U.S. patent application number 10/117351 was filed with the patent office on 2002-12-12 for wound healing.
This patent application is currently assigned to RENOVO LIMITED. Invention is credited to Ferguson, Mark William James, Foreman, David Michael, Shah, Mamta.
Application Number | 20020187149 10/117351 |
Document ID | / |
Family ID | 10692386 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187149 |
Kind Code |
A1 |
Ferguson, Mark William James ;
et al. |
December 12, 2002 |
Wound healing
Abstract
A composition for use in the treatment of wounds to inhibit scar
tissue formation during healing is disclosed, comprising an
effective activity-inhibiting amount of a growth factor
neutralising agent or agents specific against only fibrotic growth
factors together with a pharmaceutically acceptable carrier. The
method of preparation of said composition and method of
administering the composition to a host suffering from tissue
wounding is also disclosed.
Inventors: |
Ferguson, Mark William James;
(Skuupl Evczauo, GB) ; Foreman, David Michael;
(Manchester, GB) ; Shah, Mamta; (Manchester,
GB) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201
US
|
Assignee: |
RENOVO LIMITED
|
Family ID: |
10692386 |
Appl. No.: |
10/117351 |
Filed: |
April 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10117351 |
Apr 8, 2002 |
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09448891 |
Nov 29, 1999 |
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09448891 |
Nov 29, 1999 |
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08838449 |
Apr 7, 1997 |
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08838449 |
Apr 7, 1997 |
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08122508 |
Sep 27, 1993 |
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5662904 |
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Current U.S.
Class: |
424/145.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 17/00 20180101; A61P 17/02 20180101; C07K 14/71 20130101; C07K
16/22 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/145.1 |
International
Class: |
A61K 039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 1991 |
GB |
9106678.7 |
Claims
1. A composition for use in the treatment of wounds to inhibit scar
tissue formation during healing, comprising an effective
activity-inhibiting amount of a growth factor neutralising agent or
agents specific against only fibrotic growth factors together with
a pharmaceutically acceptible carrier.
2. A composition according to claim 1, wherein the growth factor
neutralising agent is a growth factor neutralising antibody.
3. A composition according to claim 2, wherein the growth factor
neutralising antibody is selected from anti-TGF-.beta.1 antibody,
anti-TGF-.beta.2-antibody and anti-PDGF-antibody.
4. A composition according to claim 1, wherein the growth factor
neutralising agent is a growth factor receptor blocking agent.
5. A composition according to claim 4, wherein the growth factor
receptor blocking agent is a peptide containing the receptor
binding site of the growth factor.
6. A composition according to claim 5, wherein the peptide contains
the receptor binding site for TGF-.beta.1 or TGF-.beta.2 or
PDGF.
7. A composition according to claim 1, wherein the growth factor
neutralising agent is a molecule which binds to the growth factor
to inhibit receptor binding.
8. A composition according to claim 7, wherein the growth factor is
selected from TGF-.beta.1 and TGF-.beta.2 and the molecule is
selected from Decorin and Biglycan.
9. A composition according to claim 1, wherein the growth factor
neutralising agent is an antisense oligonucleotide to growth factor
mRNA.
10. A composition according to claim 1, wherein the growth factor
neutralising agent is a ribosyme(s) active against growth factor
mRNA.
11. A composition according to claim 1, wherein the growth factor
neutralising agent is a soluble form of the receptor or the growth
factor binding domain of the receptor.
12. A composition according to any preceding claims wherein the
growth factor neutralising agent is present in an active form.
13. A composition according to cliams 1 to 11, wherein the growth
factor neutralising agent is present in an inactive form.
14. A compostion according to claim 13, wherein the growth factor
neutralising agent is inactivated by being encapsulated.
15. A composition according to claim 14, wherein the capsules are
degradable by an external stimulus to release the active growth
factor neutralising agent when required.
16. A composition according to claim 15, wherein the external
stimulus includes UV light, in vivo enzymes, ultrasound or
heat.
17. A composition according to claim 13, wherein the growth factor
neutralising agent is inactivated by the molecular addition of a
binding molecule.
18. A composition according to claim 17, wherein the binding
molecule is detached from and releases active growth factor
neutralising agent by an external stimulus including UV light, in
vivo enzymes, ultrasound or heat.
19. A composition according to any preceding claim, wherein the
pharmaceutically acceptable carrier comprises a neutral sterile
cream, gel, aerosol or powder for topical application.
20. A composition according to calims 1 to 18, wherein the
phamaceutically acceptable carrier comprises a sterile solution for
injection, irrigation or inhalation.
21. A composition according to claims 1 to 18, wherein the
pharmaceutically acceptable carrier comprises a sterile dressing
for topically covering a wound.
22. A composition according to claim 21, wherein the dressing
comprises a biodegradable/absorbable polymer.
23. A composition according to claims 1 to 18, wherein the
pharmaceutically acceptable carrier comprises a bioploymer/polymer
for implanting within the wound.
24. A composition according to any preceding claim, comprising
active cytokines.
25. A method of preparation of a pharmaceutical composition
containing the growth factor neutralising agent or agents specific
against only fibrotic growth factors for applying the composition
topically in a cream, gel, aerosol, powder, dressing or patch or in
a solution for injection, irrigation or inhalation, or as a control
release implant.
26. A method of preparation according to claim 25, wherein the
composition comprises active cytokines.
27. A method of inhibiting scar tissue formation during the healing
of wounds, said method consisting in administering to a host
suffering from tissue wounding a growth factor neutralising agent
or agents specific against only fibrotic growth factors in the
wound area in a dosage effective to reduce activity of one or more
growth factors involved in the process that leads to the formation
of scar tissue during healing.
Description
[0001] This invention relates to the healing of wounds and to
agents and techniques for facilitating repair and healing of animal
tissue, especially, but not exclusively, skin or other epithelial
tissue, that has been damaged by, for example, wounds resulting
from accidental injury, surgical operations or other trauma. The
invention has particular reference to the healing of wounds in
humans and other vertebrates.
[0002] As is well known, the healing of wounds in tissue such as
skin generally involves, at least in adult humans and other
mammals, a process of extra-cellular matrix (ESC) biosynthesis,
turnover and organisation which commonly leads to the production of
fibrous, connective tissue scars and consequential loss of normal
tissue function.
[0003] In the realm of surgery scar tissue formation and
contraction is a major clinical problem for which there is no
entirely satisfactory solution at present. Likewise, scarring and
fibrosis following accidental burning or other injuries or trauma,
particularly in children, often has serious results, leading to
impaired function, defective future growth, and to unsightly
aesthetic effects, and again presents a major problem.
[0004] In regard to unsightly aesthetic effects produced by scars,
there also commonly arises a need for cosmetic treatment or
operations to attempt to remove these disfigurements in order to
improve appearance. Additionally, a similar need for cosmetic
treatment often arises in connection with unwanted tattoos and
other skin blemishes. At present, however, it is difficult or
impossible to carry out such cosmetic treatment or operations
satisfactorily since a certain amount of surgery is generally
involved which in itself is likely to result in wounds producing
fresh unsightly scar tissue.
[0005] In adult humans and other mammalian vertebrates, wound
healing in tissues such as skin is generally a reparative process,
in contrast to a regenerative process which appears to take place
in healing of fetal and embryonic tissue. The outcome of a wound
repair process appears to be influenced by a number of different
factors, including both intrinsic parameters, e.g. tissue
oxygenation, and extrinsic parameters, e.g. wound dressings. There
is, however, considerable evidence indicating that the overall
process of healing and repair of wound damaged tissue, including
the necessary intercellular communication, is regulated in a
coordinated manner in adult humans and other mammals by a number of
specific soluble growth factors which are released within the wound
environment (especially by degranulating platelets and incoming
macrophages) and which, amongst other things, appear to induce
neovascularisation, leucocyte chemotaxis, fibroblast proliferation,
migration and deposition of collagen and other extracellular matrix
molecules within the wounds. Such growth factors that have been
identified and isolated are generally specialised soluble proteins
or polypeptides and include transforming growth factor alpha
(TGF-.alpha.), transforming growth factor beta (TGF-.beta.1 ,
TGF-.beta.2, TGF-.beta.3 etc), platelet derived growth factor
(PDGF), epidermal growth factor (EGF), insulin-like growth factors
I and II (IGFI and IGFII) and acidic and basic fibroblast growth
factors (acidic FGF and basic FGF). Many of these growth factors
have already been made by genetic engineering using recombinant DNA
technology.
[0006] General reviews or these growth factors are to be found in
articles by Mary H McGrath in Clinics in Plastic Surgery, Vol. 17,
No. 3, July 1990, pp 421-432, and by George A Ksander in Annual
Resorts in Medicinal Chemistry, 1989, Chap, 24 (published by
Academic Press, Inc.) of which the contents are incorporated herein
by reference.
[0007] The recognition of the importance of the role of such growth
factors in the control of wound healing has led to numerous
proposals for their clinical use and application as exogenous
growth factor agents in treatment for acceleration and promotion of
healing of wounds, especially in cases of defective wound healing
states (see for example Sporn et al, Science (1983) 219, 1329-1331;
Brown et al, J. Exp. Med. (1986) 163, 1319-1324; Mustoe et al,
Science (1987) 237, 1319-1324), and this has been the main trend in
endeavouring to develop therapeutic applications of the knowledge
acquired about these growth factors.
[0008] According to the present invention there is provided a
composition for use in the treatment of wounds to inhibit scar
tissue formation during healing, comprising an effective
activity-inhibiting amount of a growth factor neutralising agent or
agents specific against only fibrotic growth factors together with
a pharmaceutically acceptible carrier.
[0009] The TGF-.beta. growth factor family, for example, is
believed to have a particularly important regulating role in wound
repair, especially in adult animals, as a stimulant of macrophage
infiltration, fibroblast migration, and extracellular matrix
synthesis, especially collagen synthesis and deposition by
fibroblasts which are involved in the production of scar tissue.
Other growth factors, e.g. PDGF, are also important in this process
and, to some extent, are believed to act in cooperation with one
another in the complex overall regulatory process that is involved
in wound healing. Indeed, PCT/US90/05566 discloses the general use
of antibodies to TGF-.beta. to reduce fibrosis in a rat kidney
nephrosis induced model. However, it is now found that not all
TGF-.beta. growth factors are fibrotic and that supressing the
activity of TGF.beta.-3 in particular is counter-productive.
[0010] PCT/US90/05566 mentions TGF.beta.-1 and TGF.beta.-2 as
having the function of increasing extracellular matrix production,
but does not suggest that any particular TGF.beta. does not have
such an effect.
[0011] The growth factor neutralising agent may be a growth factor
neutralising antibody, for example antibodies to TGF-.beta.1,
TGF-.beta.2 and PDGF.
[0012] The growth factor neutralising agent may be a growth factor
receptor blocking agent, for example a peptide containing the
receptor binding site of the growth factors TGF-.beta.1,
TGF-.beta.2 or PDGF, for example.
[0013] The growth factor neutral;sing agent may also comprise a
molecule which binds to the growth factor to inhibit receptor
binding, for example where the growth factor is selected from
TGF-.beta.1 and TGF-.beta.2, the molecule may be selected from
Decorin and Biglycan.
[0014] The growth factor neutralising agent may also be an
antisense oligonucleotide or ribosyme(s) to growth factor mRNA,
which both act to prevent mRNA from being translated.
[0015] The growth factor neutralising agent may also be a soluble
form of the receptor or the growth factor binding domain of the
receptor.
[0016] The growth factor neutralising agent may be present in the
composition in an active form. Alternatively, the growth factor
neutralising agent may be present in an inactive form.
[0017] One method of inactivating the growth factor neutralising
agent is encapsulation, whereby the capsules may be degradable by
an external stimulus to release the active growth factor
neutralising agent when required.
[0018] The external stimulus may include UV light, in vivo enzymes,
ultrasound or heat.
[0019] A second method of inactivating the growth factor
neutralising agent may be by the molecular addition of a binding
molecule.
[0020] Again, the binding molecule may be detached from the complex
to release active growth factor neutralising agent by an external
stimulus including UV light, in vivo enzymes, ultrasound or
heat.
[0021] The pharmaceutically acceptable carrier may comprise a
neutral sterile cream, gel or powder for topical application, or a
sterile solution for injection, irrigation or inhalation or an
aerosol, or may comprise a sterile dressing for topically covering
a wound or may be in the form of a tablet or capsule for enteral
administration, or the carrier may comprise a biopolymer patch or a
slow release device for implantation.
[0022] The composition may also comprise active cytokines, for
example fibroblast growth factor or factors or other cell
proliferation or migration stimulating or glyco-aminoglycan
stimulating factors in a ratio sufficient to accelerate wound
healing in addition to the growth factor neutralising agent(s)
reducing wound scarring.
[0023] The invention also provides a method of preparation of a
pharmaceutical composition containing the growth factor
neutralising agent or agents for applying the composition topically
in a cream, gel, powder or dressing; in a solution for injection,
irrigation or inhalation or aerosol, or in the form of a tablet or
capsule for enteral administration. The pharmaceutical preparation
may also comprise a biodegradable polymer forming a patch, or an
implantable control release device, useful in surgical operations
having a large initial release followed by a slower release later.
It will be appreciated that this list is not exhaustive, many other
types of compositions being possible, such as might readily occur
to one skilled in the art.
[0024] The method of preparation of the composition may also
include a composition comprising active cytokines.
[0025] The invention also provides a method of inhibiting scar
tissue formation during the healing of wounds, said method
consisting in administering to a host suffering from tissue
wounding a growth factor neutralising agent or agents in the wound
area in a dosage effective to reduce activity of one or more growth
factors involved in the process that leads to the formation of scar
tissue during healing.
[0026] Preferably, the inhibitory agent or mixture of agents
employed for this purpose comprises a neutralising antibody or
antibodies specific to one or more of the growth factors concerned,
or to precursors of such growth factors. Advantageously such
antibody or each such antibody, as a monoclonal antibody obtained
by recombinant DNA techniques. However, polyclonal antibodies,
purified for example by affinity chromotography from antiserum
prepared by injection of relevant growth factor(s) in an
appropriate host, may also be used quite satisfactorily as an
alternative, as has been the case in most of the preliminary
experimental investigations. If desired, instead of complete
antibodies, fragments thereof (Fab's) retaining the specific
antigen binding characteristics can also be used and such fragments
are intended to be included within the scope of the term "antibody"
as used herein in this specification.
[0027] In regard to precursors of these growth factors, it is known
that in many cases the growth factors are initially present in an
inactive state as part of, or as a ligand bound to, a larger
protein molecule, and are separated from the latter, e.g. by
enzymic action, when released in their active form. Binding of a
neutralising agent such as an antibody to such inactive protein
precursors may therefore prevent or inhibit proteolytic action and
release of the active growth factors which will lead to an overall
neutralising effect and inhibition of activity in the same way as
the alternative process of a direct binding of an inhibitory agent
to the active growth factor molecules themselves or to cellular
receptor sites of such growth factors.
[0028] Instead of using growth factor neutralising antibodies, the
inhibitory agent or mixture of agents may alternatively consist of
a synthetic peptide or peptides that can act to antagonise or block
growth factor activity, e.g. by blocking binding of the growth
factor(s) at cellular receptor sites without eliciting any
intracellular "second messenger" response. Such peptide "blocking"
agents could have the advantage of being free of potential adverse
immunogenic effects, and may passs through membrane barriers more
easily than antibodies so that they would be most suitable for
making up pharmaceutical formulations or compositions for topical
application. These "blocking" peptides may readily be designed from
knowledge of the amino acid sequence of the growth factors
concerned and of that portion of this sequence which is involved in
binding to the cellular receptors since these peptides will need to
"mimic" this binding portion of the sequence. It is, for instance,
known that with TGF-.beta.1 it is the c-terminal region of the
molecule that is involved in receptor binding. Similarly, the
region(s) involved in receptor binding with TGF-.alpha. is the
region between cys 33 and cys 42, and with EGF it is the regions
between cys 20 and cys 31, between tyrosine 14 and cys 31 and
between leucine 15 and arginine 53 that are involved. With FGF's
the critical receptor binding reason is that between amino acids
105 and 115.
[0029] As a further possibility, the inhibitory growth factor
neutralising agent(s) may consist of other molecular entities that
act by binding directly to a growth factor or factors, or
precursor(s) thereof, to inactivate the latter. An example of a
neutralising or inhibitory agent of this kind is Decorin which is a
small chrondroitin-dermatan sulphate proteoglycan known to strongly
bind TGF-.beta., as reported by Yamaguchi et al, Nature (1990),
346, 281-284.
[0030] Alternatively, the inhibitory growth factor neutralising
agent(s) may be active at the molecular level and consist of
molecules active against a growth factor's mRNA. Such molecules may
include antisense oligonucleotide(s) synthesised against one or
more growth factor mRNA sequences to prevent translation thereof,
so the molecule nay be a ribosyme(s) targetted against specific
sequences of one or more growth factor mRNA sequences to destroy
the mRNA and again prevent its translation.
[0031] Although use of an antibody or other agent having a
neutralising effect in respect of only one growth factor that is
involved in the formation of scar tissue during wound healing,
especially TGF-.beta.1 and 2 or PDGF for example, may be quite
sufficient to prevent any significant amount of scar tissue from
being produced.
[0032] In some cases combined administration of two or more
different antibodies or other inhibitory agents having a
neutralising effect against two or more different growth factors
concerned may be found to be even more effective, especially for
relatively large excisional wounds for example. In this case, the
different or other inhibitory agents may be administered separately
but simultaneously or sequentially, or they may be made up into a
mixture or "cocktail" within a single pharmaceutical
formulation.
[0033] Although it is believed that a series of these growth
factors, including at least those of the TGF-.beta. family and
PDGF, normally act in cooperation with one another in an
orchestrated manner to regulate the overall process of wound
healing, including the steps leading to the production of scar
tissue, the effect on production of scar tissue of reducing or
neutralising activity of any one growth factor is likely to vary
depending on the nature or identity of that growth factor and on
the form of the resultant active growth factor profile. Thus,
whilst inhibition of the activity of TGF-.beta. and/or PDGF can
generally be very effective in this respect, inhibition of the
activity of certain of the other growth factors may, at least on
its own, be less effective under similar conditions for reducing
scar tissue formation, even though such other growth factors may
still be necessary, or may at least have a beneficial effect, in
connection with promoting wound healing.
[0034] There can therefore be a further possibility in applying the
invention of using an inhibitory or neutralising agent or agents
effective in reducing activity of a growth factor or factors e.g.
TGF-.beta. and/or PDGF, having a major role in the formation of
scar tissue in combination with a different exogeneous growth
factor or agent which does not independently promote the formation
of scar tissue to any significant extent but which, at the same
time, can independently promote wound healing or provide a
beneficial effect in respect of quality of healing. At least in
some cases, such other additional exogeneous growth factor, for use
in combination With TGF-.beta. or PDGF neutralising agent(s) for
example, may be provided by fibroblast growth factors (FGF's).
Thus, by providing a pharmaceutical preparation having a ratio of
the active cyctokine FGF to TGF-.beta. and/or PDGF neutralising
agent(s), a preparation may be obtained which not only prevents
scarring of a wound, but also accelerates the whole process of
wound healing.
[0035] It might have been anticipated that any treatment for
reducing or preventing scar tissue formation would be most
effectively applied at a relatively late stage of healing during
the phase of tissue remodelling or reorganisation that occurs
subsequent to the formation of granulation tissue which replaces
fibrin initially produced in the early stages of healing. Contrary
to such expectation, however, it has been found, surprisingly, that
in applying the present invention the treatment with the growth
factor neutralising agent or agents may need to be carried out at
an early stage of healing in order to be effective. In general, the
treatment is best carried out before and/or during the granulation
phase whilst fibrin is still present, i.e. before the fibrin has
been wholly replaced by granulation tissue. This will usually be
within a period of about 14 days after the initial occurrence of a
wound. Preferably, however, treatment will be commenced earlier,
within seven days or, if possible, within three days or less
following wounding. Indeed, it may often be most advantageous to
commence treatment on the same day as wounding, or at least on the
following day, and in the case of surgical wounds the commencement
of this treatment, say by topical or parenteral application of the
growth factor neutralising agent(s) in a pharmaceutical formulation
applied to the wound area, may well be incorporated as an integral
part of the surgical procedure and be applied before surgery or
immediately the main surgery is completed, before or after
suturing.
[0036] It has also been found, again somewhat surprisingly, that
the treatment does not necessarily need to be repetitive and to be
continued throughout all the phases of wound healing. In order to
be effective, it may often be sufficient to administer the growth
factor neutralising agent(s) in an appropriate dosage once, or only
a few times at most, during the early stages of wound healing. This
is of course important where agents such as proteins are concerned
which may tend to provoke immunological reactions, and it also
gives other practical and economic advantages.
[0037] Although it is possible that in some cases the overall time
to achieve complete healing of a wound may be somewhat extended
upon applying this treatment, any increase in overall healing time
may well be more than aequately compensated for by the improved
quality of the healed wound. A noteworthy and further surprising
feature of the experimental work so far conducted, however, is that
no really significant increase in overall healing time has been
observed, nor has there been any impairment of wound strength upon
healing. Indeed, in respect of this latter point, it would seem
that wound strength may even be improved in that the orientation
observed of the new collagen fibres or fibrils formed during
healing, at least in the case of incisional dermal wounds, more
closely resembled that of undamaged tissue, lying generally prallel
to the outer skin surface instead of at a large angle or generally
perpendicular to the outer surface as is commonly found when such
wounds heal normally with formation or scar tissue.
[0038] By way of further background explanation and description of
the invention, illustrative examples are hereinafter presented of
some of the investigations made and results obtained in the
development of the invention, from which the skilled person in the
art will more readily be able to appreciate the nature thereof and
to put the invention into practical effect.
[0039] First there follows an outline or summary of the materials,
methods and techniques which have generally been used, unless
subsequently stated otherwise, in the investigations and
illustrative examples referred to.
[0040] The preliminary experimental work in these investigations
was carried out using rats as model experimental animals, but the
results are applicable generally to humans and other animals.
[0041] Adult, male, Sprague-Dawley rats weighing between 200-250
grammes were anaesthetised with halothane/nitrous oxide/oxygen
inhalation. After locally clipping the fur, four linear
full-thickness incisions, 10 mm in length, were made on the dorsal
skin of the animal, equidistant from the midline and adjacent to
its four limbs.
[0042] In each animal one wound (control) was unmanipulated, one
(sham control) was injected with an irrelevant antibody, one (the
positive control) was injected with a growth factor detailed below,
and one (the experimental wound) was injected with a preparation of
appropriate growth factor neutralising antibody or antibodies. The
experiments were conducted on groups of these animals, and
according to which group was concerned the injections (100 .mu.l
each) were caried out daily for either a period of three
consecutive days or seven consecutive days, starting either on the
day of wounding, or on the following day, or in a few groups at a
much later stage, e.g. 7 days or 19 days post-wounding.
[0043] In each group, at least two animals were usually killed (by
chloroform overdose on post-wounding days 7, 14, 28 and 42, and in
some cases also on post-wounding days 70, 112 and 168. All four
wounds were excised (with a 0.5 cm margin on all sides) immediately
after death of each animal and were subjected to tissue analysis by
conventional immunohistochemical, histological staining and
biochemical techniques.
[0044] Generally, for carrying out this analysis, each wound was
bisected to provide two samples which were either frozen and/or
fixed and processed for immunocytochemical staining using
antibodies to collagens I, III, IV, laminin and fibronectin or
processed for routine histological examination using a variety of
connective issue stains, or they were immediately freeze-dried for
biochemical analyses after microscopic dissection.
[0045] In the immunohistochemical analyses, primary and secondary
antibodies for indirec immunostaining were used as show in the
following tables.
1TABLE 1 Primary Antibodies Raised Secondary Ab Against Host Source
Dilution (Table 2 ref) HUMAN SHEEP a 1:100 1 FIBRONECTIN MOUSE
RABBIT b 1:50 2 LAMININ RAT TYPE I RABBIT b 1:50 2 COLLAGEN RAT
TYPE III RABBIT b 1:50 2 COLLAGEN RAT TYPE IV RABBIT b 1:100
COLLAGEN RAT MOUSE a 1:200 3 & 4 MACROPHAGES RAT MONOCYTES
& MOUSE a 1:200 3 & 4 MACROPHAGES HUMAN FACTOR RABBIT c
1:200 2 VIII
[0046]
2TABLE 2 Secondary Antibodies Raised Against Host Source Dilution
(Table 1 ref) SHEEP IgG DONKEY a 1:40 1 RABBIT IgG SWINE c 1:40 2
MOUSE IgG SHEEP d 1:200 3 STREPTAVIDNE d 1:100 4 Key to Source
Codes aSEROTEC LTD, Oxford, UK. bInstitut Pasteur de Lyon, France.
cDAKOPATTS, Copenhagen1 Denmark. dAMERSHAM, INTERNATIONAL Plc,
Amersham, UK. Note : Secondary antibodies 1, 2 and 4 were FITC
conjugated (fluorescein isothiocyanate labelled) for
immunofluorometric detection and measurement; 3 was
biotynlated.
[0047] In carrying out the indirect immunostaining, the incubation
with the primary antibody was for 1 hour followed by three rinses
in phosphate buffered saline PBS. Incubation with FITC-conjugated
secondary antisera was for 1 hour followed by a further three rinse
with PBS. Immunostaining for macrophages and monocytes involved the
Biotin-Streptavidine technique, i.e. after the primary incubation
and rinsing, the sections were incubated with the biotynlated sheep
antimouse IgG for 1 hour, rinsed with PBS three times, incubated
with the fluorescein streptavidine for 20 minutes and finally
washed with PBS three times. The sections were mounted in a
non-fading medium, DABCO (1,4-diazobicyclo-(2,2,2)-octane), and
photographed using a Leitz Dialux microscope and kodak Ektachrome
400 ASA film.
[0048] For each primary antibody and each wound, control sections
were stained, substituting PBS for the primary antibody.
[0049] In carrying out the routine histology staining, cellularity
of the wounds was studied by staining cryosections of the tissue
(post-fixed in Bouins fluid) with Harris's haematoxylin and eosin,
and collagen deposition in the wounds was studied by staining
cryosections with Masson's trichrome and Hughesdon's modification
of Mallory's trichrome stains.
[0050] For the biochemical analyses, the wounds were
microscopically dissected out along with a piece of unwounded
dorsal skin from each wound and immediately freeze-dried to
constant weight. The tissue was homogenised in 1 ml of 1M guanidine
hydrochloride, 0.15 M sodium acetate, 0.01 M EDTA, pH 5.8, for 24
hours at 4.degree. C. to extract the glycosaminoglycans. The
homogenate was then centrifuged at 18,000 g for 1 hour. The pellet
was washed twice with 0.5 ml of water and the washings added to the
supernatant. The supernatant was dialysed against 100 mM phosphate
buffer with 5 mM EDTA, pH 6.5, followed by digestion with papain
2.5 mg/ml. The glycosaminoglycans were precipitated with 2% CPC and
separated using the method of Cappelletti et al, 1979.
[0051] After washing, the pellets were digested with pepsin 100
.mu.g/ml in 0.5 M acetic acid at 4.degree. C. fo 24 hours. This was
followed by centrifuging at 18,000 g for 1 hour. The pellet thus
obtained was subjected to Hydroxyproline assay as described by
Stegman and Stadler (1987); some of the supernatant was also used
for this assay. To measure the ratio of type I/III collagen, the
supernatant was subjected to SDS PAGE using the method of Sykes et
al (1976).
[0052] The growth factors used in these experiments were
commercially available reagents obtained from R & D Systems
(Mineapolis, U.S.A.) or British Biotechnology (U.K.) or Serotec
(U.K.) and included:
[0053] 1. Transforming growth factor beta-1 (TGF-.beta.1) derived
from porcine platelets--Dose 10 ng/injection.
[0054] 2. Platelet derived growth factor (PDGF) from porcine
platelets--Dose 10 ng/injection.
[0055] 3. Epidermal growth factor (EGF) derived from mouse salivary
gland--Dose 10 ng/injection.
[0056] 4. Basic Fibroblast growth factor (bFGF) derived from bovine
brain--Dose 10 ng/injection.
[0057] 5. Acidic Fibroblast growth factor (aFGF) derived from
bovine brain--Dose 10 ng/injection.
[0058] The growth factor neutralising antibodies used in these
experiments were also reagents commercially available as detailed
above and were of known neutralising potency. They included:
[0059] 1. TGF Beta neutralising antibody (raised in rabbit against
native porcine platelet TGF-.beta.1--neutralises both TGF.beta.-1
and TGF.beta.-2)--Dose 50 .mu.g/injection.
[0060] 2. PDGF neutralising antibody (raised in goats against
native human PDGF)--Dose 20 .mu.g/injection.
[0061] 3. EGF neutralising antibody (polyclonal antibody raised in
mouse against human EGF)--Dose 10 .mu.g/injection.
[0062] 4. Basic FGF neutralising antibody (raised in rabbits
against native bovine brain basic FGF)--Dose 30
.mu.g/injection.
[0063] 5. Acidic FGF neutralising antibody (raised in rabbit
against native bovine brain acidic FGF)--Dose 30
.mu.g/injection.
[0064] The irrelevant antibodies used for the sham control wounds
were either rabbit IgG or goat IgG according to the host in which
neutralising antibody to the growth factor was raised. The dose of
the irrelevant antibody was similar to that of the neutralising
antibody.
SUMMARY OF RESULTS
[0065] In all the experiments conducted, no differences were found
between the control and sham control wounds at any of the
timepoints at which the wounds were examined, thereby indicating an
absence of any major effects being produced by the introduction of
foreign proteins. Also, no wounds showed impaired healing and the
rate of epithelialisation was similar in all treatments.
[0066] However, at least in the case of the experimental wounds
treated with the neutralising antibodies to TGF-.beta. and PDGF
major effects were produced provided treatment was commenced whilst
the wounds were still fresh, preferably at or soon after the time
of wounding, before or during the granulation phase. Thus, although
no major differences were observed between the control wounds and
the experimental wounds when treatment was not commenced until the
19th day post-wounding, in other cases, especially when treatment
was commenced on the same day as wounding or on the following day,
the experimental wounds contained much less collagen I and III
compared to the other three wounds in the same animal at any
timepoint. There was much greater spacing between the collagen
fibrils but their orientation was almost identical to that of
normal skin. Indeed, in the neutralising antibody treated wounds,
it was often difficult to detect the site of the latter (except for
the loss of ectodermal hair follicles). This was in sharp contrast
to the other wounds which showed a distinct scar with vertically
orientated, parallel, and densely packed collagen fibrils. These
effects were most marked in the papillary dermis and subcutaneous
tissues. Wounds treated with neutralising antibodies to TGF-.beta.
or PDGF also showed a marked reduction in fibronectin, particularly
in the reticular dermis, with an orientation pattern similar to
that of the collagen fibrils. Although fibronectin staining was
markedly reduced throughout the wound, it was still brightest at
the dermal/epidermal function. Treatment with neutralising
antibodies to TGF-.beta. and PDGF also decreased the number of
blood vessels, monocytes and macrophages within the healing wound.
By contrast, the positive control wounds treated with TGF-.beta.
and PDGF showed a marked increase in extracellular matrix
accumulation, in the density of extracellular matrix packing and in
the number of blood vessels, monocytes and macrophages. Scarring
was more prominent in these growth factor treated wounds compared
to controls.
[0067] These results demonstrate the ability of neturalising
antibodies to selected growth factors markedly to reduce scar
tissue formation in adult dermal wound healing. Most importantly,
this advantageous effect was not accompanied by attendant problems
of delayed wound healing or delayed epithelialisation and low wound
strength.
[0068] Some improvement in reducing scar tissue formation was also
observed after administering neutralising antibodies to fibroblast
growth factors (FGF's) although in the preliminary experimental
work it was less marked than in the case of neutralising TGF-.beta.
and PDGF growth factors. Interestingly, exogenous acidic or basic
FGF itself seemed to improve scarring. It is, however, believed
that similar results to TGF-.beta. and PDGF may be achieved,
although perhaps to a somewhat lesser extent, with neutralising
agents to other growth factors administered at an appropriate
dosage level under suitable conditions.
[0069] In the case of TGF-.beta. at least, which appears to be
highly active in connection with the production and organisation of
collagen, especially collagen I, leading to the formation of scar
tissue, it is believed that normally after the initial injury the
level of this growth factor in the environment of the wound may
increase quite quickly by means of an auto-catalytic cascade
effect. Thus, not only does the TGF-.beta. present within an
initial wound from platelet degradation act as a chemoattractant to
monocytes, macrophages and fibroblasts at increasing
concentrations, but it also feeds back on its own promoter to
stimulate its own synthesis so that high levels can soon arise. The
inflammatory calls, especially macrophages, release TGF-.beta. and
exhibit this auto-inductive effect on TGF-.beta. synthesis.
TGF-.beta. also stimulates the synthesis and release of other
growth factors, e.g. TGF-.alpha., PDGF, EGF. TGF-.beta., and these
other growth factors stimulate the synthesis of extracellular
matrix molecules, e.g. collagens and glycosaminoglycans, by the
wound fibroblasts and also influence the degree of proteolytic
turnover and organisation of these extracellular matrix molecules.
As the initial fibrin slot is dense, fibroblasts from the adjacent
normal skin initially migrate up and down between the clot and the
wound margin in a direction broadly perpendicular to the basement
membrane. The collagen and other extracellular matrix molecules are
also deposited in this abnormal orientation and this eventually
gives rise to the scar
[0070] It can be hypothesised that normal wound healing in adults
is phylogenetically optimised for speed of closure in adverse
healing conditions. Consequently, the quantities of growth factor
released are generally excessive, giving the speed of the healing
process considerable buffering against external noxious factors but
with the long term disadvantage of scarring. Modern methods of
wound care, e.g. bandages, and reduction in risks of infection have
largely eliminated the necessity of this growth factor "overdrive"
so that treatment to diminish the active growth factor profile is
permissible and will minimise subsequent scarring.
[0071] Thus the autocatalytic cascade of events, described above
for TGF-.beta., is reduced by treatment at an early stage with a
neutralizing agent. The latter, however, will not be applied in an
amount sufficient to neutralize all of this growth factor, thereby
leaving sufficient to enable wound healing to proceed without
serious inhibition. A similar explanation is also applicable to at
least PDGF.
[0072] Practical Usage
[0073] It will be appreciated that the results obtained from the
investigations made in development of this invention have direct
practical application in clinical use for controlling scar tissue
formation in wound healing, either in therapeutic or cosmetic
fields. For practical use, in general an appropriate amount of
growth factor antibody or antibodies or other growth factor
inhibitory agent(s), constituting the material effective in
neutralising and/or in modifying the profile of relevant growth
factors active and responsible for scar tissue formation in the
healing of wounds, will be made up by any of the methods well-known
in the art of pharmacy as a pharmaceutical formulation or
preparation for administration in any suitable manner to a patient
in need of wound treatment. Such pharmaceutical formulations or
preparations may, moreover, be presented not only individually for
clinical use but they may also be presented as components of first
aid kit for example for non-clinical emergency use.
[0074] By way of example in relation to TGF-.beta. and PDGF growth
factors, in general the antibody or antibodies or other
neutralising agent(s) should be administered (at least for
incisional wounds) so as to effectively neutralise between 1 pg-1
.mu.g TGF-.beta. (1 and 2) and/or PDGF (but preferably an amount of
between 100 pg-10 ng) per cm linear incision per administration. As
previously indicated, application early in the wound healing
process is essential. Normally this will be before, during or
before and during, the phase of granulation tissue formation,
within about 14 days, but preferably within 7 or 3 days, or less,
following wounding.
[0075] The pharmaceutical preparations may conveniently be applied
topically by application to the surface around the wound in liquid,
gel, aerosol, cream or powder form, or in the form of a dressing,
biodegradeable patch or control release implantable device at the
time of wounding or shortly thereafter. Parenteral adminstration,
especially subcutaneous injection, may also often be preferred so
that the neutralising antibody or antibodies or other agent(s) can
be introduced directly into the wound environment for maximum
efficiency. For this purpose the pharmaceutical formulations
prepared may comprise sterile liquid preparations (e.g. in
phosphate buffered saline) of a predetermined amount of the active
material, e.g. relevant antibody or antibodies, presented in unit
dosage form and contained in sealed ampoules ready for use. For the
alternative topical mode of administration, however, which will be
preferred in some cases, the formulations may be made up with the
active material in intimate association or admixture with at least
one other ingredient constituting a compatible pharmaceutically
acceptable carrier, diluent or excipient in order to provide a
composition, such as a cream, gel, ointment or the like, which is
most suitable for topical application. The formulation may be
applied to a sterile dressing, biodegradable/absorbable patches or
dressings for topical application, or to slow release implant
systems with a high initial release decaying to a slow release.
[0076] The formulation may also consist of a neutralising agent,
for example, relevent antibody or antibodies, attached to a
carrier, for example, a biopolymer of collagen or hyaluronic acid
or a polymer, for example, PVC, from which it can be released
quickly initially and more slowly in the longer term when applied
to, or implanted within, the wound or tissue viod.
[0077] As will be seen, the invention provides a number of
different aspects and, in general, it embraces all novel and
inventive features and aspects herein disclosed, either explicitly
or implicitly and either singly or in combination with one another.
Moreover, the scope of the invention is not to be construed as
being limited by the illustrative examples or by the terms and
expressions used herein merely in a descriptive or explanatory
sense.
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