Wound healing and treatment of fibrosis
United States Patent: 7,566,446
Issued: July 28, 2009
Inventors: Ferguson; Mark
W. J. (Derbyshire, GB), O'Kane; Sharon (Derbyshire, GB)
Assignee: Renovo Limited
Appl. No.: 11/540,956
Filed: October 2, 2006
Covidien Pharmaceuticals Outsourcing
Compositions for use in the treatment of
wounds and fibrosis comprising a therapeutically effective amount of a
compound that modulates actin assembly and organization intracellularly
and/or extracellularly and a pharmaceutically acceptable vehicle; such a
compound promotes the rate of wound healing and also prevents or reduces
fibrosis. A preferred compound is gelsolin.
Description of the
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 derangements and malfunctioning of tissue.
Wound healing in adults is a complicated reparative process. The healing
process begins with the recruitment of a variety of specialised cells to
the site of the wound and involves, 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 which develops to repair the wound area.
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).
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 in 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 are a more severe form of normal scarring, are elevated
above the normal surface of the skin and contain excessive collagen
arranged in an abnormal pattern. A keloid is another form of pathological
scarring which 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 which is organised in an abnormal fashion
predominately in whirls of collagenous tissue. There are genetic
predispositions to forming both hypertrophic scars and keloids. They are
particularly common in Africo-Carribean and Mongoloid races.
There is a need to provide medicaments that promote the healing of wounds.
For example, it is often desirable to increase the rate of healing in the
case of acute wounds (such as penetrative injuries, burns, nerve damage or
even wounds resulting from elective surgery). chronic wounds (such as
diabetic, venous and decubitus ulceration) or for generally healing
compromised individuals (for example the elderly). In these examples, the
wounds can severely influence quality of life or even result in death and
therefore the rate of healing often needs to be increased as much as is
clinically possible. Where the rate of wound healing is increased, there
is often an associated increase in scar formation hut this may be of
secondary importance compared to the desired increase in the rate of
The term "wound" as used herein is exemplified 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
There are however other instances where the regulation of scar formation
is of primary importance and the rate of wound healing is only of
secondary consideration. Examples of such situations are scars of the skin
where excessive scarring may be detrimental to tissue function and
particularly when scar contracture occurs (for instance skin burns and
wounds which 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 (particularly in
Africo-Caribbean and Mongoloid races) 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
As well as scars of the skin, internal scarring or fibrosis can be highly
detrimental and specific examples include:
(i) Within the central nervous system, glial scarring can prevent neuronal
reconnection (e.g. following neuro-surgery or penetrating injuries of the
(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.
(iii) Scarring in the heart (e.g. following surgery or myocardial
infarction) can give rise to abnormal cardiac function.
(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 structures which cause occlusion
of the lumen of the guts which again can be life threatening.
(v) Scarring in the pelvis in the region of the fallopian tubes can lead
(vi) Scarring following injury to muscles can result in abnormal
contraction and hence poor muscular function.
(vii) Scarring or fibrosis following injury to tendons and ligaments can
result in serious loss of function.
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 or 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) 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.
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.
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 are intimately involved in the repair of tissues.
WO-A-92/17206 discloses the use of neutralising agents for fibrosis
promoting growth factors that may be used to inhibit scar formation during
wound healing. For instance, WO-A-92/17206 demonstrates that compositions
which specifically inhibit the activity of Transforming Growth Factors
.beta.1 and .beta.2 and Platelet Derived Growth Factor are particularly
beneficial for reducing scar formation.
WO-A-93/19769 discloses the use of non-fibrotic growth factors, such as
Transforming Growth Factors .beta.3 which was surprisingly found to
promote healing of a wound without inducing fibrosis.
GB-A-2,288,118 discloses the use of specific antibodies generated against
growth factors that improve healing by potentiating the actions of said
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.1 or
.beta.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.
Other compositions that influence growth factor efficacy and promote wound
healing are disclosed in WO-A-95/26203.
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.
As discussed more fully below, the invention relates in its broadest
aspect to the use of compounds that modulate actin assembly and
organisation for the treatment of wounds.
All eukaryotic cells, whether simple unicellular organisms or cells from
human tissue, contain actin. Actin may be found as filamentous actin (F
actin) which is a major protein component of the cytoskeleton of the cell.
The cytoskeleton influences many cellular functions and is particularly
relevant in the regulation of motility, shape changes, chemotaxis and
F actin is a polymer comprised of a tight helix of uniformly orientated
actin monomers. These monomers are expressed in the cell as single
polypeptide globular proteins known as globular actin (G actin).
Polymerisation of G actin monomers into F actin occurs within the
cytoplasm of the cell.
F actin is a dynamic structure and the size of the filament may be
regulated, according to the functional needs of the cell. For instance, by
altering the rate at which G actin monomers are added or removed from the
filament or by inhibiting or promoting F actin degradation. As well as
forming part of the cytoskeleton. F actin may also be organised into
cellular structures, such as thin filaments in muscle, each of which may
have a specific cellular function.
Many regulatory proteins influence F actin assembly and organisation. One
such class of these regulatory proteins are those which bind to actin
monomers, cap the fast growing end of actin filaments and sever actin
filaments. Examples of this class of regulatory proteins include gelsolin,
villin, CapG, adseverin, flightless-1 and advillin.
Gelsolin is an 82 kDA protein found naturally in cells such as platelets,
fibroblasts, neutrophils and macrophages and can shear in half a 1 .mu.m
actin filament in only one second. An academic paper by Witke et al. (Cell
vol. 81 p 41-51, 1995) has established using a mouse model which had been
engineered so as not to express gelsolin (also known as a Gsn mouse of
gelsolin knockout mouse) that the motility of neutrophils and fibroblasts
is impaired in vitro and in vivo in cells that do not express gelsolin.
This paper does not contemplate the use of gelsolin and similar compounds
for the treatment of wounds and surprisingly the inventors have
established that compounds which modulate actin assembly and organisation
are useful for wound healing.
According to a first aspect of the present invention there is provided the
use of a compound that modulates actin assembly and organisation
intracellularly and/or extracellularly for the manufacture of a medicament
for the treatment of wound or fibrosis.
According to a second aspect of the present invention, there is provided a
composition comprising a therapeutically effective amount of a compound
that modulates actin assembly and organisation intracellularly and/or
extracellularly and a pharmaceutically acceptable vehicle for the
treatment of wounds or fibrosis.
According to a third aspect of the present invention, there is provided a
method of treating wounds or fibrosis comprising applying to the affected
tissue a therapeutically effective amount of a compound which modulates
actin assembly and organisation intracellularly and/or extracellularly.
In accordance with the invention, the inventors have established that
compounds which modulate actin assembly and organisation may be used to
treat wounds to improve the quality of scar formed and also to improve the
rate of healing of wounds. The compounds may also be used to treat
conditions in which there is inappropriate fibrosis.
The compounds are effective for modulating assembly and organisation of
intracellular actin and/or extracellular located actin (which may be
released from dead or damaged cells for instance).
Compounds which modulate actin assembly and organisation may be used
according to the invention in situations or conditions where scarring
needs to be prevented or reduced such as:
(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);
(ii) scarring to the skin when cosmetic considerations are important;
(iii) when hypertrophic or keloid scars (particularly in Africo-Caribbean
and Mongoloid races) may occur which can cause functional and cosmetic
(iv) scarring resulting from skin grafts in both donor sites and from the
application of artificial skin;
(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;
(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;
(vii) scarring in the heart (e.g. following surgery or myocardial
infarction) which can give rise to abnormal cardiac function;
(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;
(ix) scarring in the pelvis in the region of the fallopian tubes which can
lead to infertility;
(x) scarring following injury to muscles which can result in abnormal
contraction and hence poor muscular function;
(xi) scarring or fibrosis following injury to tendons and ligaments which
can result in serious loss of function.
The compounds 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) and arthritis.
The compounds are useful for reducing or preventing fibrosis in fibrotic
diseases and for reducing or preventing the formation of fibrosis that
manifests as hypertrophic scarring or keloids (particularly of the skin).
Compounds which modulate actin assembly and organisation also increase the
rate of healing as well as improving scar quality or treating fibrosis. We
have found that these compounds are capable of accelerating the rate at
which a wound heals. Thus such compounds will be useful for acute wounds
(such as penetrative injuries, burns, nerve damage, damaged ligaments or
tendons, or even wounds resulting from elective surgery), chronic wounds
(such as diabetic, venous, decubitus ulceration and pressure sores) or for
generally healing compromised individuals (for example the elderly). The
compounds are particularly useful for treating wounds of the skin (i.e.
dermal wounds). The abovedescribed wounds can severely influence quality
of life or even result in death and therefore the rate of healing may need
to be increased as much as is clinically possible.
A most preferred compound for use according to the invention is gelsolin.
The inventors have performed studies which demonstrate that compounds
which modulate actin assembly and organisation influence wound healing.
For instance, they have found that wound healing is impaired and scar
quality reduced in a Gelsolin Knockout Mouse model. Furthermore addition
of compounds such as gelsolin from an exogenous source improves scar
quality and the rate of wound healing compared to healing observed for
Further to the Witke et al. paper the inventors have surprisingly found
that compounds which modulate actin assembly and organisation are
particularly effective for treating wounds to reduce scar formation and
also to increase the rate of wound healing. Although the applicants do not
wish to be constrained by any hypothesis they believe that the compounds,
such as the regulatory protein gelsolin, are effective for reducing
scarring because they have found that not only do these compounds modulate
the motility of cells but they also influence mobility of the cells within
a wound, effect secretion of components of the extracellular matrix and
thereby regulate fibrosis and scar formation. The inventors believe these
functions are regulated by remodelling F actin within and without these
cells. Furthermore the inventors believe that the compounds effect
fibroblast orientation within the wound. The orientation of the
fibroblasts also influences the nature, organisation and orientation of
the extracellular matrix deposited by the fibroblasts and therefore
influences the connective tissue scar which repairs the wounded area. The
inventors have found that collagen deposition within the matrix is
particularly influenced by the compounds. The inventors have also
demonstrated that soluble, extracellular gelsolin binds to and clears
extracellular actin released from degenerating cells at the wound site and
that this clearance of cellular debris may be an important mechanism in
accelerating the healing of chronic wounds. The inventors therefore
believe that the application to a wound of compounds used according to the
invention improves the final quality and appearance of the scar and
increases the rate of wound healing as described above.
Whilst gelsolin is the preferred compound other compounds such as villin,
CapG, adseverin, flightless-1 and advillin or derivatives thereof may also
be used according to the invention.
Compounds used according to the invention may be proteins. Such proteins
can easily be modified (for instance by amino acid addition, substitution
or deletion) to form derivatives which retain the ability to bind to actin
monomers, cap the fast growing end of actin filaments or sever actin
filaments. Therefore derivatives which retain functional characteristics
of naturally occurring proteins are also preferred compounds 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.
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 compounds of the invention to a subject
(person or animal) in need of treatment.
The composition may be provided on a sterile dressing or patch which may
be used to cover or even pack a wound to be treated. Alternatively the
composition of the invention may be an injectable solution or provided as
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 compound to the wound. Such a vehicle is preferably
biodegradeable, bioresolveable and/or non-inflammatory.
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.
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 a topically applied composition may need to be one
capable of going across the keratinous layer of the skin. Examples of
suitable vehicles for the purpose include dimethyl sulphoxide and acetic
acid. Such prophylactic use is a preferred use of compounds according to
The compositions are suitable to be used for accelerating healing and
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.
The compositions 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.
It will be appreciated that the amount of a compound that modulates actin
assembly and organisation 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 compound. Other factors include:
A) The half-life of the compound in the subject being treated.
B) The specific condition to be treated.
C) Whether quick healing or reduced scarring is desired.
D) The age of the subject.
The frequency of administration will also be influenced by the above
mentioned factors and particularly the half-life of the compound within
the subject being treated.
Generally when the compositions are used to treat existing wounds or
fibrotic disorders the compound 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 clinicians
satisfaction or, in the case of a fibrotic disorder, the risk or cause of
abnormal fibrous tissue formation has been removed.
Compositions for promoting the rate of wound healing should be applied to
a wound as soon as possible after the wound has formed. For acute wounds
and wounds of subjects who are healing competent (e.g. the young)
application of the composition will ideally be at the time of wounding,
preferably within hours of wounding and no longer than a few days
post-wounding. For chronic wounds or wounds in the healing compromised
(e.g. the elderly) administration should be as soon as possible.
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 compound
(such as gelsolin) even if it is administered days or even weeks after the
wound occurred or the disorder developed (or was diagnosed).
When used as a prophylactic (e.g. before surgery or when there is a risk
of developing a fibrotic disorder) the compositions should be administered
as soon as the risk of undesirable fibrosis or a potential for a poor rate
of wound healing has been recognised (as may be the case in elderly
subjects). For instance, a cream or ointment containing gelsolin may be
applied to a site on the skin of subject where elective surgery is to be
performed and an increased rate of wound healing 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
Frequency of administration will depend upon the biological half-life of
the compound used. Typically a cream or ointment containing a compound
should be administered to a target tissue such that the concentration of
the compound at the wound site or tissue affected by a fibrotic disorder
is maintained at a level suitable for having a therapeutic effect. This
may require administration daily or even several times daily.
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 compounds and the
frequency of administration).
Generally, compositions in accordance with the invention will contain
50-1000 nM of the compound (e.g. gelsolin), most preferably 100-500 nM.
Purely by way of example a composition containing 100-500 .mu.g/ml of
gelsolin is suitable for application to an existing (i.e. "open") wound.
By way of further example, a composition which is to be used
preoperatively to prevent scarring occurring or improve the rate of
healing of a wound resulting from subsequent surgery may contain 500-1000
nM of gelsolin to have the desired effect on wound healing.
A suitable daily dose of a compound which modulates actin assembly and
organisation depend 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 compound required for the treatment
of wounds or fibrotic disorders will be within the range of 1 ng to 100 g
of the active compound/24 hours depending upon the size of the wound or
extent of fibrosis amongst several other factors.
It will also be appreciated that compounds used according to the invention
may be isolated from nature or chemically synthesised. Such compounds need
not be proteins or even structurally similar to naturally occurring
proteins but nevertheless have the same effects on actin assembly and
organisation. These compounds are also preferred compounds for use
according to the invention.
Proteins and derivatives thereof which are used as compounds for
modulating actin assembly and organisation may be prepared by any
convenient method, including peptide ligation and complete protein
synthesis. Alternatively they may be purified from natural sources. For
instance gelsolin may be isolated from neutrophils, platelets or
fibroblasts expressing the protein (e.g. by the methods of Bryan J.
Methods in Enzymology vol 215 p 88-99, Academic Press, London 1992).
A preferred manner of preparing protein and derivatives thereof for use in
the invention is by expression from a recombinant DNA system.
Recombinant DNA technology provides a convenient means by which sufficient
quantities of such proteins and derivatives thereof may be formed for use
in the manufacture of medicaments in desirable quantities.
Many known methods of administering compounds to a relevant tissue have
the disadvantage that it can be difficult to achieve sustained levels of
the active compound at a wound site or site of fibrosis over the course of
even a few days because the active agents usually have very short
half-lives in vivo. The half-lives of the compounds tend to be short for a
number of reasons which include:
(i) Degradation by proteases and the like.
(ii) Clearance by binding proteins (e.g. .alpha.2 macroglobulin).
(iii) Binding and inhibition of agent activity by extracellular matrix
molecules such as decorin and fibronectin.
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.
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, wound healing amount of a compound that modulates actin
assembly and organisation by gene therapy.
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 modulates actin
assembly and organisation, said DNA molecule being capable of being
transcribed to lead to the expression of said protein.
According to a fifth aspect or 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
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 fourth aspect of the invention.
The delivery systems according to the invention are highly suitable for
achieving sustained levels of a compound which modulates actin assembly
and organisation 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.
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 conventional pharmaceutical vehicles such as
those required in ointments or creams that are contacted with the wound.
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 modulating
actin assembly and organisation. 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 modulating actin assembly and
It is preferred that the DNA molecule codes for gelsolin or a biologically
fragment or derivative thereof. The DNA molecule may also code for
compounds such as villin, CapG, adseverin, flightless-1 and advillin or
derivatives thereof. Alternatively the DNA molecule may code for a protein
which indirectly increases expression of compounds such as gelsolin. For
instance, the DNA molecule may code for an enzyme, transcription factor or
the like which promote expression of gelsolin.
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.
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.
The recombinant vector may also further comprise a promoter or regulator
to control expression of the gene as required.
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 inducable
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).
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.
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.
The protein expressed from the DNA molecule may be one which directly or
indirectly provides for wound healing reduced scarring, one which provides
an increase in the rate of wound healing whilst possibly resulting in
increased scar formation or one which serves to regulate (inhibit, prevent
or reverse) fibrosis.
Claim 1 of 5 Claims
1. A method of reducing scar formation in
incisional dermal wounds, the method comprising delivering directly to a
dermal wound in need of such treatment a therapeutically effect amount of
a regulatory protein that caps the fast growing end of actin filaments
and/or severs capped actin filaments so that said scar formation is
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