AU6177499A - Pharmaceutical composition containing stimulators of interferon-gamma - Google Patents
Pharmaceutical composition containing stimulators of interferon-gamma Download PDFInfo
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- AU6177499A AU6177499A AU61774/99A AU6177499A AU6177499A AU 6177499 A AU6177499 A AU 6177499A AU 61774/99 A AU61774/99 A AU 61774/99A AU 6177499 A AU6177499 A AU 6177499A AU 6177499 A AU6177499 A AU 6177499A
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Description
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: THE VICTORIA UNIVERSITY OF MANCHESTER Invention Title: PHARMACEUTICAL COMPOSITION CONTAINING STIMULATORS OF
INTERFERON-GAMMA
0 0 *4 *0 00S .0
P
4* S.
The following statement is a full description of this invention, including the best method of performing it known to me/us: 2 PHARMACEUTICAL COMPOSITION CONTAINING STIMULATORS OF
INTERFERON-GAMMA
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.
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) 15 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 20 fibrotic disorders include pulmonary fibrosis, glomerulonephritis, cirrhosis of the liver, and proliferative vitreoretinopathy.
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.
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.
H:\MaraR\Keep\Speci\P36185.doc 26/11/99 3 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 "kick-start" wound healing and may then be used in combination with compositions 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.
o* 15 According to the present invention there is provided a stimulator of IFN-y (Interferon-y) for use in promoting the healing of wounds and fibrotic disorders with reduced scarring.
20 IFN-Y (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-y (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.
IFN-y has been widely studied (see, for example, Kovacs, 1991, Immunology Today, 12(1): 17-23 who states that IFN-y decreases fibroblast proliferation and connective tissue production, i.e. inhibits scar tissue formation). Past studies of the effects of IFN-y at wound H:\MaraR\Keep\Speci\P36185.doc 26,11/99 4 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-y to hypertrophic scars (an abnormal thickening of muscle), most 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-y inhibits collagen types I and II 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-y is involved in macrophage 15 activation in vivo. Tamai et al (1995, J. Invest.
Dermatol., 104: 384-390) have shown that IFN-y is involved in the regulation of metalloproteases (MMP) and tissue inhibitor of metalloproteases (TIMP) in in vitro cell culture. Various uses for IFN-y 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.
It appears that IFN-y 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-y 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-y. From these results, it appears that the treatment of sites (of wounds H:\MaraR\Keep\Speci\P36185.dOC 26/11/99 5 or fibrotic disorders) with IFN-Y would result in healing with reduced scarring.
The experiments (see 'Experimental' section below) have also shown that, very surprisingly, treatment of a site with IFN-y actually promotes the deposition of collagen and healing with increased scarring and therefore may be used to promote the healing of chronic wounds.
By "stimulator" is meant anything which may stimulate agonise) the quantity or efficacy of active IFN-Y at a site or the activation of the IFN-y receptor. This may be IFN-y itself or partially modified form of IFN-y. A •partially modified form of IFN-y may, for example, have a 15 longer half-life than IFN-y. Alternatively, it may be an .inhibitor of IFN-y metabolism.
Partial modification may be by way of addition, deletion or substitution of amino acid residues. A 20 substitution may for example be a conserved substitution.
Hence a partially modified molecule may 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.
The stimulator may be used in conjunction with a pharmaceutically acceptable carrier, diluent or excipient.
The stimulator may be used in conjunction with a composition for promoting the healing of wounds or fibrotic disorders with reduced scarring.
H:\MaraR\Keep\Speci\P36185.doc 26/11/99 6 The stimulator may be used in conjunction with a composition for promoting the healing of chronic wounds.
Also provided according to the present invention is a method for promoting the healing of chronic wounds comprising stimulating IFN-y at a site. By "stimulating" is meant increasing the quantity or efficacy of active IFN-y at a site or the activation of the IFN-y receptor.
The stimulation may be achieved by administering to a site a stimulator of IFN-y. The stimulator may be a stimulator according to the present invention.
15 Between about 7,500 and 15,000 IU IFN-y may be 15 administered to stimulate a site.
The IFN-y may be stimulated immediately prior to wounding. It may be stimulated immediately after wounding, although it may also be stimulated later, for 20 example within approximately 3 or 7 days or longer of wounding.
a The method may be used in conjunction with a method for promoting the healing of wounds or fibrotic disorders with reduced scarring.
The method may be used in conjunction with a method for promoting the healing of chronic wounds.
The invention will be further apparent from the following example which shows, by way of example only, forms of stimulation of IFN-Y and promotion of healing H:\MaraR\Kee\Seci\P36185.doc 26/11/99 7 with reduced scarring, and of promotion of healing of chronic wounds.
EXPERIMENTAL
Method 84 male CD1 mice, 12 to 15 weeks old (Charles River) were anaesthetised using equal parts halothane, oxygen and nitrous oxide. 2 x 1 cm full-thickness incisions (through the panniculus carnosus) were made 3cm from the base of the skull and 1cm either side of the dorsal midline.
Test solutions used were anti-IFN-y, IFN-y and PBS Anti-IFN-y comprised monoclonal antibody against murine IFN-y (MuIFN-y; rat IgG'2a). Antibodies were obtained as 15 ascites fluid from thymusless nude-mice innoculated with the F3 hybridoma clone 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 30U/ml of MuIFN-y and 20 contained 1.25 ng/ml endotoxin. IFN-y was Chinese hamster ovary (CHO) cell-derived recombinant MuIFN-y purified by affinity chromatography on anti-IFN-y mAb. The IFN-y was at an initial concentration of 300,000 IU/ml (endotoxin: 73pg/ml).
Animals were split into several groups as follows: Group A: Animals were treated with a single intraperitonal (IP) injection (100pl) of neat anti-IFN-y antibodies prior to wounding.
H:\MarR\Ke-P\SP1Oi\P36185.d-c 26/11/99 8 Group B: Group C: Group D: Animals were treated with a single intradermal (ID) injection of 50pl or 25pl of anti-IFN-y antibodies (diluted with PBS) prior to wounding.
Animals were treated with a single ID injection of IFN-Y (15,000 or 7,500IU) prior to wounding.
Animals were treated with ID injections of IFN-y (15,000 or 7,500 IU) on day 0 prior to wounding and days 3 and 7 post-wounding.
Animals were treated with a single control IP injection of PBS (phosphate buffered saline) on day 0 prior to wounding (control).
Animals were treated with a single control ID injection of PBS on day 0 prior to wounding.
Animals were treated with an ID injection of PBS on day 0 prior to wounding and days 3 and 7 post-wounding.
a Group E: Group F: Group G: Animals were killed by chloroform overdose on days 7, 14, 70 120 post-wounding. Wounds were excised and bisected for routine histology and immunocytochemistry.
7pm 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.
H:\MaraR\Keep\Speci\P36185.doc 26/11/99 9 Results Anti-IFN-y antibodies: No difference was observed between control wounds and treated wounds at any time point in the animals treated with a single IP injection.
With a single ID injection of anti-IFN-y, 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.
Anti-IFN-y treatment is anti-scarring, improving the 15 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 20 orientated in a "basketweave" fashion. Control wounds (scarred) had compacted parallel collagen fibres throughout the wound area.
IFN-y At the early time points (7 and 14 days), all the IFN-y-treated wounds (in both injection regimes) showed increased inflammation and angiogenesis in a dosedependent manner, i.e. lower doses, although worse than control wounds, were not as bad as wounds treated with higher doses of IFN-y.
H:\MaraR\Kee\Speci\P361 8 5 .doc 26/11/99 10 By 70 and 120 days, the wounds treated on days 0, 3 and 7 post-wounding with a high dose of IFN-Y showed marked fibrosis 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 dosedependent, i.e. the greater the dose of IFN-y, the greater the scarring.
Discussion Previous work has shown that administration of IFN-y 15 to wounds inhibits collagen synthesis, suggesting that it may be useful as an anti-scarring agent. Other workers have shown that treatment of kelolds or hypertrophic scars with IFN-y decreases the size of the scar.
20 Contrary to these findings, these experiments have shown that, very surprisingly, the early treatment of wounds with IFN-y causes fibrosis with raised scars that are packed full of collagen, and therefore indicates that the use of a stimulator of IFN-y production will be useful in promoting the healing of chronic wounds.
H:\MaraR\Kee\Speci\P36185.doc 26/11/99
Claims (11)
1. The use of a stimulator of IFN-y in the manufacture of a medicament for promoting the healing of wounds.
2. The use of a stimulator of IFN-y according to claim 1 where it is selected from any one of the group of IFN-y or a partially modified form thereof, and an inhibitor of IFN-y metabolism.
3. The use of a stimulator of IFN-y according to either one of claims 1 or 2 in conjunction with a pharmaceutically acceptable carrier, diluent or excipient. 15
4. The use of a stimulator of IFN-y according to any one of claims 1 to 3 in conjunction with a composition for promoting the healing of wounds or fibrotic disorders with reduced scarring. 20
5. The use of a stimulator of IFN-y according to any one of claims 1 to 4 in conjunction with a composition for S promoting the healing of chronic wounds.
6. A method for promoting the healing of chronic wounds comprising the use of a stimulator of IFN-y according to any one of claims 1 to
7. A method according to claim 6, comprising administering to a site of wounding a stimulator of IFN-y.
8. A method according to either one of claims 6 or 7 comprising the use of between about 7,500 and 15,000 IU IFN-y. H;\MaraR\Kep\Spei\P36185.dC'c 26/11/99 12
9. A method according to any one of claims 6 to 8, comprising stimulating IFN-y either immediately prior to wounding or immediately after wounding.
10. A method according to any one of claims 6 to 9 used in conjunction with a method for promoting the healing of wounds or fibrotic disorders with reduced scarring.
11. A method according to any one of claims 6 to 10 used in conjunction with a method for promoting the healing of chronic wounds. Dated this Twenty-Sixth day of November 1999 THE VICTORIA UNIVERSITY OF MANCHESTER By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia a e o o f H:\MaraR\Keep\Speci\P36185.doc 26/11/99
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU61774/99A AU747598B2 (en) | 1995-08-18 | 1999-11-26 | Pharmaceutical composition containing stimulators of interferon-gamma |
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Application Number | Priority Date | Filing Date | Title |
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GB9516967 | 1995-08-18 | ||
AU61774/99A AU747598B2 (en) | 1995-08-18 | 1999-11-26 | Pharmaceutical composition containing stimulators of interferon-gamma |
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AU67071/96A Division AU713809B2 (en) | 1995-08-18 | 1996-08-09 | Pharmaceutical composition containing inhibitors of interferon- gamma |
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AU6177499A true AU6177499A (en) | 2000-03-30 |
AU747598B2 AU747598B2 (en) | 2002-05-16 |
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AU61774/99A Ceased AU747598B2 (en) | 1995-08-18 | 1999-11-26 | Pharmaceutical composition containing stimulators of interferon-gamma |
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WO1992014480A1 (en) * | 1991-02-22 | 1992-09-03 | Amgen Inc. | Use of gm-csf and g-csf to promote accelerated wound healing |
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Owner name: RENOVO LIMITED Free format text: THE FORMER OWNER WAS: THE VICTORIA UNIVERSITY OF MANCHESTER |
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FGA | Letters patent sealed or granted (standard patent) |