U.S. patent application number 12/624248 was filed with the patent office on 2011-03-10 for use of furin convertase inhibitors in the treatment of fibrosis and scarring.
This patent application is currently assigned to RENOVO LIMITED. Invention is credited to Georg Brunner, Mark William James FERGUSON.
Application Number | 20110059896 12/624248 |
Document ID | / |
Family ID | 9941004 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059896 |
Kind Code |
A1 |
FERGUSON; Mark William James ;
et al. |
March 10, 2011 |
USE OF FURIN CONVERTASE INHIBITORS IN THE TREATMENT OF FIBROSIS AND
SCARRING
Abstract
The present invention relates to use of convertase inhibitors
for the reduction of scarring during the healing of wounds and also
for reducing fibrosis in the treatment of fibrotic conditions.
Inventors: |
FERGUSON; Mark William James;
(Derbyshire, GB) ; Brunner; Georg; (Muenster,
DE) |
Assignee: |
RENOVO LIMITED
|
Family ID: |
9941004 |
Appl. No.: |
12/624248 |
Filed: |
November 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10522222 |
Jan 24, 2005 |
7700562 |
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PCT/GB03/03159 |
Jul 23, 2003 |
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12624248 |
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Current U.S.
Class: |
514/18.6 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/08 20130101; A61K 38/07 20130101; A61P 19/04 20180101; A61K
48/00 20130101; A61P 17/02 20180101; A61P 27/02 20180101; A61P
13/12 20180101; A61P 1/00 20180101; A61P 25/00 20180101; A61P 17/00
20180101; A61K 38/55 20130101; A61P 9/00 20180101; A61P 1/16
20180101 |
Class at
Publication: |
514/18.6 |
International
Class: |
A61K 38/07 20060101
A61K038/07; A61P 17/02 20060101 A61P017/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 2002 |
GB |
0217136.1 |
Claims
1-17. (canceled)
18. A method for reducing scarring during the healing of wounds,
reducing fibrosis in the treatment of fibrotic conditions, or for
preventing or inhibiting scar formation or fibrosis, comprising
applying a furin inhibitor to a site of a wound or fibrotic
disorder or to a site where a wound may form or fibrosis may
occur.
19. The method defined in claim 18 wherein the inhibitor is a
serine protease inhibitor.
20. The method defined in claim 18 wherein the inhibitor is lipid
soluble.
21. The method defined in claim 19 wherein the inhibitor is a
peptidyl chloroalkylketone having a peptide moiety which mimics at
least one convertase enzyme cleavage site.
22. The method defined in claim 19 wherein the inhibitor is
decanoyl-RVKR-cmk (SEQ ID NO: 1).
23. The method defined in claim 18 wherein the inhibitor is water
soluble.
24. The method defined in claim 23 wherein the inhibitor is
hexa-arginine (SEQ ID NO: 5).
25. The method defined in claim 18 for reducing fibrosis in the
treatment of fibrotic conditions.
26. The method defined in claim 25 wherein the fibrotic condition
is a fibrotic disorder selected from glomerulonephritis, cirrhosis
of the liver, fibrotic disease, adhesions or restenosis.
27. A composition comprising an effective amount of a furin
inhibitor for reducing scarring during the healing of wounds,
reducing fibrosis in the treatment of fibrotic conditions, or for
preventing scar formation or fibrosis, and a pharmaceutically
acceptable carrier.
28. The method of claim 18 wherein said injury is to a muscle,
tendon, ligament or skin.
29. The method of claim 18 wherein the wound is a surgical
wound.
30. The method of claim 18 wherein said applying is topical.
31. The method of claim 18 wherein said applying is by injection.
Description
[0001] The present invention relates to wound healing and also to
regulating fibrosis in the treatment of conditions in which
fibrosis is a major mechanism of tissue repair or where excessive
fibrosis leads to pathological derangement and malfunctioning of
tissue.
[0002] Wound healing in adults is a complicated reparative process.
The term "wound" as used herein is exemplified by, but not limited
to, injuries to the skin. Other types of wound can involve damage,
injury or trauma to an internal tissue or organ such as the lung,
kidney, heart, gut, tendons or liver.
[0003] The healing process in skin wounds typically begins with a
haemostatic response initiated by damage to blood vessels in the
skin. During this process platelets and a number of factors present
in the blood contribute to the formation of a clot that prevents
further blood loss. Factors released during this process,
particularly by the degranulation of platelets, then cause
recruitment of a variety of specialised cells to the site of the
wound that are in turn involved in extracellular matrix and
basement membrane deposition, angiogenesis, selective protease
activity and re-epithelialisation. An important component of the
healing process in adult mammals is the stimulation of fibroblasts
to generate the extracellular matrix. This extracellular matrix
constitutes a major component of the connective tissue that
develops to repair the wound area.
[0004] The connective tissue that forms during the healing process
is often fibrous in nature and commonly forms into a connective
tissue scar (a process known as fibrosis).
[0005] A scar is an abnormal morphological structure resulting from
a previous injury or wound (e.g. an incision, excision or trauma).
Scars are composed of a connective tissue which is predominately a
matrix of collagen types 1 and 3 and fibronectin. The scar may
consist of collagen fibres with an abnormal organisation (as seen
in scars of the skin) or it may be an abnormal accumulation of
connective tissue (as seen in scars of the central nervous system).
Most scars consist of abnormally organised collagen and also excess
collagen. In man, in the skin, scars may be depressed below the
surface or elevated above the surface of the skin. Hypertrophic
scars represent a severe form of normal scarring. They are elevated
above the normal surface of the skin and contain excessive collagen
arranged in an abnormal pattern. Keloids are another form of
pathological scarring in which the scar is not only elevated above
the surface of the skin but also extends beyond the boundaries of
the original injury. In a keloid there is excessive connective
tissue that is organised in an abnormal fashion predominately in
whirls of collagenous tissue. There are genetic predispositions to
the formation of both hypertrophic scars and keloids. These
aberrant forms of scarring are particularly common in
Afro-Caribbean and Mongoloid races.
[0006] There are many instances where the regulation of scar
formation is of primary importance when considering the outcome of
wound healing. Examples of such situations are scars of the skin
where excessive scarring may be detrimental to tissue function,
particularly in contexts where scar contracture occurs (for
instance skin burns and wounds that impair flexibility of a joint).
The reduction of scarring to the skin when cosmetic considerations
are important is also highly desirable. In the skin hypertrophic or
keloid scars can cause functional and cosmetic impairment and there
is a need to prevent their occurrence. Scarring resulting from skin
grafts in both donor sites and from the application of artificial
skin can also be problematic and need to be minimised or
prevented.
[0007] As well as scars of the skin, internal scarring or fibrosis
can be highly detrimental and specific examples include:
[0008] (i) Within the central nervous system, glial scarring can
prevent neuronal reconnection (e.g. following neuro-surgery or
penetrating injuries of the brain).
[0009] (ii) Scarring in the eye can be detrimental. In the cornea,
scarring can result in abnormal opacity and lead to problems with
vision or even blindness. In the retina, scarring can cause
buckling or retinal detachment and consequently blindness. Scarring
following wound healing in operations to relieve pressure in
glaucoma (e.g. glaucoma filtration surgery) results in the failure
of the surgery whereby the aqueous humour fails to drain and hence
the glaucoma returns.
[0010] (iii) Scarring in the heart (e.g. following surgery or
myocardial infarction) can give rise to abnormal cardiac
function.
[0011] (iv) Operations involving the abdomen or pelvis often result
in adhesion between viscera. For instance, adhesions between
elements of the gut and the body wall may form and cause twisting
in the bowel loop leading to ischaemia, gangrene and the necessity
for emergency treatment (untreated they may even be fatal).
Likewise, trauma or incisions to the guts can lead to scarring and
scar contracture to strictures which cause occlusion of the lumen
of the guts which again can be life threatening.
[0012] (v) Scarring in the pelvis in the region of the fallopian
tubes can lead to infertility.
[0013] (vi) Scarring following injury to muscles can result in
abnormal contraction and hence poor muscular function.
[0014] (vii) Scarring or fibrosis following injury to tendons and
ligaments can result in serious loss of function.
[0015] Related to the above is the fact that there are a number of
medical conditions known as fibrotic disorders in which excessive
fibrosis leads to pathological derangement and malfunctioning of
tissue. Fibrotic disorders are characterised by the accumulation of
fibrous tissue (predominately collagens) in an abnormal fashion
within the tissue. Accumulation of such fibrous tissues may result
from a variety of disease processes. These diseases do not
necessarily have to be caused by surgery, traumatic injury or
wounding. Fibrotic disorders are usually chronic. Examples of
fibrotic disorders include cirrhosis of the liver, liver fibrosis,
glomerulonephritis, pulmonary fibrosis, scleroderma, myocardial
fibrosis, fibrosis following myocardial infarction, central nervous
system fibrosis following a stroke or neuro-degenerative disorders
(e.g. Alzheimer's Disease), proliferative vitreoretinopathy (PVR),
restenosis (for example following angioplasty) and arthritis. There
is therefore also a need for medicaments which may be used for the
treatment of such conditions by regulating (i.e. preventing,
inhibiting or reversing) fibrosis/scarring in these fibrotic
disorders.
[0016] Whilst the above considerations mainly apply to conditions,
disorders or diseases of man it will be appreciated that wound
healing, scarring and fibrotic disorders can also be problematic in
other animals, particularly veterinary or domestic animals (e.g.
horses, cattle, dogs, cats etc). For instance abdominal wounds or
adhesions are a major reason for having to put down horses
(particularly race horses), as are tendon and ligament damage
leading to scarring or fibrosis.
[0017] There have been several recent developments in the fields of
wound healing, scarring and fibrotic disorders. Some of these
developments revolve around the recent understanding that an array
of cytokines and growth factors is intimately involved in the
repair of wounded tissue. In particular, members of the
Transforming Growth Factor .beta.(TGF-.beta.) superfamily have been
found to play an important role in wound healing. At least 25
molecules are known to be members of the TGF-.beta. superfamily.
These include a number of cytokines such as TGF-.beta.s 1 to 5, the
DVR group (e.g. dpp and Vg1), Bone Morphogenetic Proteins, Nodal,
Activin and Inhibin.
[0018] TGF-.beta.s are often secreted from cells in an inactive
form known as latent TGF-.beta.. Latent TGF-.beta. consists of an N
terminal Latency Associated Peptide (LAP) and the TGF-.beta. and is
also referred to as the Small Latent Complex. Additionally the
Small Latent Complex can bind to another peptide (derived from a
different gene) of variable size called Latent TGF-.beta. Binding
Protein (LTBP) in which case the entire complex is known as the
Large Latent TGF-.beta. Complex.
[0019] Latent TGF-.beta. is activated when the TGF-.beta. is caused
to be dissociated from the LAP. This dissociation may be
co-ordinated at a mannose-6-phosphate/Insulin Like Growth Factor II
receptor (M6P-R) and involve proteases such as plasmin, the
substrates being associated at the cell surface by tissue
transglutaminase. Free radicals and reactive oxygen species can
also activate TGF-.beta. by causing dissociation from the LAP.
[0020] TGF-.beta. (particularly TGF-.beta..sub.1 and
TGF-.beta..sub.2) promotes wound healing but is also associated
with increased scar formation and fibrosis. Clinical interest in
the modulation of TGF-.beta. has been associated with inhibiting
its activity in order to reduce scar formation (although this may
compromise the rate of wound healing). For instance, WO 92/17206
discloses neutralising agents that inhibit the activity of
TGF-.beta..sub.1 and TGF-.beta..sub.2 and are particularly
beneficial for reducing scar formation.
[0021] Another development involves the use of mannose-6-phosphate
for use in treating fibrotic disorders associated with accumulation
of extracellular matrix and with elevated levels of Transforming
Growth Factors .beta..sub.1 or .beta..sub.2 (GB-A-2,265,310).
Mannose-6-phosphate is believed to interfere with the conversion of
latent forms of these Transforming Growth Factors into their active
form.
[0022] Despite such advances there remains a need to continue to
develop medicaments that may be used to modulate the healing of
wounds, scarring and fibrosis. In particular there is a need for
medicaments which do not compromise the rate of wound healing or
quality of scar in favour of one or the other.
[0023] As discussed more fully below, the invention relates in its
broadest aspect to the use of convertase inhibitors for the
treatment of wounds.
[0024] According to a first aspect of the present invention there
is provided the use of a convertase inhibitor in the manufacture of
a medicament for reducing scarring during the healing of wounds or
reducing fibrosis in the treatment of fibrotic conditions wherein
the medicament is topically applied to the site of a wound or
fibrotic disorder.
[0025] According to a second aspect of the present invention, there
is provided a composition comprising a therapeutically effective
amount of a convertase inhibitor and a pharmaceutically acceptable
vehicle for the treatment of wounds or fibrosis.
[0026] According to a third aspect of the present invention, there
is provided a method of treating a subject to reduce or prevent
scarring during the healing of wounds; or reduce or prevent
fibrosis in the treatment of fibrotic conditions comprising
topically administering to a subject in need of such treatment a
therapeutically effective amount of a convertase inhibitor.
[0027] Convertases are a family of Ca.sup.2+-dependant serine
proteases, otherwise known as SPCs (subtilisin-like pro-protein
convertases; see Dubois et al., 1995, Journ. Biol. Chem.,
270(18):10618-10624; Sha, X., et al., 1989, Mol. Endocrinology, 3:
1090-1098; Chan, Si., et al., 1992, Proc. Natl. Acad. Sci. USA 89:
6678-6682; and references therein). The inventors have found that
the convertase enzyme furin is particularly involved in the
activation of mature latent TGF-.beta. at the site of a wound or
fibrotic disorder. Although the inventors do not wish to be bound
by any hypothesis they believe that convertase activity is able to
indirectly stimulate TGF-.beta. activation by modifying the
activity of other enzyme(s) with TGF-.beta. activating properties.
The inventors believe that the convertase activity contributing to
TGF-.beta. activation initially occurs intracellularly, within the
platelet, and then continues extracellularly as the platelet
contents are released on de-granulation. Accordingly convertase
inhibitors used according to the present invention are believed to
modify activity of this enzyme such that TGF-.beta. activation is
reduced.
[0028] For the purposes of the specification references to
intracellular activity should also be taken to encompass activity
within the membranes of cell fragments, such as platelets, except
where the context requires otherwise.
[0029] The inventors believe that the convertases involved in
TGF-.beta. activation are furin-like proprotein convertases. Furins
comprise a family of seven transmembrane proprotein convertases
produced as an inactive precursor. They must be activated
intracellularly, and are involved in pre-protein processing in the
trans-Golgi network, at the cell surface, extracellularly and in
endosomes. Furins have their effect at arginine-containing cleavage
sites, the minimal site being Arg-X-X-Arg. Relevant reviews include
Molloy et al 1.999; Shapiro et al. 1997 (J. Histochem. Cytochem.
45:3-12) and Pearton et al. 2001 (Experimental Dermatology
10:193-203).
[0030] Platelets are a major source of TGF-.beta. in the
circulation and release latent TGF-.beta. when the platelet is
activated (e.g. in response to injury). During the healing process,
various forms of TGF-.beta. are to be found at a wound site or site
of a fibrotic disorder. These different forms are active TGF-.beta.
(which is in its free form), the small latent complex
(TGF-.beta.-latency associated peptide), and the large latent
complex (TGF-.beta.-latency associated peptide-latent TGF-.beta.
binding protein). The different complexes undergo different fates
and perform different roles during the healing process. In
particular, the large and small latent complexes are activated by
cleaving in order to release active TGF-.beta. whilst the healing
process occurs.
[0031] The prior art suggests that cleavage of the large and small
latent complexes at a wound site is mediated by plasmin.
Furthermore, convertases such as furin are believed to be
responsible for the intracellular processing of pro-TGF-.beta.
within megakaryocytes (which give rise to platelets) in the bone
marrow. This processing of pro-TGF-.beta. involves cleavage and
folding of the pro-protein to produce the mature form. The mature
form produced is not, however, "active" TGF-.beta., and may be
associated with the large or small latent complexes. Accordingly
convertases have not previously been thought to play a part in the
activation of latent TGF-.beta. (such as TGF-.beta. in the small
latent complex) from platelets in the blood at a site of a wound or
fibrotic disorder.
[0032] However, the inventors have established (as described in
more detail in the Example) that, surprisingly, activity of
convertase enzymes such as furin effects the extracellular
activation of TGF-.beta. at a wound site. Hence by inhibiting the
activity of convertase enzymes at a wound site or site of a
fibrotic disorder it is possible to reduce the amount of active
TGF-.beta. at such a site and thereby reduce scarring and/or
fibrosis. It is interesting to note that this activity of
convertases appears to occur intracellularly and extracellularly.
Furthermore the activity does not appear to be associated with, or
controlled by, transcriptional regulation (in contrast to known
convertase activity in cells such as megakaryocytes) and thus is
able to take place in the anuclear platelet, or even
extracellularly.
[0033] The novel observation that convertase enzymes are involved
in activation of TGF-.beta. is in contrast to the previously
reported role of these enzymes in TGF-.beta. processing and
maturation, and opens a range of therapeutic possibilities that
could not have been envisaged before.
[0034] Although the prior art recognises that platelets contain
latent TGF-.beta. it provides no indication that it is possible to
prevent activation of this TGF-.beta. by inhibiting convertase
activity.
[0035] Instead the prior art only suggests that inhibitors of
convertase are able to inhibit the processing and maturation that
produces latent TGF-.beta.. This activity of convertases is
transcriptionally regulated. Thus the skilled person would have
recognised that once latent TGF-.beta. is present in circulating
platelets convertase inhibitors would not be able to influence its
state.
[0036] It is only with the present invention that it can be seen
that convertase inhibitors are able to prevent the activation, and
undesirable effects, of this platelet-borne latent TGF-.beta. at a
wound site.
[0037] The efficacy of convertase inhibitors for reducing scarring
or fibrosis is enhanced by the fact that the latent TGF-.beta.
released by platelets is almost entirely composed of
TGF-.beta..sub.1 (associated with the small latent complex). It is
well known that TGF-.beta..sub.1 is a key factor in wound healing
and is pro-fibrotic favouring scar formation. The preponderance of
the pro-fibrotic TGF-.beta. isoforms during the early phases of
wound healing (which causes local conditions that favour scar
formation and fibrosis) is in large measure due to
platelet-mediated growth factor release. With time the ratio of
TGF-.beta. isoforms changes as levels of platelet-derived
TGF-.beta..sub.1 decrease and there is an increase in the levels of
anti-fibrotic TGF-.beta..sub.3 derived from fibroblasts. Preventing
the activation of latent TGF-.beta..sub.1 according to the present
invention can therefore dramatically reduce the degree of scarring
associated with wound healing.
[0038] Several classes of compound may be used according to the
invention as convertase inhibitors. These compounds include: [0039]
(i) compounds that bind to convertase enzymes and inhibit its
activity (e.g. competitive inhibitors or allosteric inhibitors);
[0040] (ii) compounds which prevent the transcription, translation
or expression of convertase enzymes (e.g. ribozymes or antisense
DNA molecules); [0041] (iii) compounds which increase the rate of
degradation of convertase enzymes; [0042] (iv) compounds which
inhibit the interaction of convertase enzymes with latent
TGF-.beta. and/or with TGF-.beta. activating proteins; [0043] (v)
compounds which inhibit the proteolytic activation of the inactive
furin precursor; and [0044] (vi) compounds which inhibit a
potential intracellular translocation of convertase enzymes, such
as furin or PACE-4, to subcellular sites of activity.
[0045] In one embodiment of the invention it is preferred that the
convertase inhibitor is an inhibitor of furin.
[0046] In a further embodiment of the invention it is preferred
that the convertase inhibitor is an inhibitor of furin-like
proprotein convertases, and more preferably an inhibitor of
PACE-4.
[0047] The convertase inhibitor may be a serine protease inhibitor
and is preferably a thiol inhibitor. The thiol inhibitor may be a
peptidyl chloroalkylketone having a peptide moiety which mimics at
least one convertase enzyme cleavage site. It has been found that
peptidyl chloroalkylketones with peptide moieties that mimic the
convertase enzyme cleavage site are specific inhibitors of the
enzymatic activity. A preferred inhibitor is decanoyl-RVKR-cmk and
derivatives thereof.
[0048] Further convertase inhibitors suitable for use according to
the invention include: [0049] (i) alpha 1-antitrypsin (.alpha.-1
PDX), or nucleic acids encoding the same; [0050] (ii) derivatives
of alpha 1-antitrypsin such as those comprising the amino acid
sequences arg-val-pro-arg, ala-val-arg-arg or arg-val-arg-arg, or
nucleic acids encoding the same; [0051] (iii)
p-chloromercuribenzoate; [0052] (iv) tosylamido-phenylethyl
chloromethyl ketone (TPCK); [0053] (v) D-polyarginines (e.g.
hexa-arginine and its derivatives); [0054] (vi)
Acetyl-leu-leu-arg-aldehyde hemisulfate; [0055] (vii)
S-carboxyphenylethyl-carbamoyl-arg-val-arg-aldehyde; [0056] (viii)
Threodimercaptobutanediol; and [0057] (ix)
Tos-Lys-chloromethylketone.
[0058] Alternatively and/or in addition, the convertase inhibitor
may sequester Ca.sup.2+. Furthermore a Ca2+ sequester (such as EDTA
or EGTA) may be used in conjunction with inhibitors such as those
mentioned above.
[0059] The inventors have established that convertase enzymes act,
both extracellularly and intracellularly, to cause the activation
of latent TGF-.beta. in the extracellular space at the site of a
wound or a fibrotic condition. This led them to realise that it is
possible to use convertase inhibitors applied topically to prevent
the activation of latent TGF-.beta. associated with wound healing
or fibrosis. The prior art suggests that the activity of
convertases such as furin takes place in the megakaryocytes in the
bone marrow that give rise to platelets, and that this activity is
limited to the processing that produces latent TGF-.beta..
Accordingly the prior art contains no teaching that suggests that
local inhibition of furin activity would inhibit the conversion of
latent TGF-.beta. to active TGF-.beta.. Furthermore the prior art
suggest that therapeutic manipulation of furin would require an
agent that may be delivered to, and achieve its action in, the bone
marrow.
[0060] The surprising finding that extracellular convertase
activity contributes to TGF-.beta. activation led the inventors to
realise that water-soluble convertase inhibitors may be used to
decrease TGF-.beta. activation, and thus reduce scarring. The use
of water-soluble inhibitors, which cannot penetrate the cell
membrane, is particularly advantageous since such inhibitors
generally exhibit low levels of cytotoxicity. Water-soluble
convertase inhibitors can also be readily formulated into
compositions that induce minimal inflammatory reactions, an
important consideration when designing anti-scarring agents. A
preferred water-soluble convertase inhibitor suitable for use
according to the invention is L-hexaarginine.
[0061] The effects of localised inhibition of convertase activity
are very different from those that would arise as a result of
systemic administration of convertase inhibitors. Even were a
skilled person to suggest that systemic use of convertase
inhibitors would indirectly reduce levels of active TGF-.beta. by
inhibiting processing of pro-TGF-.beta. in the bone marrow they
would also understand that such an approach would have a number of
deleterious effects. One such effect would be that systemic
administration of convertase inhibitors would also reduce the level
of anti-fibrotic TGF-.beta..sub.3 that is important in the later
stages of wound healing. A second problem is that systemic use of
convertase inhibitors would have detrimental, and possibly toxic,
effects since convertases are involved in the normal processing of
proteins other than TGF-.beta..
[0062] Neither of these disadvantages is applicable to the topical
application of convertase inhibitors according to the invention. In
this use the effect of the inhibitors is directed to the region of
the wound, or site of fibrosis, and the administration of the
inhibitors may be timed so that only the initial quantities of
TGF-.beta. released from platelets are affected.
[0063] As set out above, the inventors believe that the convertase
activity contributing to TGF-.beta. activation is initiated within
the platelets, and that the extracellular convertase activity
occurs as a result of convertases being released into the
extracellular space on de-granulation of the platelets. As a
result, inhibitors of convertase activity for use according to the
invention may be inhibitors that are able to cross the cell
membrane and act intracellularly. Such inhibitors are able to
reduce convertase activity prior to de-granulation. They may be
able to decrease TGF-.beta. activation occurring both
intracellularly and extracellularly. That said, it appears that the
majority of convertase activity contributing to TGF-.beta.
activation occurs extracellularly after platelet degranulation.
Thus water-soluble inhibitors may be used to effectively reduce
TGF-.beta. activation and thus reduce scarring and/or fibrosis.
[0064] In a preferred embodiment of the invention the medicament
containing the convertase inhibitor may be applied
prophylactically. Thus the medicament may be applied to a site
where a wound may be formed or fibrosis may occur (e.g. before
elective surgery).
[0065] The inventors find that the inhibitors according to the
present invention are highly suited for topical application to
dermal wounds or dermal fibrotic conditions.
[0066] Convertase inhibitors used according to the invention may be
proteins or have peptidyl components. Such proteins can easily be
modified (for instance by amino acid addition, substitution or
deletion) to form derivatives that retain the ability to inhibit
enzymes such as furin. Therefore derivatives that retain functional
characteristics of naturally occurring proteins are also preferred
inhibitors of the invention. Examples of such derivatives include
functionally active fragments of naturally occurring proteins and
even precursors of naturally occurring proteins (e.g. proproteins)
which are activated in situ.
[0067] Convertase inhibitors may be used according to the invention
in situations or conditions where scarring needs to be prevented or
reduced such as:
[0068] (i) where scars of the skin may be excessive and/or
detrimental to tissue function and particularly when scar
contracture occurs or may occur (for instance skin burns and wounds
which impair flexibility of a joint and particularly scarring in
children);
[0069] (ii) scarring to the skin when cosmetic considerations are
important;
[0070] (iii) when hypertrophic or keloid scars (particularly in
Afro-Caribbean and Mongoloid races) may occur which can cause
functional and cosmetic impairment;
[0071] (iv) scarring resulting from skin grafts in both donor sites
and from the application of artificial skin;
[0072] (v) scarring within the central nervous system (e.g.
following neuro-surgery or penetrating injuries of the brain), for
example glial scarring can prevent reconnection of severed
neurons;
[0073] (vi) scarring in the eye and particularly of the cornea
(scarring can result in abnormal opacity and lead to problems with
vision or even blindness), in the retina (scarring can cause
buckling or retinal detachment and consequently blindness) and
scarring following wound healing in operations to relieve pressure
in glaucoma (e.g. glaucoma filtration surgery) which can result in
the failure of the surgery whereby the aqueous humour fails to
drain and hence the glaucoma returns;
[0074] (vii) scarring in the heart (e.g. following surgery or
myocardial infarction) which can give rise to abnormal cardiac
function;
[0075] (viii) scarring of the gut such as may occur following
operations involving the abdomen or pelvis that result in adhesion
between viscera (adhesions between elements of the gut and the body
wall can form and cause twisting in the bowel loop leading to
ischaemia, gangrene and the necessity for emergency
treatment--untreated they may even be fatal); likewise, trauma or
incisions to the guts can lead to scarring and scar contracture or
strictures which cause occlusion of the lumen of the guts which
again can be life threatening;
[0076] (ix) scarring in the pelvis in the region of the fallopian
tubes which can lead to infertility;
[0077] (x) scarring following injury to muscles which can result in
abnormal contraction and hence poor muscular function;
[0078] (xi) scarring or fibrosis following injury to tendons and
ligaments which can result in serious loss of function.
[0079] The convertase inhibitors may also be used for the treatment
or prevention of fibrosis. For instance the compounds may be used
to treat fibrotic disorders such as cirrhosis of the liver, liver
fibrosis, glomerulonephritis, pulmonary fibrosis, scleroderma,
myocardial hibernation, fibrosis following myocardial infarction,
central nervous system fibrosis following a stroke or
neuro-degenerative disorders (e.g. Alzheimer's Disease),
proliferative vitreoretinopathy (PVR), restenosis and
arthritis.
[0080] The convertase inhibitors are useful for reducing or
preventing fibrosis in fibrotic diseases and for reducing or
preventing the formation of fibrosis that manifests as hypertrophic
scarring (particularly of the skin) or keloids.
[0081] Wound healing compositions used according to the invention
may take a number of different forms depending, in particular on
the manner in which they are to be used. Thus, for example, they
may be in the form of a liquid, ointment, cream, gel, hydrogel,
powder or aerosol. All of such compositions are suitable for
topical application to skin, which is a preferred means of
administering convertase inhibitors to a subject (person or animal)
in need of treatment.
[0082] The convertase inhibitors may be provided on a sterile
dressing or patch which may be used to cover or even pack a wound
to be treated.
[0083] A preferred composition of the invention may be in the form
of an injectable solution (e.g. for intradermal injection around
the margins of a wound or a site to be wounded).
[0084] It will be appreciated that the vehicle of the composition
of the invention should be one which is well tolerated by the
patient and allows release of the active convertase inhibitor to
the wound. Such a vehicle is preferably biodegradeable,
bioresolveable and/or non-inflammatory.
[0085] The composition of the invention may be used in a number of
ways. Thus, for example, a composition may be applied in, and/or
around a wound of a patient to regulate wound healing. If the
composition is to be applied to an "existing" wound, then the
pharmaceutically acceptable vehicle will be one which is relatively
"mild" i.e. a vehicle which is biocompatible, biodegradable,
bioresolvable and non-inflammatory.
[0086] It is also possible to use compositions in accordance with
the invention prior to surgery (particularly elective surgery) so
as to provide for regulation of healing of the subsequently formed
surgical wound. In this case the vehicle of the topically applied
composition may need to be one capable of going across the
keratinous layer of the skin. Examples of suitable vehicles for
this purpose include dimethyl sulphoxide and acetic acid. Such
prophylactic use is a preferred use of convertase inhibitors
according to the invention.
[0087] The compositions are suitable to be used for reducing or
controlling scarring resulting form surgical operations on the eye
(e.g. laser surgery on the cornea). In this case the composition or
medicament may be in the form of an eye drop.
[0088] Convertase inhibitors may be used in a range of internal
wound healing applications. Thus for example, the composition may
be formulated for inhalation for use in wound healing of the lungs
or for the prevention or treatment of fibrosis and strictures in
the lung.
[0089] It will be appreciated that the amount of a convertase
inhibitor to be applied to the wound site depends on a number of
factors such as the biological activity and bioavailability of the
compound, which in turn depends on the mode of administration and
the physicochemical properties of the inhibitor. Other factors may
include:
[0090] A) The half-life of the inhibitor in the subject being
treated.
[0091] B) The specific condition to be treated.
[0092] C) The age of the subject.
[0093] The frequency of administration will also be influenced by
the above mentioned factors and particularly the half-life of the
convertase inhibitor within the subject being treated.
[0094] Generally when the compositions are used to treat existing
wounds or fibrotic disorders the convertase inhibitor should be
administered as soon as the wound has occurred or the disorder has
been diagnosed. Therapy with the composition should continue until
the wound has healed to a clinician's satisfaction or, in the case
of a fibrotic disorder, the risk or cause of abnormal fibrous
tissue formation has been removed.
[0095] Compositions which modulate scarring and/or fibrotic
disorders should also be applied to a wound as soon as possible
after the wound has formed. However scars and fibrosis can develop
over days or even weeks. Therefore the subject being treated may
well benefit by administration of a convertase inhibitor even if it
is administered days or even weeks after the wound occurred or the
disorder developed (or was diagnosed).
[0096] When used as a prophylactic (e.g. before surgery or when
there is a risk of developing a fibrotic disorder) the convertase
inhibitors should be administered as soon as the risk of
undesirable fibrosis has been recognised. For instance, a cream or
ointment containing a convertase inhibitor may be applied to a site
on the skin of a subject where elective surgery is to be performed
and decreased scar formation is subsequently desired. In this case,
the composition may be applied during the preoperative preparation
of the subject or it may even be desirable to apply the composition
in the hours or days preceding the surgery (depending upon the
health status and age of subject as well as the size of the wound
to be formed).
[0097] Frequency of administration will depend upon the biological
half-life of the inhibitor used. Typically a cream or ointment
containing a convertase inhibitor should be administered to a
target tissue such that the concentration of the inhibitor at the
wound site or tissue affected by a fibrotic condition is maintained
at a level suitable for having a therapeutic effect. This may
require administration daily or even several times daily.
[0098] Known procedures, such as those conventionally employed by
the pharmaceutical industry (e.g. in vivo experimentation, clinical
trials etc), may be used to establish specific formulations of
compositions and precise therapeutic regimes (such as daily doses
of the convertase inhibitor and the frequency of
administration).
[0099] Generally, compositions for use in accordance with the
invention should be formulated such that when administered to a
wound site or site of a fibrotic disorder that a convertase
inhibitor concentration of between 0.01 .mu.M and 10 mM is achieved
at the site.
[0100] Purely by way of example an injectable solution containing
between 0.1 .mu.M and 10 mM of decanoyl-RVKR-cmk is suitable for
application to an existing (i.e. "open") wound.
[0101] A suitable daily dose of a compound which inhibits
convertase activity depends upon the factors discussed above as
well as upon the size of the wound, or amount of tissue effected by
fibrosis, which is to be treated. Typically the amount of a
convertase inhibitor required for the treatment of wounds or
fibrotic disorders will be within the range of 0.01 .mu.g to 100 mg
of the active compound/24 hours depending upon the size of the
wound or extent of fibrosis amongst several other factors.
[0102] It will also be appreciated that convertase inhibitors may
be isolated from nature or chemically synthesised.
[0103] Many known methods of administering convertase inhibitors to
a relevant tissue have the disadvantage that it can be difficult to
achieve sustained levels of the active convertase inhibitor at a
wound site or site of fibrosis over the course of even a few days
because convertase inhibitors may have short half-lives in vivo.
The half-lives of the convertase inhibitors may be short for a
number of reasons which include:
[0104] (i) Degradation by proteases and the like.
[0105] (ii) Clearance by binding proteins (e.g. .alpha.2
macroglobulin).
[0106] (iii) Binding and inhibition of agent activity by
extracellular matrix molecules such as decorin and fibronectin.
[0107] Furthermore, compounds for wound healing and/or treatment of
scarring/fibrosis need to be administered in a suitable vehicle and
are often provided as a composition comprising the compound and the
vehicle. As outlined above, such vehicles are preferably
non-inflammatory, biocompatible, bioresorbable and must not degrade
or inactivate the active compound (in storage or in use). However,
it can often be difficult to provide a satisfactory vehicle for
delivering specific compounds to a tissue to be treated.
[0108] A convenient way in which these problems can be obviated or
mitigated is to provide at a wound site (or site of fibrosis) a
therapeutically effective amount of protein or peptide convertase
inhibitor by gene therapy.
[0109] According to a fourth aspect of the present invention there
is provided a delivery system for use in a gene therapy technique,
said delivery system comprising a DNA molecule encoding for a
protein which inhibits convertase activity, said DNA molecule being
capable of being transcribed to lead to the expression of said
protein.
[0110] According to a fifth aspect of the present invention there
is provided the use of a delivery system as defined in the
preceding paragraph for use in the manufacture of a medicament for
use in the treatment of wounds or fibrosis.
[0111] According to a sixth aspect of the present invention there
is provided a method of treating a wounds or fibrosis comprising
administering to a patient in need of treatment a therapeutically
effective amount of a delivery system as defined for the fifth
aspect of the invention.
[0112] The delivery systems according to the invention are highly
suitable for achieving sustained levels of a convertase inhibitor
at a wound site or site of fibrosis over a longer period of time
than is possible for most conventional delivery systems. Protein
may be continuously expressed from cells at the wound site or site
of fibrosis that have been transformed with the DNA molecule of the
invention. Therefore, even if the protein has a very short
half-life as an agent in vivo, therapeutically effective amounts of
the protein may be continuously expressed from the treated
tissue.
[0113] Furthermore, the delivery system of the invention may be
used to provide the DNA molecule (and thereby the protein which is
an active therapeutic agent) without the need to use conventional
pharmaceutical vehicles such as those required in ointments or
creams that are contacted with the wound.
[0114] The delivery system of the present invention is such that
the DNA molecule is capable of being expressed (when the delivery
system is administered to a patient) to produce a protein which
directly or indirectly has activity for wound healing and/or
treatment of fibrosis or scarring by inhibiting convertase
activity. By "directly" we mean that the product of gene expression
per se has the required activity for wound healing and/or
regulating fibrosis or scarring. By "indirectly" we mean that the
product of gene expression undergoes or mediates (e.g. as an
enzyme) at least one further reaction to provide an agent effective
for wound healing and/or regulating fibrosis or scarring by
inhibiting convertase activity.
[0115] The DNA molecule may be contained within a suitable vector
to form a recombinant vector. The vector may for example be a
plasmid, cosmid or phage. Such recombinant vectors are highly
useful in the delivery systems of the invention for transforming
cells with the DNA molecule.
[0116] Recombinant vectors may also include other functional
elements. For instance, recombinant vectors may be designed such
that the vector will autonomously replicate in the nucleus of the
cell. In this case, elements which induce DNA replication may be
required in the recombinant vector. Alternatively the recombinant
vector may be designed such that the vector and recombinant DNA
molecule integrates into the genome of a cell. In this case DNA
sequences which favour targeted integration (e.g. by homologous
recombination) are desirable. Recombinant vectors may also have DNA
coding for genes that may be used as selectable markers in the
cloning process.
[0117] The recombinant vector may also further comprise a promoter
or regulator to control expression of the gene as required.
[0118] The DNA molecule may (but not necessarily) be one which
becomes incorporated in the DNA of cells of the subject being
treated. Undifferentiated cells may be stably transformed leading
to the production of genetically modified daughter cells. When this
is the case, regulation of expression in the subject may be
required e.g. with specific transcription factors, gene activators
or more preferably with inducible promoters which transcribe the
gene in response to a signal specifically found at a wound site.
Alternatively, the delivery system may be designed to favour
unstable or transient transformation of differentiated cells in the
subject being treated. In this instance, regulation of expression
may be less important because expression of the DNA molecule will
stop when the transformed cells die or stop expressing the protein
(ideally when the wound, fibrosis or scarring has been treated or
prevented).
[0119] The delivery system may provide the DNA molecule to the
subject without it being incorporated in a vector. For instance,
the DNA molecule may be incorporated within a liposome or virus
particle. Alternatively the "naked" DNA molecule may be inserted
into a subject's cells by a suitable means e.g. direct endocytotic
uptake.
[0120] The DNA molecule may be transferred to the cells of a
subject to be treated by transfection, infection, microinjection,
cell fusion, protoplast fusion or ballistic bombardment. For
example, transfer may be by ballistic transfection with coated gold
particles, liposomes containing the DNA molecule, viral vectors
(e.g. adenovirus) and means of providing direct DNA uptake (e.g.
endocytosis) by application of plasmid DNA directly to the wounded
area topically or by injection.
[0121] The protein expressed from the DNA molecule may be one which
directly or indirectly provides for wound healing with reduced
scarring or one which serves to regulate (inhibit, prevent or
reverse) fibrosis.
[0122] It will be appreciated that the delivery system according to
the fifth aspect of the invention may be used according to the
sixth or seventh aspects of the invention to treat any of the
conditions hereinbefore described.
[0123] The present invention will further be described in the
following non-limiting Example which refers to the accompanying
drawing, in which:
[0124] FIG. 1 illustrates the results of analysis of the ability of
different inhibitors, and putative inhibitors, of TGF-.beta.
activation to prevent TGF-.beta. activation by platelets;
[0125] FIG. 2 illustrates the effect of Dec-RVKR-cmk and
hexaarginine in: A platelet releasates; and B platelet-free
releasates as referred to in experimental results section 2 of the
example. In A .box-solid. indicates active hexaarginine and
.quadrature. indicates total hexaarginine whereas indicates active
dec-RVKR-cmk and .largecircle. indicates total dec-RVKR-cmk. In
panel B .largecircle. indicates hexaarginine whereas indicates
dec-RVKR-cmk; and
[0126] FIG. 3 illustrates the effect of furin inhibitors on furin
activity in cell lysates and releasates for control samples
(.box-solid.); dec-RVKR-cmk (); and hexaarginine (.quadrature.) in
experimental results section 2 of the example.
EXAMPLE
[0127] The ability of inhibitors of furin activity to inhibit
activated platelets' production of active TGF-.beta. was
demonstrated by the ability of the inhibitors to abrogate the
platelet's release and activation of TGF-.beta. in response to
stimulation with thrombin. The experimental protocol was as set out
below.
1. Protocols.
[0128] Collection and Preparation of Human Platelets:
[0129] Peripheral venous blood samples from healthy adult
volunteers (aged 21 to 45) were taken into 20-gauge S-Monovettes.
EDTA was used to prevent coagulation of the samples. Human
platelets were isolated by differential centrifugation according to
the following protocol:
[0130] Blood samples were centrifuged at 300 g for 10 minutes to
produce a supernatant of platelet rich plasma. Platelet rich plasma
was then centrifuged at 2,500 g for 15 minutes to produce a pellet
of platelets. Platelets were harvested from the pellet and
resuspended prior to use.
[0131] Production of Platelet Hypotonic Lysates:
[0132] Platelets collected as described above were resuspended in
distilled water and incubated at room temperature for 5 minutes.
Lysis was stopped by addition of an equal volume of 2.times.
serum-free DME medium containing 0.2% pyrogen poor bovine serum
albumen. The lysate was cleared by centrifugation at 12,000 g for
10 minutes at room temperature before use.
2. Experimental Results 1.
[0133] 2.1. Platelets Activated with Thrombin Release and Activate
TGF-.beta.:
[0134] Human platelets collected as outlined above were activated
by the addition of thrombin (0.1 u/ml for 30 minutes at 37.degree.
C.). This activation caused the release of large amounts of
TGF-.beta. from the platelets (total TGF-.beta. 60.2 ng/ml
average), and also triggered the generation of significant amounts
of active TGF-.beta. (159 pg/ml on average if the platelets were
static during activation, 896 pg/ml on average if the platelets
were subjected to agitation during activation) in the platelet
releasates as assayed using the PAI/L bioassay for TGF-.beta.
described by Abe et al. ("An assay for transforming growth
factor-.beta. using cells transfected with a plasminogen activator
inhibitor-1 promoter-luciferase construct" 1994, Anal. Biochem.
216: 276-284). Pan-specific neutralising anti-TGF-.beta. antibodies
completely abolished the signal obtained in the PAI/L assay
verifying that active TGF-.beta. was measured. Activation of latent
TGF-.beta. was platelet-mediated, since exogenous thrombin added to
platelet releasates was unable to activate latent TGF-.beta.
directly (data not shown). Antibody inhibition experiments
confirmed that platelets expressed exclusively TGF-.beta..sub.1
(data not shown).
2.2. Platelets Contain Intracellular Active TGF-.beta.:
[0135] The presence of active TGF-.beta. within platelets was
confirmed by both immuno-localisation and bioassay studies.
[0136] Confocal microscope immuno-fluorescence studies of the
localisation of active TGF-.beta. within platelets were carried out
using the chicken derived active TGF-.beta.1-specific IgY AF-101-NA
(R&D Systems) and an antibody specifically reactive with the
membrane marker CD41 (BD Biosciences). Subcellular localisation of
these proteins was investigated in optical sections collected at
0.5 .mu.m intervals along the z-axis. TGF-.beta.1 was demonstrated
to be present, and to have an intracellular localisation (lying
within that of the membrane marker CD41).
[0137] The PAI/L bioassay for TGF-.beta. was used to validate the
presence of active TGF-.beta. in platelets through assaying for the
presence of active TGF-.beta. in hypotonic lysates of human
platelets (prepared as described above).
[0138] In lysates derived from resting platelets 99% of the total
TGF-.beta. present (total TGF-.beta. 34.7 ng/ml) was the latent
form, although a significant proportion of active TGF-.beta. (104
pg/ml) was detected. In lysates derived from thrombin activated
platelets the mean total TGF-.beta. was 19.5 ng/ml, of which 151
pg/ml was active.
[0139] The results of the bioassay thereby confirmed the
immuno-localisation finding that platelets contain active
TGF-.beta..
2.3. Inhibition of Furin Activity Inhibits TGF-.beta. Activation by
Thrombin Activated Platelets:
[0140] In a comparative experiment the ability of inhibitors of
known activators of TGF-.beta. (such as TSP-1, plasmin, M6P/IGF-II
receptor) and putative activators of latent TGF-.beta. such as
other serine proteinases, cysteine proteinases, calpain I and II,
caspase-3, and furin) to block latent TGF-.beta. activation in
human platelets was assessed.
[0141] Human platelets were pre-incubated with the inhibitors
(listed below) prior to stimulation with thrombin (as above), and
active and total TGF-.beta. levels were determined in the platelet
releasates using the PAI/L assay (as above). The results are shown
in FIG. 1, panels A to C.
[0142] The results showed that latent TGF-.beta. activation was not
significantly affected by the presence of inhibitors specific for
TSP-1 (LSKL peptide), plasmin (neutralising monoclonal antibody,
PG19--a neutralising antibody provided by Dr. Michael Kramer), or
M6P/IGF-II receptor (M6P--mannose-6-phosphate) (panel A). Moreover,
a number of inhibitors (aprotinin, pefabloc, .alpha. 1-antitrypsin,
E-64, pepstatin, leupeptin, caspase-3 inhibitor and calpain
inhibitor) of other proteinases potentially involved in
platelet-mediated latent TGF-.beta. activation also proved to be
ineffective (panel B).
[0143] In comparison platelet incubation with a membrane-permeable
inhibitor of furin-like proprotein convertases, dec-RVKR-cmk
(decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone--Bachem), drastically
reduced the generation of active TGF-.beta. in releasates as well
as intracellularly in a dose-dependent fashion (panel C).
(intracellular measurements were taken from hypotonic lysates). The
inventors believe that the residual TGF-.beta. present
(approximately 20-30%) was activated prematurely during platelet
preparation prior to the addition of the inhibitor.
2.4. Summary.
[0144] The above results indicate that platelet-mediated latent
TGF-.beta. activation surprisingly occurs extracellularly at the
site of platelet activation (i.e. a wound site or a site of a
fibrotic condition) and involves proteolytic processing by a
furin-like convertase enzyme. They further show that the activation
of latent TGF-.beta. by platelets can be successfully inhibited by
treatment of the platelets with an inhibitor of furin activity
which may be applied topically.
[0145] As skilled person will appreciate from these results that
convertase inhibitors may be used to inhibit TGF-.beta..sub.1
activation and will thereby be effective as anti-scarring or
anti-fibrotic agents.
3. Experimental Results 2.
3.1. Furin-Like Enzymes are Involved in Platelet-Mediated Latent
TGF-activation.
[0146] Platelets were activated with thrombin in the absence or
presence of furin inhibitors. Platelets were pre-incubated with
hexaarginine, whereas dec-RVKR-cmk was added 5 min after thrombin
addition because of interference with platelet activation at higher
concentrations. Active and total TGF-.beta. in releasates were
determined in the PAI/L bioassay. The results are shown in panel A
of FIG. 2. Active TGF-.beta. levels in the controls were 82 pg/ml
(dec-RVKR-cmk data) and 61 pg/ml (hexaarginine data), total
TGF-.beta. levels were 33.4 ng/ml (dec-RVKR-cmk data) and 39.5
ng/ml (hexaarginine data).
[0147] In a further experiment, furin inhibitors were added to
platelet-free releasates of activated platelets, and activation was
allowed to continue in the absence of platelets for 30 min at
37.degree. C. The results of this experiment are shown in panel B
of FIG. 2. Active TGF-.beta. levels in the controls were 77 pg/ml
(dec-RVKR-cmk data) and 104 pg/ml (hexaarginine data). Incubation
on ice reduced activation in the controls to approximately 56%
(data not shown). Data represent the mean values of three
independent experiments assayed in triplicate.
[0148] The results illustrate that incubation of
thrombin-stimulated platelets with the membrane-permeable protease
inhibitor, dec-RVKR-cmk, considerably reduces the generation of
active TGF-.beta. in releasates (FIG. 2 panel A). Dec-RVKR-cmk is a
specific and potent inhibitor of subtilisin/Kex2p-like proprotein
convertases, with its peptide sequence being based on the substrate
recognition sequence of these enzymes.
[0149] The most prominent and ubiquitously expressed member of this
endoprotease family is furin, which typically cleaves at the
consensus sequence motif R-X-(K/R)-R. The membrane-impermeable
furin inhibitor, hexa-L-arginine, also significantly reduced active
TGF-.beta. in releasates (FIG. 2 panel A) indicating that at least
part of the activation occurred extracellularly following latent
TGF-.beta. release.
[0150] Latent TGF-.beta. activation appeared to be enzymatic and
independent of the continuous presence of platelets, since
incubation of platelet-free releasates on ice (as compared to
37.degree. C.) reduced active TGF-.beta. levels to approximately
56%. As observed for platelet suspensions, activation in releasates
was inhibited, in a dose-dependent fashion, by the furin
inhibitors, dec-RVKR-cmk and hexa-L-arginine (FIG. 2B). This
indicates that the furin-like enzyme involved in latent TGF-.beta.
activation is released from activated platelets.
3.2. Platelets Contain and Release Furin-Like Enzyme Activity.
[0151] Releasates or hypotonic lysates of activated platelets were
assayed using the furin substrate, pyr-RTKR-amc in the absence or
presence of the furin inhibitors, hexaarginine (200 .mu.M) or
dec-RVKR-cmk (150 .mu.M). Values were corrected for
substrate-independent endogenous fluorescence (control without
substrate) as well as for spontaneous substrate hydrolysis (buffer
control). Mean values.+-.S.E.M. of 2-3 separate experiments assayed
in duplicate are shown.
[0152] The presence of furin-like enzyme activity in both hypotonic
lysates and releasates of human platelets was analysed using the
fluorogenic furin substrate, pyr-RTKR-amc. Platelet lysates
contained a furin-like enzyme activity, part of which
(approximately 12%) was released upon thrombin stimulation. Enzyme
activity in cell lysates and releasates was inhibited by
dec-RVKR-cmk and hexa-L-arginine (FIG. 3).
[0153] The extracellular generation of active TGF-.beta. by
thrombin-stimulated human platelets was significantly reduced in
the presence of inhibitors of furin-like proprotein
convertases.
3.3 Summary.
[0154] Furin-like proprotein convertases catalyze the maturation of
pro-TGF-.beta. precursor to heat-activatable latent growth factor
complex. Our data indicate, however, that platelet .beta.-granules
contain and release mature, heat-activatable latent TGF-.beta., and
that the levels are not affected by furin inhibitors. Thus,
pro-TGF-.beta. processing in the megakaryocytic lineage occurs in
the megakaryocytes. These data therefore identify a novel function
of furin-like enzymes, namely involvement in the extracellular
activation of platelet large latent TGF-.beta..sub.1 complex under
physiological conditions.
[0155] In summary, the inventors found that platelets are not only
major storage sites for latent TGF-.beta. but also activate part of
it following degranulation. While the mechanism of activation does
not require any of the well-characterized activators, TSP-1,
M6P/IGF-II receptor, or plasmin, the platelet latent TGF-.beta.
complex appears to be activated via a sequence of events by a
furin-like convertase released by the platelets. Following release
in vivo, this enzyme appears to continue to operate, independently
of the presence of platelets, in the surrounding tissue (e.g. the
wound area), leading to the activation of extracellular-matrix
associated latent TGF-.beta. complex. Therefore, this novel
mechanism of activation represents a target to modulate TGF-.beta.
activity in pathologic conditions involving platelet degranulation,
such as wound repair, fibrosis, arteriosclerosis, and cancer.
Therefore the inventors have found that inhibitors according to the
invention (such as decanoyl-RVKR-cmk and hexa-arginine may be used
according to the invention).
Sequence CWU 1
1
714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Val Lys Arg124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Arg
Val Pro Arg134PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Ala Val Arg Arg144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Arg
Val Arg Arg156PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Arg Arg Arg Arg Arg Arg1
564PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Leu Ser Lys Leu174PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Arg
Thr Lys Arg1
* * * * *