The excessive, chronic
bronchial secretion results in expectoration, and can last from a
few days to many years. The mucus hypersecretion of COPD results in
small airway obstruction producing reduced maximal respiratory flow
and slow forced lung emptying. There is minimal reversal of the
impaired airway function of COPD by bronchodilators and currently
no effective therapy for the mucus hypersecretion.
[0006] Mucus hypersecretion is also a significant contributing
factor to the pathophysiology of asthma. It is a key component in
status asthmaticus, and contributes to the chronic symptoms and
morbidity of asthma. The mucus hypersecretion component of asthma
is not well controlled by current therapies, particularly in severe
and chronic cases.
[0007] It would accordingly be desirable to treat, reduce or
prevent the mucus hypersecretion that causes or leads to these
disease conditions.
SUMMARY OF THE INVENTION
[0008] Accordingly, the invention provides a method of treating
mucus hypersecretion comprising inhibiting mucus secretion by mucus
secreting cells and/or inhibiting neurotransmitter release from
neuronal cells that control or direct mucus secretion. The
invention further provides, in a second aspect, a compound, for use
in the treatment of mucus hypersecretion, which inhibits mucus
secretion by (i) inhibiting mucus secretion by mucus secreting
cells, or (ii) inhibiting neurotransmitter release from neuronal
cells controlling or directing mucus secretion.
[0009] An advantage of the invention is that an agent for effective
treatment of mucus hypersecretion and associated disease states is
now provided and used, offering a relief to sufferers where
hitherto there was no such agent available.
[0010] The present invention thus represents a new different
approach to treatment of mucus hypersecretion by inhibiting
secretory processes, namely one or other or both of the mucus
secretion by mucus secretory cells and the secretion of
neurotransmitters regulating mucus secretion. Agents of the present
invention reduce mucus secretion and/or prevent the hypersecretion
of COPD and asthma, and any other disease in which mucus
hypersecretion is a causative element.
DETAILED DESCRIPTION OF THE INVENTION
[0011] A compound of the invention typically inhibits exocytosis in
mucus secreting cells or neurones that control or direct mucus
secretion. This compound is administered to a patient suffering
from mucus hypersecretion and inhibition of exocytosis in the cells
specified results in reduction of secretion of mucus. Specific
disease states caused by or exacerbated by hypersecretion are
localised to the airways, and hence an embodiment of the invention
comprises topical administration to the airways or to a selected
region or to a selected portion of the airways of a compound that
inhibits exocytosis in mucus secreting cells or in neurones that
control or direct mucus secretion.
[0012] A compound of embodiments of the invention is a polypeptide
that consists of or comprises an inhibiting domain which inhibits
exocytosis in the mucus secreting cell or inhibits exocytosis in a
neuronal cell, thereby directly inhibiting exocytosis of mucus or
one or more mucus components or indirectly inhibiting mucus
secretion by inhibiting exocytosis of neurotransmitter which would
in turn lead to or otherwise stimulate mucus secretion. The
inhibiting domain can suitably comprise a light chain of a
clostridial neurotoxin, or a fragment or variant thereof which
inhibits exocytosis.
[0013] The compound preferably further comprises a translocating
domain that translocates the inhibiting domain into the cell. This
domain may comprise a H.sub.N region of a botulinum polypeptide, or
a fragment or variant thereof that translocates the inhibiting
domain into the cell.
[0014] The compound preferably comprises a targeting domain which
binds to (i) a mucus secreting cell, or (ii) a neuronal cell
controlling or directing mucus secretion. The compound is thus
rendered specific for these cell types. It is also optional for the
compound to be relatively non-specific but for inhibition of mucus
secretion to be achieved via targeting of the compound through
choice of route of administration--the compound is hence preferably
administered to mucus secreting epithelial cells in the airways,
specifically in the lungs. Whilst a non-specific compound of the
invention may affect exocytosis in many cells of a wide range of
types, generally only those cells that are stimulated will be
affected and these stimulated cells will in typical disease states
be those that are secreting mucus and contributing to disease.
[0015] When present, suitable targeting domains include, but are
not restricted to, a domain selected from Substance P, VIP,
beta.sub.2, adrenoreceptor agonists, gastrin releasing peptide and
calcitonin gene related peptide. The precise cells targeted in
preferred embodiments of the invention are selected from (a) cells
that secrete mucins, such as epithelial goblet cells and submucosal
gland mucus secreting cells, (b) cells that secrete aqueous
components of mucus, such as Clara cells and serous cells, and (c)
cells that control or direct mucus secretion, such as
"sensory-efferent" C-fibres, or NANC neural system fibres. The
compound may be administered as a substantially pure preparation
all targeted to the same cell type, or may be a mixture of
compounds targeted respectively to different cells.
[0016] The compound of specific embodiments of the invention
comprises first, second and third domains. The first domain is
adapted to cleave one or more vesicle or plasma-membrane associated
proteins essential to exocytosis. This domain prevents exocytosis
once delivered to a targeted cell. The second domain translocates
the compound into the cell. This domain delivers the first domain
into the cell. The third domain binds to the target cell, ie binds
to (i) a mucus secreting cell, or (ii) a neuronal cell controlling
or directing mucus secretion, and may be referred to as a targeting
moiety ("TM"). The compound may be derived from a toxin and it is
preferred that such a compound is free of clostridial neurotoxin
and free of any clostridial neurotoxin precursor that can be
converted into toxin. Botulinum and tetanus toxin are suitable
sources of domains for the compounds of the invention.
[0017] In use, the agent of specific embodiments of the invention
has a number of discrete functions. It binds to a surface structure
(the Binding Site {BS}) which is characteristic of, and has a
degree of specificity for, the relevant secretory cells and or
neurones in the airways responsible for secretion of mucus and or
regulation of said secretion. It enters the cell to which it binds
by a process of endocytosis. Only certain cell surface BSs can
undergo endocytosis, and preferably the BS to which the agent binds
is one of these. The agent enters the cytosol, and modifies
components of the exocytotic machinery present in the relevant
secretory cells and or neurones in the airways responsible for
secretion of mucus and or regulation of said secretion.
[0018] Surprisingly, agents of the present invention for treatment
of mucus hypersecretion can be produced by modifying a clostridial
neurotoxin or fragment thereof. The clostridial neurotoxins share a
common architecture of a catalytic L-chain (LC, ca 50 kDa)
disulphide linked to a receptor binding and translocating H-chain
(HC, ca 100 kDa). The HC polypeptide is considered to comprise all
or part of two distinct functional domains. The carboxy-terminal
half of the HC (ca 50 kDa), termed the H.sub.C domain, is involved
in the high affinity, neurospecific binding of the neurotoxin to
cell surface receptors on the target neuron, whilst the
amino-terminal half, termed the H.sub.N domain (ca 50 kDa), is
considered to mediate the translocation of at least some portion of
the neurotoxin across cellular membranes such that the functional
activity of the LC is expressed within the target cell. The H.sub.N
domain also has the property, under conditions of low pH, of
forming ion-permeable channels in lipid membranes, this may in some
manner relate to its translocation function.
[0019] For botulinum neurotoxin type A (BoNT/A) these domains are
considered to reside within amino acid residues 872-1296 for the
H.sub.C, amino acid residues 449-871 for the H.sub.N and residues
1-448 for the LC. Digestion with trypsin effectively degrades the
H.sub.C domain of the BoNT/A to generate a non-toxic fragment
designated L H.sub.N, which is no longer able to bind to and enter
neurons. The LH.sub.N fragment so produced also has the property of
enhanced solubility compared to both the parent holotoxin and the
isolated LC.
[0020] It is therefore possible to provide functional definitions
of the domains within the neurotoxin molecule, as follows: [0021]
(A) clostridial neurotoxin light chain: [0022] A metalloprotease
exhibiting high substrate specificity for vesicle and/or plasma
membrane associated proteins involved in the exocytotic process. In
particular, it cleaves one or more of SNAP-25, VAMP
(synaptobrevin/cellubrevin) and syntaxin. [0023] (B) clostridial
neurotoxin heavy chain H.sub.N domain: [0024] A portion of the
heavy chain which enables translocation of that portion of the
neurotoxin molecule such that a functional expression of light
chain activity occurs within a target cell. [0025] The domain
responsible for translocation of the endopeptidase activity,
following binding of neurotoxin to its specific cell surface
receptor via the binding domain, into the target cell. [0026] The
domain responsible for formation of ion-permeable pores in lipid
membranes under conditions of low pH. [0027] The domain responsible
for increasing the solubility of the entire polypeptide compared to
the solubility of light chain alone. [0028] (C) clostridial
neurotoxin heavy chain H.sub.C domain. [0029] A portion of the
heavy chain which is responsible for binding of the native
holotoxin to cell surface receptor(s) involved in the intoxicating
action of clostridial toxin prior to internalisation of the toxin
into the cell.
[0030] The identity of the cellular recognition markers for these
toxins is currently not understood and no specific receptor species
have yet been identified although Kozaki et al. have reported that
synaptotagmin may be the receptor for botulinum neurotoxin type B.
It is probable that each of the neurotoxins has a different
receptor.
[0031] By covalently linking a clostridial neurotoxin, or a hybrid
of two clostridial neurotoxins, in which the H.sub.C region of the
H-chain has been removed or modified, to a new molecule or moiety,
the Targeting Moiety (TM), that binds to a BS on the surface of the
relevant secretory cells and or neurones in the airways responsible
for secretion of mucus and or regulation of said secretion, a novel
agent capable of inhibiting mucus secretion is produced. A further
surprising aspect of the present invention is that if the L-chain
of a clostridial neurotoxin, or a fragment of the L-chain
containing the endopeptidase activity, is covalently linked to TM
which can also effect internalisation of the L-chain, or a fragment
of the L-chain containing the endopeptidase activity, into the
cytoplasm of the relevant secretory cells and or neurones in the
airways responsible for secretion of mucus and or regulation of
said secretion, this also produces a novel agent capable of
inhibiting mucus secretion.
[0032] Accordingly, the invention may thus provide a compound
containing a first domain equivalent to a clostridial toxin light
chain and a second domain providing the functional aspects of the
H.sub.N of a clostridial toxin heavy chain, whilst lacking the
functional aspects of a clostridial toxin H.sub.C domain, and a
third domain which binds to the target mucus secreting or mucus
secretion controlling cell.
[0033] For the purposes of the invention, the functional property
or properties of the H.sub.N of a clostridial toxin heavy chain
that are to be exhibited by the second domain of the polypeptide of
the invention is translocation of the first domain into a target
cell once the compound is proximal to the target cell. References
hereafter to a H.sub.N domain or to the functions of a H.sub.N
domain are references to this property or properties. The second
domain is not required to exhibit other properties of the H.sub.N
domain of a clostridial toxin heavy chain. A second domain of the
invention can thus be relatively insoluble but retain the
translocation function of a native toxin--this is of use if
solubility is not essential to its administration or if necessary
solubility is imparted to a composition made up of that domain and
one or more other components by one or more of said other
components.
[0034] The translocating domain may be obtained from a microbial
protein source, in particular from a bacterial or viral protein
source. It is well documented that certain domains of bacterial
toxin molecules are capable of forming such pores. It is also known
that certain translocation domains of virally expressed membrane
fusion proteins are capable of forming such pores. Such domains may
be employed in the present invention.
[0035] Hence, in one embodiment, the translocating domain is a
translocating domain of an enzyme, such as a bacterial or viral
toxin. One such molecule is the heavy chain of a clostridial
neurotoxin, for example botulinum neurotoxin type A. Other sources
of bacterial toxin translocating domains include diphtheria toxin
and domain II of pseudomonas exotoxin.
[0036] Other sources of translocating domains include certain
translocating domains of virally expressed membrane fusion
proteins. For example, Wagner et al. (1992) and Murata et al.
(1992) describe the translocation (i.e. membrane fusion and
vesiculation) function of a number of fusogenic and amphiphilic
peptides derived from the N-terminal region of influenza virus
haemagglutinin. Other virally expressed membrane, fusion proteins
known to have the desired translocating activity are a
translocating domain of a fusogenic peptide of Semliki Forest Virus
(SFV), a translocating domain of vesicular stomatitis virus (VSV)
glycoprotein G, a translocating domain of SER virus F protein and a
translocating domain of Foamy virus envelope glycoprotein. Virally
encoded "spike proteins" have particular application in the context
of the present invention, for example, the E1 protein of SFV and
the G protein of the G protein of VSV.
[0037] Preferably it has been found to use only those portions of
the protein molecule capable of pore-formation within the endosomal
membrane.
[0038] Methodology to enable assessment of membrane fusion and thus
identification of translocation domains suitable for use in the
present invention are provided by Methods in Enzymology Vol 220 and
221, Membrane Fusion Techniques, Parts A and B, Academic Press
1993.
[0039] Examples of preferred translocating domains for use in the
present invention are listed in the table below. The below-listed
citations are all herein incorporated by reference.
TABLE-US-00001 Translocation Amino acid domain source residues
References Diphtheria toxin 194-380 Silverman et al., 1994, J.
Biol. Chem. 269, 22524-22532 London E., 1992, Biochem. Biophys.
Acta., 1113, 25-51 Domain II of 405-613 Prior et al., 1992,
pseudomonas exotoxin Biochemistry 31, 3555- 3559 Kihara &
Pastan, 1994, Bioconj Chem. 5, 532- 538 Influenza virus
GLFGAIAGFIENGW Plank et al., 1994, J. haemagglutinin EGMIDGWYG (SEQ
Biol. Chem. 269, ID NO: 1), and 12918-12924 variants thereof Wagner
et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry
31, 1986- 1992 Semliki Forest virus Translocation domain Kielian et
al., 1996, J fusogenic protein Cell Biol. 134(4), 863-872 Vesicular
Stomatitis 118-139 Yao et al., 2003, virus glycoprotein G Virology
310(2), 319- 332 SER virus F protein Translocation domain Seth et
al., 2003, J Virol 77(11) 6520- 6527 Foamy virus envelope
Translocation domain Picard-Maureau et al., glyoprotein 2003, J
Virol. 77(8), 4722-4730
[0040] Use of the translocating domains listed in the above table
includes use of sequence variants thereof. A variant may comprise
one or more conservative nucleic acid substitutions and/or nucleic
acid deletions or insertions, with the proviso that the variant
possesses the requisite translocating function. A variant may also
comprise one or more amino acid substitutions and/or amino acid
deletions or insertions, so long as the variant possesses the
requisite translocating function.
[0041] The only functional requirement of the translocating domain
is that it is capable of forming appropriate pores in the endosomal
membrane. A number of routine methods are available for confirming
that a particular translocating domain has the requisite
translocating activity, and thus to determine the presence of a
translocating domain. Shone et al. (1987), and Blaustein et al.
(1987) provide details of two very simple assays to confirm that
any particular bacterial translocating domain has the requisite
translocating activity. Shone (1987) describes a simple in vitro
assay employing liposomes, which are challenged with a test
molecule. The presence of a molecule having the requisite
translocating function is confirmed by release from the liposomes
of K.sup.+ and/or labelled NAD. Blaustein (1987) describes a simple
in vitro assay employing planar phospholipid bilayer membranes,
which are challenged with a test molecule. The presence of a
molecule having the requisite translocation function is confirmed
by an increase in conductance across the phospholipid membrane.
[0042] The polypeptide of the invention may be obtained by
expression of a recombinant nucleic acid, preferably a DNA, and is
a single polypeptide, that is to say not cleaved into separate
light and heavy chain domains. The polypeptide is thus available in
convenient and large quantities using recombinant techniques.
[0043] The first domain optionally comprises a fragment or variant
of a clostridial toxin light chain. The fragment is optionally an
N-terminal, or C-terminal fragment of the light chain, or is an
internal fragment, so long as it substantially retains the ability
to cleave the vesicle or plasma-membrane associated protein
essential to exocytosis. Domains necessary for the activity of the
light chain of clostridial toxins are described in J. Biol. Chem.,
Vol. 267, No. 21, July 1992, pages 14721-14729. The variant has a
different peptide sequence from the light chain or from the
fragment, though it too is capable of cleaving the vesicle or
plasma-membrane associated protein. It is conveniently obtained by
insertion, deletion and/or substitution of a light chain or
fragment thereof. In embodiments of the invention described below a
variant sequence comprises (i) an N-terminal extension to a
clostridial toxin light chain or fragment (ii) a clostridial toxin
light chain or fragment modified by alteration of at least one
amino acid (iii) a C-terminal extension to a clostridial toxin
light chain or fragment, or (iv) combinations of 2 or more of
(i)-(iii).
[0044] In an embodiment of the invention described in an example
below, the toxin light chain and the portion of the toxin heavy
chain are of botulinum toxin type A. In a further embodiment of the
invention described in an example below, the toxin light chain and
the portion of the toxin heavy chain are of botulinum toxin type B.
The polypeptide optionally comprises a light chain or fragment or
variant of one toxin type and a heavy chain or fragment or variant
of another toxin type.
[0045] In a polypeptide according to the invention said second
domain preferably comprises a clostridial toxin heavy chain H.sub.N
portion or a fragment or variant of a clostridial toxin heavy chain
H.sub.N portion. The fragment is optionally an N-terminal or
C-terminal or internal fragment, so long as it retains the function
of the H.sub.N domain. Teachings of regions within the H.sub.N
responsible for its function are provided for example in
Biochemistry 1995, 34, pages 15175-15181 and Eur. J. Biochem, 1989,
185, pages 197-203. The variant has a different sequence from the
H.sub.N domain or fragment, though it too retains the function of
the H.sub.N domain. It is conveniently obtained by insertion,
deletion and/or substitution of a H.sub.N domain or fragment
thereof. In embodiments of the invention, described below, it
comprises (i) an N-terminal extension to a H.sub.N domain or
fragment, (ii) a C-terminal extension to a H.sub.N domain or
fragment, (iii) a modification to a H.sub.N domain or fragment by
alteration of at least one amino acid, or (iv) combinations of 2 or
more of (i)-(iii). The clostridial toxin is preferably botulinum
toxin or tetanus toxin.
[0046] These polypeptides of the invention thus typically contain
two or more polypeptide first and second domain, linked by
di-sulphide bridges into composite molecules, and further linked to
a third domain.
[0047] The TM provides specificity for the BS on the relevant
neuronal and or secretory cells responsible for secretion of mucus
in the airways. The TM component of the agent can comprise one of
many cell binding molecules, including, but not limited to,
antibodies, monoclonal antibodies, antibody fragments (Fab,
F(ab)'.sub.2, Fv, ScFv, etc.), lectins, hormones, cytokines, growth
factors or peptides.
[0048] It is known in the art that the H.sub.C portion of the
neurotoxin molecule can be removed from the other portion of the
H-chain, known as H.sub.N, such that the H.sub.N fragment remains
disulphide linked, to the L-chain of the neurotoxin providing a
fragment known as LH.sub.N. Thus, in one embodiment of the present
invention the LH.sub.N fragment of a clostridial neurotoxin is
covalently linked, using linkages which may include one or more
spacer regions, to a TM.
[0049] The H.sub.C domain of a clostridial neurotoxin may be
mutated or modified, eg by chemical modification, to reduce or
preferably incapacitate its ability to bind the neurotoxin to
receptors at the neuromuscular junction. This modified clostridial
neurotoxin is then covalently linked, using linkages which may
include one or more spacer regions, to a TM.
[0050] The heavy chain of a clostridial neurotoxin, in which the
H.sub.C domain is mutated or modified, eg by chemical modification,
to reduce or preferably incapacitate its ability to bind the
neurotoxin to receptors at the neuromuscular junction, may be
combined with the L-chain of a different clostridial neurotoxin.
This hybrid, modified clostridial neurotoxin is then covalently
linked, using linkages which may include one or more spacer
regions, to a TM.
[0051] In another embodiment of the invention, the H.sub.N domain
of a clostridial neurotoxin is combined with the L-chain of a
different clostridial neurotoxin. This hybrid LH.sub.N is then
covalently linked, using linkages which may include one or more
spacer regions, to a TM. In a further embodiment of the invention,
the light chain of a clostridial neurotoxin, or a fragment of the
light chain containing the endopeptidase activity, is covalently
linked, using linkages which may include one or more spacer
regions, to a TM which can also effect the internalisation of the
L-chain, or a fragment of the L-chain containing the endopeptidase
activity, into the cytoplasm of the relevant secretory and/or
neuronal cells in the airways responsible for secretion of mucus
and or regulation of said secretion.
[0052] The agent is optionally expressed recombinantly as a fusion
protein which includes an appropriate TM in addition to any desired
spacer regions. The recombinantly expressed agent may be derived
wholly from the gene encoding one serotype of neurotoxin or may be
a chimaera derived from genes encoding one or more serotypes. In
another embodiment of the invention the required LH.sub.N, which
may be a hybrid of an L and H.sub.N from different clostridial
types, is expressed recombinantly as a fusion protein with the TM,
and may include one or more spacer regions.
[0053] The light chain of a clostridial neurotoxin, or a fragment
of the light chain containing the endopeptidase activity, may be
expressed recombinantly as a fusion protein with a TM which can
also effect the internalisation of the L-chain, or a fragment of
the L-chain containing the endopeptidase activity, into the
cytoplasm of the relevant secretory and or neuronal cells in the
airways responsible for secretion of mucus and or regulation of
said secretion. The expressed fusion protein may also include one
or more spacer regions.
[0054] A neurotoxin fragment as described in the present invention
can be prepared by methods well known in the protein art,
including, but not limited to, proteolytic cleavage or by genetic
engineering strategies. Said fragment is preferably a non-toxic
fragment. The conjugation may be chemical in nature using chemical
or covalent linkers. Conjugates according to the present invention
may be prepared by conventional methods known in the art.
[0055] In a third aspect, the invention provides a composition for
use in treating mucus hypersecretion, comprising: [0056] a compound
according to any of the second aspect of the invention; and [0057]
at least one of a pharmaceutically acceptable excipient, adjuvant
and/or propellant, [0058] wherein the composition is for
administration to the airways of a patient.
[0059] Aerosol administration is a preferred route of
administration, though the present invention encompasses also any
administration that delivers the compound to epithelia in the
airways. Nasal administration is optional though buccal is
preferred. The compound may thus be formulated for oral
administration via aerosol or nebuliser or as a dry powder for
inhalation using conventional excipients, adjuvants and/or
propellants. The invention therefore further provides a
pharmaceutical composition comprising a compound of the invention
and a pharmaceutically acceptable carrier.
[0060] In use the compound will generally be employed in a
pharmaceutical composition in association with a human
pharmaceutical carrier, diluent and/or excipient, although the
exact form of the composition will depend on the mode of
administration. The compound may, for example, be employed in the
form of an aerosol or nebulisable solution.
[0061] In a specific embodiment of the invention, described in
further detail below, a polypeptide according to the invention
comprises Substance P, and an L chain and a heavy chain H.sub.N
region of botulinum toxin A. In use, this may be administered to a
patient by aerosol. A solution of the polypeptide is prepared and
converted into an aerosol using a nebuliser for inhalation into the
lungs of nebulised particles of diameter 1-5 microns.
[0062] The dosage ranges for administration of the compounds of the
present invention are those to produce the desired therapeutic
effect. It will be appreciated that the dosage range required
depends on the precise nature of the conjugate, the route of
administration, the nature of the formulation, the age of the
patient, the nature, extent or severity of the patient's condition,
contraindications, if any, and the judgement of the attending
physician. Wide variations in the required dosage, however, are to
be expected depending on the precise nature of the conjugate.
Variations in these dosage levels can be adjusted using standard
empirical routines for optimisation, as is well understood in the
art.
[0063] Fluid unit dosage forms are prepared utilising the compound
and a pyrogen-free sterile vehicle. The compound, depending on the
vehicle and concentration used, can be either dissolved or
suspended in the vehicle. In preparing solutions the compound can
be dissolved in the vehicle, the solution being made isotonic if
necessary by addition of sodium chloride and sterilised by
filtration through a sterile filter using aseptic techniques before
filling into suitable sterile vials or ampoules and sealing.
Alternatively, if solution stability is adequate, the solution in
its sealed containers may be sterilised by autoclaving.
Advantageously additives such as buffering, solubilising,
stabilising, preservative or bactericidal, suspending or
emulsifying agents and/or local anaesthetic agents may be dissolved
in the vehicle.
[0064] Dry powders which are dissolved or suspended in a suitable
vehicle prior to use may be prepared by filling pre-sterilised drug
substance and other ingredients into a sterile container using
aseptic technique in a sterile area. Alternatively the drug and
other ingredients may be dissolved into suitable containers using
aseptic technique in a sterile area. The product is then freeze
dried and the containers are sealed aseptically.
[0065] Compositions suitable for administration via the respiratory
tract include aerosols, nebulisable solutions or microfine powders
for insufflation. In the latter case, particle size of less than 50
microns, especially less than 10 microns, is preferred. Such
compositions may be made up in a conventional manner and employed
in conjunction with conventional administration devices.
[0066] In further aspects of the invention, there is provided use
of a compound that inhibits exocytosis in mucus secreting cells or
neurones that control or direct mucus secretion in manufacture of a
medicament for treating mucus hypersecretion, asthma or COPD.
[0067] The invention yet further provides a method of manufacture
of a pharmaceutical composition, comprising: [0068] obtaining a
clostridial neurotoxin and modifying it so as to remove or disable
its H.sub.C portion; or [0069] obtaining a clostridial neurotoxin
the H.sub.C portion of which has been removed or disabled; [0070]
linking the toxin with a targeting moiety that binds to (i) a mucus
secreting cell, or (ii) a neuronal cell that controls or directs
mucus secretion. The invention still further provides a method of
manufacture of a pharmaceutical composition, comprising obtaining a
first component having the domains: [0071] an inhibiting domain
which inhibits exocytosis in a mucus secreting cell or neuronal
cell that controls or directs mucus secretion; [0072] a
translocating domain which translocates the inhibiting domain into
the cell; and [0073] linking the first component to a second
component that binds to (i) a mucus secreting cell, or (ii) a
neuronal cell that controls or directs mucus secretion.
[0074] The first and second components are preferably formulated in
an orally administrable composition in combination with one or more
or an excipient, an adjuvant and a propellant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] Specific embodiments of the invention are now illustrated in
the following examples with reference to the accompanying drawings
in which:--
[0076] FIG. 1 illustrates the preparation of the substance
P-LH.sub.N/A conjugate of Example 1;
[0077] FIG. 2 shows Western blot detection of conjugated substance
P-LH.sub.N/A;
[0078] FIG. 3 shows SDS-PAGE analysis of a WGA-LH.sub.N/A
purification scheme;
[0079] FIGS. 4-6 show inhibition of neurotransmitter release from
cultured neuronal cells; and
[0080] FIG. 7 shows WGA-LH.sub.N/A inhibits release from, but does
not have specificity for, eDRG neurons.
EXAMPLES
Example 1
Method for the Preparation of Substance P-LH.sub.N/A Conjugates
[0081] The lyophilised peptide was rehydrated in 0.1%
trifluoroacetic acid (TFA) to a final concentration of 10 mM.
Aliquots of this solution were stored at -20 degrees C. until use.
The LH.sub.N/A was desalted into PBSE (PBS containing 1 mM EDTA).
The resulting solution (3-5 mg/ml) was reacted with a three- or
four-fold molar excess of SPDP by addition of a 10 mM stock
solution of SPDP in DMSO. After 3 hours at room temperature the
reaction was terminated by desalting over a PD-10 column into
PBSE.
[0082] A portion of the derivatised LH.sub.N/A was removed from the
solution and reduced with DTT (5 mM, 30 min). This sample was
analysed spectrophotometrically at 280 nm and 343 nm to determine
the degree of derivatisation. The degree of derivatisation achieved
was typically 2 mol/mol.
[0083] The bulk of the derivatised LH.sub.N/A and the substance P
peptide were mixed in proportions such that the peptide was in
four-fold molar excess. The conjugation reaction was allowed to
proceed for >16 hours at 4.degree. C.
[0084] The product mixture was centrifuged to clear any precipitate
that had developed. The supernatant was applied to a PD-10 column
equilibrated in PBS and protein fractions were eluted by addition
of PBS. Peptide and reaction by-products eluted after the main peak
of protein and were discarded.
[0085] The conjugate mixture was concentrated to >1 mg/ml by
centrifugation through concentrators (with 10000 molecular weight
exclusion limit). The concentrated conjugate mixture was analysed
by SDS-PAGE and Western blotting (probed with anti-substance P
antibody) to confirm linkage of substance P peptide to
LH.sub.N/A.
[0086] The method described is for linkage of substance P peptide
covalently to LH.sub.N/A via a SPDP linker. A sulphydryl residue is
incorporated into the C-terminus of the substance P residue, in
this case by the addition of a Cys residue. Alternative linkers are
available, including linkers utilising similar chemistry of
derivatisation but generating non-reducible covalent bonds between
the linked species.
[0087] The Substance P peptide sequence used in this particular
example is RPKPQQFFGLMC (SEQ ID NO:2), though alternative sequences
are also suitable, e.g. CRPKPQQFFGLM (SEQ ID NO:3), i.e. substance
P with an N-terminal Cys.
[0088] The method described does not make use of any tagging system
(e.g. poly His) to purify the conjugate from free LH.sub.N/A. This
has been demonstrated to be a successful method for the preparation
of opioid peptide LH.sub.N/A such the receptor binding function of
the peptide was not compromised. A similar approach can be applied
to the synthesis of subP-LH.sub.N/A.
[0089] FIG. 1 illustrates the preparation of the substance
P-LH.sub.N/A conjugate of Example 1. In the results shown in FIG.
1, LH.sub.N/A and substance P-LH.sub.N/A samples at the
concentrations indicated were applied to nitrocellulose and probed
with rabbit anti-substance P antibody (upper two rows). The
emergence of cross-reaction with the conjugate (second row), rather
than the LH.sub.N/A (first row), is indicative of substance P
conjugated to LH.sub.N/A. The lower control row illustrates the
presence of LH.sub.N/A.
[0090] FIG. 2 shows Western blot detection of conjugated substance
P-LH.sub.N/A. Samples of substance P-LH.sub.N/A (lane 3) and
LH.sub.N/A (lane 2) were electrophoresed alongside molecular weight
markers (lane 1). Detection of substance P by rat anti-substance P
antisera indicated protein of approx. 100 kDa molecular weight in
the conjugate lane, but no such band in the LH.sub.N/A only lane.
Thus the conjugated LH.sub.N/A does contain substance P.
Example 2
Method for the Preparation of a Broad Specificity Agent
[0091] Conjugation and purification of WGA-LH.sub.N/A. WGA (10
mg/ml in phosphate-buffered saline (PBS)) was reacted with an equal
concentration of SPDP (10 mM in dimethyl sulphoxide (DMSO)) for one
hour at ambient temperature. Reaction by-products were removed by
desalting into PBS prior to reduction of the cross-linker with
dithiothreitol. The thiopyridone and DTT were then removed by
desalting into PBS to result in derivatised WGA (dWGA) with 1 mole
-SH incorporated per mole of WGA.
[0092] LH.sub.N/A at a concentration of 3-5 mg/ml in PBSE (PBS
containing 1 mM EDTA) was reacted with a three or four-fold molar
excess of SPDP (10 mM in DMSO). After 3 h at ambient temperature
the reaction was terminated by desalting over a PD-10 column into
PBSE.
[0093] The derivatised WGA (dWGA) and the derivatised LH.sub.N/A
(dLH.sub.N/A) were mixed in a 3:1 molar ratio. After 16 h at
4.degree. C. the mixture was centrifuged to clear any precipitate
that had developed. The supernatant, was concentrated by
ultrafiltration before application to a Superose.TM. 12 column on
an FPLC.RTM. chromatography system (Pharmacia). The column was
eluted with PBS and the fractions containing high molecular weight
conjugate material (separated from free dWGA) were pooled and
applied to PBS-washed N-acetylglucosamine-agarose (GlcNAc-agarose).
WGA-LH.sub.N/A conjugate bound to the GlcNAc-agarose and was eluted
from the column by the addition of 0.3M N-acetylglucosamine in PBS.
The elution profile was followed at 280 nm and fractions containing
conjugate were pooled, dialysed against PBS, and stored at
4.degree. C. until use.
[0094] FIG. 3 shows SDS-PAGE analysis of WGA-LH.sub.N/A
purification scheme. Protein fractions were subjected to 4-20%
polyacrylamide SDS-PAGE prior to staining with Coomassie blue.
Lanes 6-8 were run in the presence of 0.1M DTT Lanes 1 (&7) and
2 (& 8) represent derivatised WGA and derivatised LH.sub.N/A
respectively. Lanes 3-5 represent conjugation mixture,
post-Superose-12 chromatography and post GlcNAc-affinity
chromatography respectively. Lanes 6 represents a sample of reduced
final material. Approximate molecular masses (kDa) are indicated on
the Figure.
Example 3
Preparation and Maintenance of Neuronal Cultures and Inhibition of
Neurotransmitter Release
[0095] PC12 cells were seeded at a density of 4.times.10.sup.5
cells/well onto 24 well (matrigel coated) plates (NUNC.TM.) from
stocks grown in suspension. The cells were cultured for 1 week
prior to use in RPMI, 10% horse serum, 5% foetal bovine serum, 1%
L-glutamine. SH-SY5Y cells were seeded at a density of
5.times.10.sup.5 cells/well onto 24 well plates (FALCON.TM.). The
cells were cultured in HAM-F12:MEM (1:1 v/v), 15% foetal bovine
serum, 1% MEM non-essential amino acids, 2 mM L-glutamine for 1
week prior to use. Embryonic spinal cord (eSC) neurons were
prepared from spinal cords dissected from 14-15 day old foetal
Sprague Dawley rats and were used after 21 days in culture using a
modification of previously described method.
[0096] Inhibition of transmitter release. PC12 cells or SH-SY5Y
cells were washed with a balanced salt solution (BSS: 137 mM NaCl,
5 mM KCl, 2 mM CaCl.sub.2, 4.2 mM NaHCO.sub.3, 1.2 mM MgCl.sub.2,
0.44 mM KH.sub.2PO.sub.4, 5 mM glucose, 20 mM HEPES, pH7.4) and
loaded for 1 hour with [.sup.3H]-noradrenaline (2 .mu.Ci/ml, 0.5
ml/well) in BSS containing 0.2 mM ascorbic acid and 0.2 mM
pargyline. Cells were washed 4 times (at 15 minutes intervals for 1
hour) then basal release determined by a 5 minute incubation with
BSS (5 mM K.sup.+). Cells were then depolarised with 100 mM K.sup.+
(BSS with Na.sup.+ reduced accordingly) for 5 minutes to determine
stimulated release. Superfusate (0.5 ml) was removed to tubes on
ice and briefly centrifuged to pellet any detached cells. Adherent
cells were solubilised in 2M acetic acid/0.1% trifluoroacetic acid
(250 .mu.g/well). The quantity of released and non-released
radiolabel was determined by liquid scintillation counting of
cleared superfusates and cell lysates respectively. Total uptake
was calculated by addition of released and retained radioactivity
and the percentage release calculated ((released counts/total
uptake counts).times.100).
[0097] eSC neurons were loaded with [.sup.3H]-glycine for 30
minutes prior to determination of basal and potassium-stimulated
release of transmitter. A sample of 0.2M NaOH-lysed cells was used
to determine total counts, from which % release could be
calculated.
[0098] FIGS. 4-6 show inhibition of neurotransmitter release from
cultured neuronal cells. PC12 (FIG. 4), SH-SY5Y cells (FIG. 5) and
eSC neurons (FIG. 6) exposed for three days to a range of
concentrations of WGA-LH.sub.N/A (filled symbols) and LH.sub.N/A
(open symbols) were assessed for stimulated [.sup.3H]-noradrenaline
release (SH-SY5Y and PC12 cells) or [.sup.3H]-glycine release (eSC)
capability. Results are expressed as percentage inhibition compared
to untreated controls. Each concentration was assessed in
triplicate. For each cell type the dose response curve is
representative of at least three experiments. Each point shown is
the mean of at least three determinations.+-. SE of the mean.
[0099] FIG. 7 shows dose-response curves of WGA-LH.sub.N/A
inhibition of eDRG substance P and eSC [.sup.3H]-glycine release.
Cells were exposed to conjugate for three days. Representative
curves are shown. Mean IC.sub.50 eDRG: 0.32.+-.0.05 .mu.g/ml (n=4),
eSC: 0.06.+-.0.01 .mu.g/ml (n=3).
Example 4
Method for the Preparation of LC/B-Epidermal Growth Factor with a
Translocation Domain from Diphtheria Toxin by Recombinant
Expression
[0100] Using standard DNA manipulation procedures, the DNA encoding
LC/B, diphtheria toxin amino acids 194-380, and epidermal growth
factor are assembled in frame and inserted into an appropriate
expression vector. Inserted between the LC/B DNA and the diphtheria
toxin translocation domain is DNA encoding a short spacer sequence
with a specific cleavable peptide bond (.dwnarw.), bounded by a
pair of cysteine amino acids. Examples of specific enzymes that may
be used to activate the fusion protein include factor Xa
(IEGR.dwnarw.) (SEQ ID NO:4), enterokinase (DDDDK.dwnarw.) (SEQ ID
NO:5), TEV protease (EXXYXQS.dwnarw.G) (SEQ ID NO:6), precission
(LEVLFQ.dwnarw.GP) (SEQ ID NO:7), Thrombin (LVPR.dwnarw.GS) (SEQ ID
NO:8) and genenase (HY or YH). Expression of a single polypeptide
of the form LC/B-DT.sub.194-380-EGF is achieved in E. coli using
standard techniques. The expressed fusion protein is isolated from
E. coli by standard purification techniques and cleaved by the
specific activation enzyme prior to assessment in an in vitro cell
model.
Example 5
Method for the Preparation of LC/C-Epidermal Growth Factor with a
Translocation Domain from Pseudomonas Exotoxin by Recombinant
Expression
[0101] Using standard DNA manipulation procedures, the DNA encoding
LC/C, pseudomonas exotoxin amino acids 405-613, and epidermal
growth factor are assembled in frame and inserted into an
appropriate expression vector. Inserted between the LC/C DNA and
the pseudomonas exotoxin translocation domain is DNA encoding a
short spacer sequence with a specific cleavable peptide bond,
bounded by a pair of cysteine amino acids. Examples of specific
enzymes that may be used to activate the fusion protein include
factor Xa (IEGR.dwnarw.), (SEQ ID NO:4), enterokinase
(DDDDK.dwnarw.) (SEQ ID NO:5), TEV protease (EXXYXQS.dwnarw.G) (SEQ
ID NO:6), precission (LEVLFQ.dwnarw.GP) (SEQ ID NO:7), Thrombin
(LVPR.dwnarw.GS) (SEQ ID NO:8) and genenase (HY or YH). Expression
of a single polypeptide of the form LC/C-PE.sub.405-613-EGF is
achieved in E. coli using standard techniques. The expressed fusion
protein is isolated from E. coli by standard purification
techniques and cleaved by the specific activation enzyme prior to
assessment in an in vitro cell model.
Example 6
Method for the Preparation of LC/A-Epidermal Growth Factor with a
Translocation Domain from Influenza Virus Haemagglutinin by
Recombinant Expression
[0102] Using standard DNA manipulation procedures, the DNA encoding
LC/A, GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO:1) from influenza virus
haemagglutinin (HA), and epidermal growth factor are assembled in
frame and inserted into an appropriate expression vector. Inserted
between the LC/A DNA and the haemagglutinin sequence is DNA
encoding a short spacer sequence with a specific cleavable peptide
bond, bounded by a pair of cysteine amino acids. Examples of
specific enzymes that may be used to activate the fusion protein
include factor Xa (IEGR.dwnarw.) (SEQ ID NO:4), enterokinase
(DDDDK.dwnarw.) (SEQ ID NO:5), TEV protease (EXXYXQS.dwnarw.G) (SEQ
ID NO:6), precission (LEVLFQ.dwnarw.GP) (SEQ ID NO:7), Thrombin
(LVPR.dwnarw.GS) (SEQ ID NO:8) and genenase (HY or YH). Expression
of a single polypeptide of the form LC/A-HA-EGF is achieved in E.
coli using standard techniques. The expressed fusion protein is
isolated from E. coli by standard purification techniques and
cleaved by the specific activation enzyme prior to assessment in an
in vitro cell model.
[0103] The agent described in this invention can be used in vivo,
either directly or as a pharmaceutically acceptable salt, for the
treatment of conditions involving mucus hypersecretion, including
COPD and asthma.
Sequence CWU 1
1
8123PRTInfluenza virus 1Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp Tyr Gly
20212PRTUnknownSubstance P peptide sequence 2Arg Pro Lys Pro Gln
Gln Phe Phe Gly Leu Met Cys1 5 10312PRTUnknownSubstance P peptide
sequence with N-terminal Cys 3Cys Arg Pro Lys Pro Gln Gln Phe Phe
Gly Leu Met1 5 1044PRTUnknownFactor Xa 4Ile Glu Gly
Arg155PRTUnknownEnterokinase 5Asp Asp Asp Asp Lys1 568PRTUnknownTEV
protease 6Glu Xaa Xaa Tyr Xaa Gln Ser Gly1 578PRTUnknownPrecission
7Leu Glu Val Leu Phe Gln Gly Pro1 586PRTUnknownThrombin 8Leu Val
Pro Arg Gly Ser1 5
* * * * *