U.S. patent application number 10/722573 was filed with the patent office on 2004-10-21 for pharmaceutical composition containing inhibitors of interferon-gamma.
This patent application is currently assigned to RENOVO LIMITED. Invention is credited to Ferguson, Mark William James.
Application Number | 20040208871 10/722573 |
Document ID | / |
Family ID | 33301255 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040208871 |
Kind Code |
A1 |
Ferguson, Mark William
James |
October 21, 2004 |
Pharmaceutical composition containing inhibitors of
interferon-gamma
Abstract
The present invention provides compositions and methods for
promoting the healing of wounds or fibrotic disorders with reduced
scarring, comprising inhibitors and inhibiting IFN-.gamma.,
together with compositions and methods for promoting the healing of
chronic wounds, comprising stimulating and stimulators of
IFN-.gamma..
Inventors: |
Ferguson, Mark William James;
(Furness Vale, GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
RENOVO LIMITED
Manchester
GB
|
Family ID: |
33301255 |
Appl. No.: |
10/722573 |
Filed: |
November 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10722573 |
Nov 28, 2003 |
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09459979 |
Dec 14, 1999 |
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09459979 |
Dec 14, 1999 |
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09029098 |
May 13, 1998 |
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09029098 |
May 13, 1998 |
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PCT/GB96/01949 |
Aug 9, 1996 |
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Current U.S.
Class: |
424/145.1 |
Current CPC
Class: |
C07K 2317/70 20130101;
A61P 43/00 20180101; A61K 2039/505 20130101; A61K 38/217 20130101;
C07K 16/249 20130101 |
Class at
Publication: |
424/145.1 |
International
Class: |
A61K 039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 1995 |
GB |
9516967.8 |
Claims
1-24. (Cancel).
25. A method for promoting the healing of a chronic wound in a
patient comprising administering to said patient an amount of a
stimulator of IFN-.gamma. sufficient to effect the promotion of
healing of said chronic wound.
26. The method according to claim 25, wherein said stimulator of
IFN-.gamma. is administered to a site of wounding.
27. The method according to claim 25, wherein between 7,500 and
15,000 IU IFN-.gamma. is administered.
28. The method according to claim 25 wherein said stimulator of
IFN-.gamma. is selected from the group consisting of IFN-.gamma.
and a partially modified form of IFN-.gamma..
29. The method according to claim 25 wherein the stimulator of
IFN-.gamma. is administered in combination with a pharmaceutically
acceptable carrier, diluent or excipient.
Description
[0001] The present invention concerns pharmaceutical preparations
for promoting the healing of wounds or fibrotic disorders, in
particular for promoting the healing of wounds or fibrotic
disorders with reduced scarring, and for promoting the healing of
chronic wounds.
[0002] By "wounds or fibrotic disorders" is meant any condition
which may result in the formation of scar tissue. In particular,
this includes the healing of skin wounds, the repair of tendon
damage, the healing of crush injuries, the healing of central
nervous system (CNS) injuries, conditions which result in the
formation of scar tissue in the CNS, scar tissue formation
resulting from strokes, and tissue adhesion, for example, as a
result of injury or surgery (this may apply to e.g. tendon healing
and abdominal strictures and adhesions). Examples of fibrotic
disorders include pulmonary fibrosis, glomerulonephritis, cirrhosis
of the liver, and proliferative vitreoretinopathy.
[0003] In particular, there is a lack of compositions for promoting
the healing of wounds or fibrotic disorders with reduced scarring.
Scar tissue formation, although providing mechanical strength to a
healed wound, can be unsightly and may impair the function of the
tissue.
[0004] This is particularly the case in wounds which result in scar
tissue formation in the CNS, the scar tissue inhibiting the
reconnection of severed or re-growing nerve ends, so significantly
affecting their function.
[0005] There is also a lack of compositions for use in the
treatment of chronic wounds, for example venous ulcers, diabetic
ulcers and bed sores (decubitus ulcers), especially in the elderly
and wheel chair bound patients. Such compositions may be extremely
useful in patients where wound healing is either slow or in whom
the wound healing process has not yet started. Such compositions
may be used to "kick-start" wound healing and may then be used in
combination with compositions (e.g. those of PCT/GB93/00586) which
promote the healing of wounds or fibrotic disorders with reduced
scarring. Hence not only may a chronic wound be healed, but it may
be healed with reduced scarring.
[0006] According to the present invention there is provided an
inhibitor of IFN-.gamma. (Interferon-.gamma.) for use in promoting
the healing of wounds and fibrotic disorders with reduced
scarring.
[0007] IFN-.gamma. (Type II or immune interferon) is produced
primarily by T lymphocytes upon mitogen or antigen stimulation
(Trinchieri et al., 1985, Immunology Today, 6: 131). IFN-.gamma.
(both murine and human) exert their effects through specific,
saturable, binding to a single class of high affinity receptors
found on a variety of cells including fibroblasts, endothelial
cells and monocytes/macrophages.
[0008] IFN-.gamma. has been widely studied (see, for example,
Kovacs, E. J., 1991, Immunology Today, 12(1): 17-23--who states
that IFN-.gamma. decreases fibroblast proliferation and connective
tissue production, i.e. inhibits scar tissue formation). Past
studies of the effects of IFN-.gamma. at wound sites have shown
(Pittel, B. et al., 1994, Plastic and Reconstructive Surgery, 93:
1224-1235) that in studies on the effect of intralesional injection
of IFN-.gamma. to hypertrophic scars (an abnormal thickening of
muscle), most (6/7) patients showed relief of symptoms, and all
patients showed reduced lesion size during treatment, although
there was no change in the total collagen content. Duncan et al
(1985, J. Exp. Med., 162: 516-527) and Amento et al (1985, J. Clin.
Invest., 76: 836-848) have shown that IFN-.gamma. inhibits collagen
types I and III and fibronectin synthesis by dermal and synovial
fibroblasts and collagen type II by chondrocytes in a
dose-dependent manner. Murray et al (1985, J. Immunol., 134:
1619-1622) have also shown that IFN-.gamma. is involved in
macrophage activation in vivo. Tamai et al (1995, J. Invest.
Dermatol., 104: 384-390) have shown that IFN-.gamma. is involved in
the regulation of metalloproteases (MMP) and tissue inhibitor of
metalloproteases (TIMP) in in vitro cell culture. Various uses for
IFN-.gamma. and antagonists of same are proposed in EP 0304291, EP
0528469, WO 92/06115, WO 91/02005, WO 88/07869, EP 0328255, WO
92/14480, WO 87/07842, WO 94/07497, and Lorat-Jacobs, H. et al.,
1994, Path. Res. Pract. 190: 920-922.
[0009] It appears that IFN-.gamma. is a multi-potent molecule with
many actions depending on the conditions of the environment to
which it is added. Several groups have reported decreased collagen
synthesis in vitro on addition of IFN-.gamma. to cultures, and
Granstein et al (1989, J. Invest. Dermatol., 93: 18-27) have shown
inhibition of collagen deposition and hence healing with reduced
scarring in wounds treated with IFN-.gamma.. From these results, it
appears that the treatment of sites (of wounds or fibrotic
disorders) with IFN-.gamma. would result in healing with reduced
scarring.
[0010] Experiments undertaken (see `Experimental` section below)
have shown that, very surprisingly, the inhibition of IFN-.gamma.
actually promotes healing with reduced scarring, despite the
teachings of the prior art.
[0011] The inhibitor may, for example, be a neutralising antibody.
It may be a monoclonal antibody, a polyclonal antibody, a
phage-derived antibody, a genetically engineered antibody (e.g.
diabody), or antibody derived from a transgenic mouse.
[0012] Alternatively, the inhibitor may be anything which inhibits
IFN-.gamma. from interacting with its receptor (i.e. antagonises
IFN-.gamma. receptor activation) or which inhibits the receptor's
activation. It may, for example, be a molecule which mimics the
IFN-.gamma. receptor binding sequence and which binds to the
receptor but does not activate it, thereby competitively inhibiting
the binding of IFN-.gamma. to the receptor and inhibiting the
activation of the receptor.
[0013] The inhibitor may be used in conjunction with a
pharmaceutically acceptable carrier, diluent or excipient.
[0014] The inhibitor may be used in conjunction with a composition
for promoting the healing of wounds or fibrotic disorders with
reduced scarring.
[0015] The inhibitor may be used in conjunction with a composition
for promoting the healing of chronic wounds.
[0016] Also provided according to the present invention is a method
for promoting the healing of wounds or fibrotic disorders with
reduced scarring comprising inhibiting IFN-.gamma..
[0017] The inhibition may be achieved by administering to a site an
inhibitor of IFN-.gamma.. By "site" is meant a site of wounding or
fibrotic disorder. The inhibitor may be an inhibitor according to
the present invention.
[0018] Between about 300 and about 30,000 IU IFN-.gamma. may be
inhibited.
[0019] The IFN-.gamma. may be inhibited immediately prior to
wounding/onset (by "onset" is meant the onset of a fibrotic
disorder). It may be inhibited immediately after wounding/onset,
although it may also be inhibited later, for example within
approximately 3 or 7 days of wounding/onset.
[0020] The method may be used in conjunction with a method for
promoting the healing of wounds or fibrotic disorders with reduced
scarring.
[0021] The method may be used in conjunction with a method for
promoting the healing of chronic wounds.
[0022] According to a further aspect of the present invention there
is also provided a stimulator of IFN-.gamma. for use in promoting
the healing of chronic wounds.
[0023] The experiments (see `Experimental` section below) have also
shown that, very surprisingly, treatment of a site with IFN-.gamma.
actually promotes the deposition of collagen and healing with
increased scarring and therefore may be used to promote the healing
of chronic wounds.
[0024] By "stimulator" is meant anything which may stimulate (i.e.
agonise) the quantity or efficacy of active IFN-.gamma. at a site
or the activation of the IFN-.gamma. receptor. This may be
IFN-.gamma. itself or partially modified form of IFN-.gamma.. A
partially modified form of IFN-.gamma. may, for example, have a
longer half-life than IFN-.gamma.. Alternatively, it may be an
inhibitor of IFN-.gamma. metabolism.
[0025] Partial modification may be by way of addition, deletion or
substitution of amino acid residues. A substitution may for example
be a conserved substitution. Hence a partially modified molecule be
a homologue of the molecule from which it was derived. It may have
at least 40%, for example 50, 60, 70, 80, 90 or 95%, homology with
the molecule from which it is derived.
[0026] The stimulator may be used in conjunction with a
pharmaceutically acceptable carrier., diluent or excipient.
[0027] The stimulator may be used in conjunction with a composition
for promoting the healing of wounds or fibrotic disorders with
reduced scarring.
[0028] The stimulator may be used in conjunction with a composition
for promoting the healing of chronic wounds.
[0029] Also provided according to the present invention is a method
for promoting the healing of chronic wounds comprising stimulating
IFN-.gamma. at a site. By "stimulating" is meant increasing the
quantity or efficacy of active IFN-.gamma. at a site or the
activation of the IFN-.gamma. receptor.
[0030] The stimulation may be achieved by administering to a site a
stimulator of IFN-.gamma.. The stimulator may be a stimulator
according to the present invention.
[0031] Between about 7,500 and 15,000 IU IFN-.gamma. may be
administered to stimulate a site.
[0032] The IFN-.gamma. may be stimulated immediately prior to
wounding. It may be stimulated immediately after wounding, although
it may also be stimulated later, for example within approximately 3
or 7 days or longer of wounding.
[0033] The method may be used in conjunction with a method for
promoting the healing of wounds or fibrotic disorders with reduced
scarring.
[0034] The method may be used in conjunction with a method for
promoting the healing of chronic wounds.
[0035] The invention will be further apparent from the following
example which shows, by way of example only, forms of inhibition of
IFN-.gamma. and promotion of healing with reduced scarring, and of
promotion of healing of chronic wounds.
EXPERIMENTAL
[0036] Method
[0037] 84 male CD1 mice, 12 to 15 weeks old (Charles River) were
anaesthetised using equal parts halothane, oxygen and nitrous
oxide. 2.times.1 cm full-thickness incisions (through the
panniculus carnosus) were made 3 cm from the base of the skull and
1 cm either side of the dorsal midline.
[0038] Test solutions used were anti-IFN-.gamma., IFN-.gamma. and
PBS. Anti-IFN-.gamma. comprised monoclonal antibody against murine
IFN-.gamma. (MuIFN-.gamma.; =rat IgG'2a). Antibodies were obtained
as ascites fluid from thymusless nude-mice innoculated with the F3
hybridoma clone (J. Immunol., 1987, 138: 4178) and purified by
affinity chromatography on an anti-rat kappa-chain mAb. The
neutralisation potential of the antibody was 1/1,000,000 against 30
U/ml of MuIFN-.gamma. and contained 1.25 ng/ml endotoxin.
IFN-.gamma. was Chinese hamster ovary (CHO) cell-derived
reconbinant MuIFN-.gamma. purified by affinity chromatography on
anti-IFN-.gamma. mAb. The IFN-.gamma. was at an initial
concentration of 300,000 IU/ml (endotoxin: 73 pg/ml).
[0039] Animals were split into several groups as follows:
[0040] Group A: Animals were treated with a single intraperitonal
(IP) injection (100 .mu.l) of neat anti-IFN-.gamma. antibodies
prior to wounding.
[0041] Group B: Animals were treated with a single intradermal (ID)
injection of 50 .mu.l or 25 .mu.l of anti-IFN-.gamma. antibodies
(diluted with PBS) prior to wounding.
[0042] Group C: Animals were treated with a single ID injection of
IFN-.gamma. (15,000 or 7,500 IU) prior to wounding.
[0043] Group D: Animals were treated with ID injections of
IFN-.gamma. (15,000 or 7,500 IU) on day 0 prior to wounding and
days 3 and 7 post-wounding.
[0044] Group E: Animals were treated with a single control IP
injection of PBS (phosphate buffered saline) on day 0 prior to
wounding (control).
[0045] Group F: Animals were treated with a single control ID
injection of PBS on day 0 prior to wounding.
[0046] Group G: Animals were treated with an ID injection of PBS on
day 0 prior to wounding and days 3 and 7 post-wounding.
[0047] Animals were killed by chloroform overdose on days 7, 14, 70
& 120 post-wounding. Wounds were excised and bisected for
routine histology and immunocytochemistry. 7 .mu.m wax sections
were cut and stained for Haemotoxylin and Eosin to assess cell
invasion and re-epithelialisation, and for Masson's Trichome to
assess collagen deposition and orientation.
[0048] Results
[0049] Anti-IFN-.gamma. Antibodies:
[0050] No difference was observed between control wounds and
treated wounds at any time point in the animals treated with a
single IP injection.
[0051] With a single ID injection of anti-IFN-.gamma., there were
no differences compared to controls at 7 and 14 days. However, by
70 and 120 days, marked differences in the orientation of the
collagen fibres within the treated wound were observed.
[0052] Anti-IFN-.gamma. treatment is anti-scarring, improving the
quality of dermal architecture, despite the prior art observations.
While the fibres were still relatively small and compacted
immediately under the epidermis, they are randomly orientated,
whereas in the mid and deep dermis the collagen fibres were less
compacted and were orientated in a "basketweave" fashion. Control
wounds (scarred) had compacted parallel collagen fibres throughout
the wound area.
[0053] IFN-.gamma.
[0054] At the early time points (7 and 14 days), all the
IFN-.gamma.-treated wounds (in both injection regimes) showed
increased inflammation and angiogenesis in a dose-dependent manner,
i.e. lower doses although worse than control wounds, were not as
bad as wounds treated with higher doses of IFN-.gamma..
[0055] By 70 and 120 days, the wounds treated on days 0, 3 and 7
post-wounding with a high dose of IFN-.gamma. showed marked
fibrosis (i.e. scarring). Macroscopically, the wounds were raised
and, microscopically, densely packed collagen in large swirling
bundles within the wound margins was observed. These treated wounds
also showed residual inflammation at the base of the wound,
compared to control wounds. Again, this scarring was
dose-dependent, i.e. the greater the dose of IFN-.gamma., the
greater the scarring.
[0056] Discussion
[0057] Previous work has shown that administration of IFN-.gamma.
to wounds inhibits collagen synthesis, suggesting that it may be
useful as an anti-scarring agent. Other workers have shown that
treatment of keloids or hypertrophic scars with IFN-.gamma.
decreases the size of the scar.
[0058] Contrary to these findings, these experiments have shown
that, very surprisingly, the early treatment of wounds with
IFN-.gamma. causes fibrosis with raised scars that are packed full
of collagen, whereas treatment of incisional wounds with antibodies
to IFN-.gamma. results in improved healing with collagen fibres
orientated in a "basketweave" fashion resembling normal dermis
(i.e. scarring is reduced).
* * * * *