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PCX 

INTERNATIONAL 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




APPLICATION PUBLISHED TINDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 ; 
A61K 38/22 



Al 



(11) Internationa) Publication Number: 
(43) International Publication Date: 



WO 97AS321 

1 May 1997 (01.05.97) 



(21) International Application Number: PCT/GB96/02559 

(22) International Filing Date: 17 October 1996 (17.10.96) 



(30) Priority Data: 

9521608.1 



21 October 1995 (21.10.95) GB 



(71) Applicant (for all designated States except US): ™ Y^™" 
RIA UNIVERSITY OF MANCHESTER [GB/GB]; Oxford 
Road, Manchester M13 9PL (GB). 

WSSSSt^ (for ™ only): FERGUSON, Mark, 
William, James [GB/GB]; 13 Peelmoat Road, Stockport, 
Cheshire SK4 4PL (GB). 

(74)Aeents: McNEIGHT, David. Leslie et al.; McNeight & 
Lawrence, Regent House, Heaton Lane, Stockport, Cheshire 
SK4 IBS (GB). 



(81) Designated States: AL, AM AT AU AZ, BB BG BR BY, 
CA f CH, CN, CZ. DE. DK, EE, ES R, GB, GE, HU IL, 
ic jp KE KG KP KR, KZ, LK, LR, LS, LT, LU, LV, 
KS ^ W, MX. NCUJZ PL PT, RO, RU, 
SD, SE, SG, SI, SK. TJ. TM, TR, TT, UA, UG, US, UZ, 
VN f ARIPO patent (KE. LS, MW, SD, SZ, UG) Eurasian 
patent (AM, AZ, BY, KG. KZ. ^.RU TJ ™>. European 
patent (AT, BE, CH. DE, DK. ES, FI FR, GB GR IE, £ 
LU MC, NL. PT, SE). OAP1 patent (BF, BJ. CF, CG, CI, 
CM, GA, GN, ML, MR, NE, SN, TD, TG). 



Published 

With international search report 
Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 




(57) Abstract 

The present invention concerns stimulators of ^ ^''^^^^^^^^^^^^^"i^jj^g'o/ ^wnS' Mri^fitwti^diMrien^witfi'^^^d 
with reduced scarring, together with medicaments and methods for promoting the healing 01 wou 

scarring. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States 
applications under the PCT. 



AM 


Armenia 


AT 


Austria 


AU 


Australia 


BB 


Barbados 


BE 


Belgium 


BF 


Burkina Faso 


BG 


Bulgaria 


Bj 


Benin 


BR 


Brazil 


BY 


Belarus 


CA 


Canada 


CF 


Central African Republic 


CG 


Congo 


CH 


Switzerland 


CI 


Cote d'l voire 


CM 


Cameroon 


CN 


China 


CS 


Czechoslovakia 


CZ 


Czech Republic 


D£ 


Germany 


DK 


Denmark 


EE 


Estonia 


ES 


Spain 


Fl 


Finland 


FR 


France 


GA 


Gabon 



to the PCT on the front pages 



GB 


United Kingdom 


GE 


Georgia 


GN 


Guinea 


GR 


Greece 


HU 


Hungary 


IE 


Ireland 


IT 


Italy 


JP 


Japan 


KE 


Kenya 


KG 


Kyrgystan 


KP 


Democratic People's Republic 




of Korea 


KR 


Republic of Korea 


KZ 


Kazakhstan 


U 


Liechtenstein 


LK 


Sri Lanka 


LR 


Liberia 


LT 


Lithuania 


LU 


Luxembourg 


LV 


Latvia 


MC 


Monaco 


MD 


Republic of Moldova 


MG 


Madagascar 


ML 


Mali 


MN 


Mongolia 


MR 


Mauritania 



pamphlets publishing international 



MW 


Malawi 


MX 


Mexico 


NE 


Niger 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


SG 


Singapore 


SI 


Slovenia 


SK 


Slovakia 


SN 


Senegal 


sz 


Swaziland 


TD 


Chad 


TG 


Togo 


TJ 


Tajikistan 


TT 


Trinidad and Tobago 


UA 


Ukraine 


UG 


Uganda 


US 


United States of America 


uz 


Uzbekistan 


VN 


Viet Nam 



WO 97/15321 



PCT/GB96/02559 



- 1 - 



PHARMACEUTICAL COMPOSITION CONTAINING AN ACTIVIN OR INHIBIN STIMULATOR 

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 eye wounds, 
including wounds to the cornea, 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, systemic sclerosis, scleroderma, proliferative vitreoretinopathy, repair following 
myocardial infarction, including myocardial hibernation. 

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. 



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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. 

According to the present invention there is provided a stimulator of Activin 
and/or Inhibin for use in promoting the healing of wounds and fibrotic disorders with 
reduced scarring. 

By 'stimulator' is meant anything which may stimulate the quantity or 
efficacy of active Activin and/or active Inhibin at a site. This may be Activin or Inhibin 
itself (or a pharmaceutical^ acceptable salt thereof) or a fragment or a partially modified 
form thereof. Partial modification may for example be by way of addition, deletion or 
substitution of amino acid residues. A substitution may for example be a conserved 
substitution. Partially modified molecules may, for example, have a longer half-life than 
their parent molecule, or they may have a different binding affinity for their receptors. 
A fragment may comprise at least that part of Activin or Inhibin which is required to 
allow it to stimulate its receptors. Alternatively, a stimulator may, for example, be an 
inhibitor of Activin metabolism, or it may be a stimulator of Activin synthesis, or it may 
be a bioprecursor of activin or inhibin. For example, it may be an analogue of a fragment 
of activin or inhibin which is bound by a degradative enzyme, for example a mimotope 
(Geysen, H.M. et al., 1987, Journal of Immunological Methods, XQ2: 259-274) made to 
a fragment of Activin or Inhibin which is bound by an enzyme which degrades it. Such 
a mimotope can bind to the receptor site of the enzyme, competitively inhibiting the 



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binding of Activin or Inhibin (as appropriate) to the enzyme and thereby inhibiting its 
degradation. 

It may be an antagonist of an antagonist of Activin or Inhibin. For example, 
it may be an antagonist of Follistatin. 

Activin is a member of the TGF p superfamily, and like the other members 
of this family, activins are dimeric proteins, composed of disulphide linked beta A or 
beta B subunits. Three different forms of Activin have been identified in vivo: Activin 
A (beta a, beta a), Activin B (beta b, beta b) and Activin AB (beta a, beta b). Herein, by 
"Activin" is meant all possible forms of activin. Inhibins are heterodimers of beta a or 
beta b chains together with a common alpha chain and are called Inhibin A (alpha beta 
a) and Inhibin B (alpha beta b). Herein, by "Inhibin" is meant all possible forms of 
inhibin (Massague, J., 1990, "The Transforming Growth Factor Beta Family", Annual 
Review of Cellular Biochemistry, 6_: 587-641. Vale, W. et aL, 1990, "The Inhibin 
/Activin Family of Hormones and Growth Factors" in Peptide Growth Factors and Their 
Receptors, Volume II, M.B. Spom and A.B. Roberts (eds), Springer-Verlag, pages 21 1- 
248). 

The biological response to Activins or Inhibins is transduced by receptors 
which exist as heteromeric complexes of type 1 receptors (called Activin receptor like 
kinases (Alk) 2 and 4) and type 2 receptors which are transmembrane serine threonine 
kinases (Matthews, L.S. and Vale, W.W., 1993, "Molecular and Functional 
Characterisation of Activin Receptors", Receptor Volume 3, pages 173-181). Follistatin 
is an Activin binding protein which acts as an Activin antagonist in vitro, but in vivo may 
present Activins to their receptors (Michael, U. et aU 1993, "Follistatins: more than 
follicle stimulating hormone suppressing proteins", Molecular and Cellular 
Endocrinology, Volume 91, pages 1-1 1). 



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PCT/GB96/02S59 



Activin increases the number of gonadotrophs in the pituitary and causes 
differentiation of ovarian granulosa cells (May, K.E., 1994, "Inhibin and Activin: 
Molecular Aspects of Regulation and Function", TEM 5_: 407-415). Activin A also 
enhances the differentiation or neuronal cells (Schubert, D. et al., 1990, "Activin is a 
nerve cell survival molecule", Nature, 244: 868-870), stimulates differentiation of 
megakaryocytes and erythroid cells (Nishimura, M. et al., 1991, "Effect of erythroid 
differentiation factor on megakaryocytic differentiation of L8057, a murine 
megakaryoblastic leukaemia cell line", Biochem Biophysics Research Communication, 
181 : 1Q42-1047) and induces mesoderm formation during early Xenopus development 
(Smith, J.C. etal, 1990, "Identification of a potent Xenopus mesoderm inducing factor 
as a homologue of Activin A", Nature, 245.: 729-73 1 ). 

Targeted disruption of the Activin beta A chain resulted in mice with 
craniofacial defects which died within 24 hours after birth (Matzuk, M.M. et al., 1995, 
"Functional analysis of activins during mammalian development", Nature, 224: 354- 
356). These mice also lacked whiskers and had abnormal whisker follicles. Activin beta 
A chain has been detected in the mesenchyme of developing hair follicles and embryonic 
skin, but not new bom or adult skin (Roberts, V.J. et al., 1991, "Expression of 
Inhibin/Activin sub-unit messenger ribonucleic acids during rat embryogenesis", 
Endocrinology 12& 3122-3129; Roberts, V.J. and Barth, S.L., 1994, "Expression of 
messenger ribonucleic acids encoding the Inhibin/Activin system during mid and late 
gestation rat embryogenesis", Endocrinology, 124: 914-923), in addition to the activin 
receptors Alk2 and Alk4 (Verschueren, K. et al., 1995, "Expression of type 1 and type 
IB receptors for activin in mid-gestation mouse embryos suggests distinct functions in 
organogenesis", Mechanisms of Development, 52: 109-123). Disruption of the activin- 
binding protein, follistatin, in transgenic mice results in abnormal whisker development 
and hyperkeratotic skin. (Matzuk, M.M. et al., 1995, "Multiple defects and perinatal 
death in mice deficient in follistatin", Nature, 224: 360-363). Disruption of the gene for 
the Activin/lnhibin beta b subunit resulted in subtle defects to eyelid development 



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(Vassaiil, A. etai, 1994, "Activin/Inhibin beta b subunit chain disruption leads to defects 
in eyelid development and female reproduction", Genes and Development, £: 414-427), 
whilst targeted disruption of the Inhibin alpha chain caused tumour formation in the 
gonads (Matzuk, M.M. et a!., 1992, "Inhibin is a tumour suppressor gene with gonadal 
specificity in mice", Nature, 2fiQ: 3 1 3-319). 

There have been no reports of the role of either Activin, Inhibin or 
follistatin during wound healing, scarring or fibrosis. 

However, the present inventor has found that Activin and Inhibin in fact 
play roles in wound healing as non-f.brotic growth factors. High levels of expression of 
Activin and of Activin and Inhibin receptors have been found post-wounding at wound 
sites, similar to TGF-p 3 (see PCT/GB93/00586). This observation is particularly 
surprising in light of the prior belief that Activin and Inhibin are predominantly 
reproductive /erythroid /neurological /mesoderm inducing factors. 

Activin and Inhibin have been found to be structurally similar to TGF-p 3 , 
the similarity being greater than that with TGF-p, and TGF-fc. It appears that Activin 
and Inhibin may in fact bind to receptors similar to those bound by TGF-p 3 and as such 
mediate the control of scarring via that route. 

It has also been found that the Act 2a receptor, which is bound by Activin 
and which is believed to be bound by TGF-p 3 , is upregulated in wound healing, 
especially on day 7 post-wounding. Table 1 details further the binding of the informs 
of the TGF-p receptor family. 

Hence Activin and Inhibin have similar anti-scarring properties to those of 
TGF-p 3 and as such Activin and Inhibin may be used to similar effect (see, for example, 
PCT/GB93/00586). 



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The stimulator may be used in conjunction with a pharmaceutical^ 
acceptable carrier, diluent or excipient. It may be used in conjunction with a 
pharmaceutical^ acceptable carrier, diluent or excipient in the preparation of a 
medicament for promoting the healing of wounds and fibrotic disorders with reduced 
scarring. Accordingly, the present invention also provides the use of a stimulator of 
activin and/or inhibin in the preparation of a medicament for use in promoting the 
healing of wounds and fibrotic disorders with reduced scarring. 

Two or more stimulators according to the present invention may of course 
be included in a single composition or medicament or used in a single treatment. 

Pharmaceutically acceptable carriers, diluents and excipients are well 
known - see for example Remington's Pharmaceutical Sciences and US Pharmacopeia 
( 1 984) Mack Publishing Company, Easton, PA. 

Pharmaceutically acceptable carriers may for example 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. 

Stimulators of activin and/or inhibin and medicaments manufactured or 
prepared according to the present invention may be in the form Of a composition for 
topical administration as 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. They 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 



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PCT/GB96/02559 



exhaustive, many other types of compositions being possible, such as might readily occur 
to one skilled in the art. 

Other forms of the present invention in which are included a stimulator of 
activin and/or inhibin also include bandages; biocompatible, biodegradable, non- 
innammatory delivery vehicles such as hyaluronic acid; implants; intradermal injections; 
systemic therapy for e.g. fibrosis or severe trauma or bums, for example by 
intraperitoneal, intravenous or oral administration; eye drops for corneal wounds or 
scarring; films and barriers for treating adhesions. 

Application for compositions and agents for promoting the healing of 
wounds and fibrotic disorders with reduced scarring are well known (see for example 
PCT/GB93/00586, PCT/GB92/00570 and US 5,520,926) and the present invention 
incorporates them accordingly. 

The stimulator may be used in conjunction with a composition for 
promoting the healing of wounds or fibrotic disorders with reduced scarring. 

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 wounds or fibrotic disorders with reduced scarring comprising stimulating 
Activin and/or Inhibin. 

The stimulation may be achieved by administering to a site activin and/or 
inhibin itself or a stimulator of Activin and/or Inhibin. By 'site' is meant a site of 
wounding or fibrotic disorder. The stimulator may be a stimulator according to the 
present invention. It may, for example, be an antagonist of Follistatin. 



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-8- 

Activin and/or Inhibin may be stimulated immediately prior to wounding. 
It may be preferably stimulated immediately after wounding. It may be stimulated within 
14 days of wounding, preferably within 7 days of wounding, more preferably within 3 
days of wounding. 

The method may be for use in conjunction with a method for promoting 
the healing of wounds or fibrotic disorders with reduced scarring. 

The method may be for use in conjunction with a method for promoting 
the healing of chronic wounds. 



The invention will be further apparent from the following description 
which show, by way of example only, forms of promotion of the healing of wounds and 
fibrotic disorders with reduced scarring. 



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FYPFRTMKNTAL 

Initial studies were undertaken to determine the expression profile of Activin in wounded 
tissue, relative to control tissue. These resulted in the conclusion that exogenous addition 
of Activin, or its related molecule Inhibin (which binds to similar receptors to Activin) 
or antagonism of the binding protein of Activin (Follistatin) could have anti-scarring 
activity. This was then tested in two sets of experiments, the first involving the use of 
Activin A and the second using Inhibin. The conclusions of the experiments were that 
Activin and Inhibin have an anti-scarring effect. 

fvperiment 1 
Wounding 

Adult male CD1 mice were anaesthetized using halothane nitrous oxide and oxygen. 
Four wounds were placed on each animal, approximately one centimetre from the mid 
line, 20 and 40 centimetres from the base of the skull respectively. The wounds were 1 
centimetre in length down to and through the panniculus camosus. Animals were killed 
and wounds recovered on days 1, 3, 7, 14, 28, 60 and 80, post wounding. At least 4 
wounds from 4 separate animals were analysed for each experiment. Wounds were 
excised, fixed in paraformaldehyde, dehydrated and embedded in wax in preparation for 
in situ hybridisation (under RNAase free conditions), or frozen in OCT (Miles 
Scientific), cryosectioned and utilized for immunocytochemistry. 

For in situ hybridisation, antisense riboprobes were constructed against the Act 2a 
receptor, Act Rl (Alk 2) and Act RIB (Alk 4). 

For immunocytochemistry, a primary antibody recognising Activin was used and 
delected using streptavadin biotin amplification using an FITC (fluorescein 
isothiocyanate) labelled secondary antibody. 



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- 10- 

As controls, non wounded adult and fetal El 6 (embryonic day 16) skin were used. 
Results 

On days 3 and 7 post wounding, enhanced staining for Activin was detected in the wound 
site, predominantly in fibroblasts of the wound margin and granulation tissue. Staining 
had returned to near normal levels by 14 days post wounding. As the antibody 
predominantly recognises the Activin beta A chain, it is assumed that this is the 
predominant isoform in the granulation tissue. 

The messenger RNA for the Act 2a receptor was up-regulated in the wound margin and 
granulation tissue on seven days post wounding. The Alk 2 (Act Rl) receptor was 
expressed in the mesenchyme of normal skin, but no significant elevation was detected 
in the wound edge or granulation tissue. By contrast, Act RIB (Alk 4) receptor was 
present at a much lower level in the normal skin dermis but was up-regulated in the 
dermal wound margin and granulation tissue of the wounds, particularly on days 7 and 
14, post wounding. 

In normal adult mouse skin, Alk 2 and Alk4 were expressed predominantly in the dermis 
and epidermis, respectively. Staining for Activin in the normal adult skin was at a 
marked low level in the dermis. However, fetal skin from embryonic day 16 mice 
showed marked staining for activin, particularly in the fetal dermis. 

These staining patterns suggest that Activin and its receptors are present in fetal skin and 
reinducedduring wound healing in adult skin. As fetal wounds heal without scarring at 
embryonic day 16 (Whitby, D.J. and Ferguson, M.W.J., "The extracellular matrix of lip 
wounds in fetal, neonatal and adult mice", Development, 112: 651-668, 1991) and with 
reduced levels of inflammation, and hence TGFpl and TGFp2, but enhanced 
endogenous dermal levels of TGFp3 (Whitby, D.J. and Ferguson, M.W.J., 1991, 
"Immunohistochemical localisation of growth factors and fetal wound healing", 



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PCT/GB96/02559 



Developmental Biology, 142: 207-215), it might reasonably be assumed that Activin 
plays a role in this scarless fetal wound healing. Hence, exogenous addition of Activin, 
or its related molecule Inhibin (which binds to similar receptors to Activin) or 
antagonism of the binding protein of Activin (Follistatin) could have anti-scarring 
activity. 

In order to test this, the following experiment was undertaken: 

fiyppriment 2 
Materials and Methods 

Recombinant bovine Activin A (4 ug) was obtained from lnnogenetics, Belgium (Cat. 
No. CY-035). Activin A was prepared by initially reconstituting the lyophilised powder 
in sterile phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) 
and then diluting with PBS/BSA to give three doses: 100 ng/ml; 50 ng/ml; and 25 ng/ml. 

Twelve adult male Sprague-Dawley rates, age- and weight-matched (220g - 250g), were 
anaesthetised using a mixture of equal parts halothane, nitrous oxide and oxygen. The 
dorsal surfaces were shaved and swabbed with 70% alcohol. Four 1 cm linear full 
thickness (down to and including the panniculus camosus) incisions were made at 
defmed anatomical positions 5 cm and 8 cm from the base of the skull, and 1cm each 
side of the midline. 

Of the four wounds per animal, two were treated with a 100 ul dose of Activin A, one 
with 100 ul of PBS, and the other remained unmanipulated. All injections were 
intradermal, approximately 50 ul delivered down each side of the incision as close as 
possible to the wound without rupturing it, and were administered one* daily for three 
days, starting immediately prior to wounding (Day 0). 



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-12- 

The twelve animals were divided into three groups according to the dose administered. 
Four animals received daily 100 ul injections of 100 ng/ml Activin A (i.e. 10 ng/100 ul 
injection), four received 50 ng/ml (i.e. 5 ng/100 ul injection) and the remaining four were 
treated with 25 ng/ml (i.e. 2.5 ng/100 ul injection). The wounds were uncovered and 
unsutured. Six animals, two from each treatment group, were killed 7 days pw (post- 
wounding) and the remaining six killed at 80 days post-wounding, all by chloroform 
overdose followed by dislocation of the neck. A PC based image capture system was 
used to save macroscopic images of the intact, shaved skin. The dorsal skin was removed 
and the full thickness wounds excised with a margin of approximately 0.5 cm of normal 
skin around the wound. One half of the tissue was fixed in formal saline and processed 
for routine wax histology and the other half immersed in OCT embedding medium and 
snap frozen over liquid nitrogen for immunocytochemical analysis. 

Wax histology 

7 urn sections were cut on a standard microtome and the sections stained with 
Haematoxylin & Eosin to examine cellularity and angiogenesis, and Masson's Trichrome 
stain for collagen organisation. 

Results 
Macroscopic 

A visual analogue scoring system was devised which ranged from 0 representing normal, 
unwounded skin, to 10 representing hypertrophic scarring. A 10 cm unmarked line was 
drawn on a blank piece of paper and the four scars on the dorsal surface of each freshly 
killed rat were scored by placing a mark along the line between 0 and 10, with a separate 
line for each scar. Only the 80 day scars were scored (i.e. 6 rats). 

The macroscopic appearances of the wounds treated with Activin A were very good. The 
scars were quite variable but the lowest doses produced the best quality macroscopic 
scars when compared to the controls. 



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PCT/GBM/02559 



Microscopic 

10 ng/100 fil injection: 

At 7 days pw, the wounds were re-epithelial ised and the epithelium had flattened out, 
similar to unwounded epithelium. One consistent observation at 7 days post-wounding 
(pw) was that there were not many inflammatory cells at the top of the wound, but quite 
a lot at the base. The control wounds (unmanipulated and PBS treated) were also re- 
epithelialised but had more inflammatory cells distributed throughout the wound. 

At 80 days pw, the microscopic appearance of the scars was very good. Another visual 
analogue scoring system was used, ranging from 0 representing normal skin to 10 
representing hypertrophic scarring. The average scores are shown in Table 2. The 
average score for treated wounds was 2.65, PBS treated 3.3, and unmanipulated 3.65. 
The orientation of collagen in the treated wounds was more like that of normal skin, the 
collagen bundles being less densely packed, larger, and having a more basket-weave 
appearance (unwounded dermis has collagen bundles arranged in a basket-weave 
architecture), particularly towards the epidermis. 

5 ng/100 pi injection: 

At 7 days pw, the wounds were all re-epithelialised and quite cellular throughout and 
treated wounds appeared similar to control wounds. There was some variation in the 
treated wounds, with some being very cellular, and others not containing as many 
inflammatory cells. 

At 80 days pw, the dermal architecture was^ood, averaging a score of 3.13, compared 
to PBS and unmanipulated control wounds which averaged 5.5 and 4. 1 respectively. The 
collagen was more open and there were thicker bundles, again particularly at the top of 
the wound site near the epidermis. 



2.5 ng/100 fil injection: 



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-14- 

At 7 days pw, the treated wounds resembled the control wounds. 

At 80 days pw, the treated wounds had a reasonable collagen architecture, and averaged 
a score of 5.25, compared to PBS and unmanipulated control wounds which averaged 
5.45 and 4.25 respectively. 

Conclusions 

These experiments show that the TGF-p family member Activin A has an anti-scarring 
effect. 

Both 5 ng/100 ul injection and 10 ng/100 ul injection treatment regimes showed 
considerable improvement in scarring relative to control wounds. The 2.5 ng/100 ul 
injection treatment regime was probably too low. It is interesting that the highest dose 
appears to reduce the influx of inflammatory cells into the wound - an effect similar to 
that achieved with TGF-p 3 . The microscopic appearance of wounds at 80 days pw which 
had been treated with 10 ng/100 ul injection Activin A was better than the controls, and 
5 ng/100 ul injection was also better than controls. Comparison between 10 ng/100 ul 
injection and 5 ng/100 ul injection treatments showed that the 10 ng/100 ul injection 
treatment was superior but that the control wounds in these animals were also better, 
possibly the result of systemic effects of the high dose of Activin A. The lowest dose (2.5 
ng/100 pi) also slightly improved scarring although the microscopic results were closer 
to the control wounds. 

The macroscopic appearance of the wounds was quite variable although the wounds 
treated with the middle (5 ng/100 pi injection) and lowest (2.5 ng/100 ul injection) doses 
appeared to be better than their respective controls. The wounds treated with the highest 
(10 ng/100 pi injection) dose were macroscopically quite variable, but an obvious effect 
may have been negated by the quality of the control wounds which were on the same 



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PCT/GB96/02559 



animal and therefore perhaps improved by systemic effects of the high dose of Activin 
A. 

F«pgP ment 3 
Materials and Methods 

Porcine Inhibin (20 ug per vial) was obtained from the National Institute for Biological 
Standards and Control, Potters Bar, UK (Cat No 86/690) 

The inhibin was reconstituted in sterile PBS with 0.1% BSA to a stock solution of 20 
ug/ml and further diluted to 0.1, 1 and 5 ug/ml (i.e. 10, 100 and 500 ng/100 ul injection). 

Pilot Experiment 

Twelve adult male Sprague-Dawley rats, age- and weight-matched (220g - 250g), were 
anaesthetised using a mixture of equal parts halothane, nitrous oxide and oxygen. The 
dorsal surfaces were shaved and swabbed with 70% alcohol. Four 1cm linear foil 
thickness (down to and including the panniculus carnosus) incisions were made at 
defined anatomical positions: 5 cm and 8 cm from the base of the skull, and 1cm each 
side of the midline. 

Two wounds per animal were treated with a 100 ul dose of Inhibin, one with 100 ul of 
either PBS/BSA or PBS alone, and one remained unmanipulated. All injections were 
intradermal. The first injection was administered at the wound site immediately prior to 
wounding (day 0) and then for two days following wounding. 50 ul was delivered down 
each side of the incision as close as possible to the wound without rupturing it. 

The 12 animals were divided into three groups according to the dose administered. Four 
animals received daily injections of 10 ng/100 ul, four received 100 ng/100 ul and the 
remaining four were treated with 500 ng/100 ul. The wounds were uncovered and 
unsutured. Six animals, two from each treatment group, were killed 7 days post 



WO 97/15321 



- 16- 



PCT/GB96/02559 



wounding and the remaining six killed at 80 days post-wounding, all by chloroform 
overdose followed by dislocation of the neck. A PC based image capture system was 
used to save macroscopic images of the intact, shaved skin. The dorsal skin was removed 
and the full thickness wounds excised with a margin of approximately 0.5 cm of normal 
skin around the wound. One half of the tissue was fixed in formal saline and processed 
for routine wax histology and the other half immersed in OCT embedding medium and 
snap frozen over liquid nitrogen for immunocytochemical analysis. 

Wax Histology: 7 um sections were cut on a standard microtome and the sections stained 
with Haematoxylin & Eosin to examine cellularity and angiogenesis, and Masson's 
Trichrome stain for collagen organisation. 



Results 
Macroscopic 

A visual analogue scoring system was devised which ranged from 0 representing normal, 
unwounded skin, to 10 representing hypertrophic scarring. A 10 cm unmarked line was 
drawn on a blank piece of paper and the four scars on the dorsal surface of each freshly 
killed rat were scored by placing a mark along the line between 0 and 10, with a separate 
line for each scar. Only the 80 day scars were scored (i.e. 6 rats, 24 wounds). 

The appearances of the wounds treated with Inhibin were variable at 80 days. Of the 
wounds treated with the highest dose (500 ng/injection), one was an extremely good, fine 
linear scar and the others were similar to controls. Wounds treated with PBS/BSA were 
also similar to unmanipulated controls. The wounds treated with the middle dose of 
Inhibin were also quite similar to control wounds. Two of the wounds treated with the 
lowest dose had very fine linear scars, barely discernible from the surrounding 
unwounded dermis, while the other scars were similar in appearance to controls. Overall, 
the macroscopic results suggested that the lowest or highest doses of Inhibin may 
improve scarring. 



WO 97/15321 



-17- 



PCT/GB96/02559 



Histology 

7 days post-wounding 

Overall, the treated wounds resembled unmanipulated or PBS treated control wounds at 
7 days. The wounds contained a lot of inflammatory cells, had re-epithelialised and were 
variable in width. The control wounds treated with PBS/BSA were very cellular, were 
very wide and in one case had not re-epithelialised. 

80 days post-wounding 

A visual analogue scoring system also ranging from 0 representing normal, unwounded 
skin, to 10 representing hypertrophic scarring, was used to score the histology slides at 
80 days post-wounding (Table 3). Some of the wounds treated with the highest dose of 
Inhibin (500 ng/100 ul injection) had a good dermal architecture, the collagen bundles 
were thick and in a random organisation resembling the normal basket-weave pattern of 
unwounded dermis. In most of the other wounds the collagen was dense and in parallel 
alignment, resembling the control wounds. The average score was 4.49 for wounds 
treated with 500 ng Inhibin/100 ul injection, 5.6 for untreated control wounds and 5.08 
for PBS control wounds. Wounds treated with the middle dose (100 ng/100 ul injection) 
were similar to PBS controls (scores were 7.58 and 7.9 respectively), the collagen fibres 
were thick but densely packed, mostly at the top of the wound. (The unmanipulated 
controls had a good score of 4.9 in this group). The collagen in the wounds treated with 
the lowest dose of Inhibin (10 ng/100 ul injection) was orientated in an open, random 
fashion but once again at the top of the wound, the collagen was quite densely packed. 
These wounds scored similarly to the controls (see Table 3). 

Conclusions 

The highest dose of Inhibin appeared to have a slight anti-scarring effect, with the 
microscopic results correlating with macroscopic data. The PBS/BSA control appeared 
to produce worse scars at 80 days post-wounding and at 7 days post-wounding the 



WO 97/15321 



PCT/GB96/02559 



-18- 

wounds contained much larger numbers of inflammatory cells. Reconstituting the Inhibin 
in PBS alone may have a more marked anti-scarring effect. 

pvperiment 4 
Follow-up Experiment 



Materials and Methods 

Porcine Inhibin (20 ug per vial) was obtained from the National Institute for Biological 
Standards and Control, Potters Bar, UK (Cat. No. 86/690). 

The Inhibin was reconstituted in sterile PBS with 0.1% BSA to a stock solution of 20 
ug/ml and further diluted to 2.5, 10 and 15 ug/ml (i.e. 250, 1000 and 1500 ng/100 ul 
injection). 

The surgical technique used was as before except for the numbers of animals used (18; 
n=72) and there was an extra time point at 40 days post-wounding. 

Two wounds per animal were treated with a 100 ul dose of Inhibin, one with 100 ul of 
either PBS/BSA or PBS alone, and one remained unmanipulated. All injections were 
intradermal. The first injection was administered at the wound site immediately prior to 
wounding (day 0) and then for two days following wounding. 50 ul was delivered down 
each side of the incision as close as possible to the wound without rupturing it. 

The 1 8 animals were divided into three groups according to the dose administered. Six 
animals received daily 100 ul injections of 250 ng, six received 1000 ng and the 
remaining six were treated with 1500 ng. The wounds were uncovered and unsutured. 
Six animals, two from each treatment group were killed 7 days post-wounding, six at 40 
days post-wounding and the remaining six killed at 80 days post-wounding, all by 
chloroform overdose followed by dislocation of the neck. A PC based image capture 



WO 97/15321 



-19- 



PCT/GB96/02559 



system was used to save macroscopic images of the intact, shaved skin. The dorsal skin 



removed and the full thickness wounds excised with a margin of approximately 0.5 
of normal skin around the wound. One half of the tissue was fixed in formal saline 
and processed for routine wax histology and the other half immersed in OCT embedding 
medium and snap frozen over liquid nitrogen for immunocytochemical analysis. 



was 
cm 



Wax Histology 

7 um sections were cut on a standard microtome and the sections stained with 
Haematoxylin & Eosin to examine cellularity and angiogenesis, and Masson's Trichrome 
stain for collagen organisation. 

Results 
Macroscopic 

The standard visual analogue scoring system was used which ranged from 0 representing 
normal unwounded skin, to 10 representing hypertrophic scarring. A 10 cm unmarked 
line was drawn on a blank piece of paper and the four scars on the shaved dorsal surface 
of each freshly killed rat were scored by placing a mark along the line between 0 and 10, 
with a separate line for each scar. Only the 40 and 80 day scars were scored (i.e. 6 rats, 
24 wounds at each time point). 

Macroscopic analysis at 40 days suggested that the wounds treated with the highest dose 
of inhibin (1500 ng/100 ul injection had the least obvious scars (average score 4.1) 
compared to wounds which had been treated with 1000 ng/100 ul injection or 250 ng/100 
ul injection (average scores of 5 and 4.6 respectively). However, at 80 days, the average 
macroscopic score (4.51) for the wounds treated with the lowest dose of inhibin (250 
ng/100 M l injection) was considerably better than scores for the two higher doses (1500 
ng/100 Ml injection and 1000 ng/100 ul injection), which had similar average scores of 
5.275 and 5.375 respectively. 



WO 97/15321 



-20- 



PCT/CB96/02559 



Histology 

7 days post-wounding 

All wounds had re-epithelialised at 7 days post-wounding. There were no differences 
between PBS treated and unmanipulated control wounds. There were high numbers of 
inflammatory cells at the base of the wounds treated with 1500 ng/100 ul injection and 
1000 ng/100 ul injection and there was not a large amount of new collagen compared to 
control wounds. The wounds treated with 250 ng/100 ul injection were narrow, did not 
contain many inflammatory cells and had a lot of new collagen. 

40 and 80 days post-wounding 

The standard visual analogue scoring system was used to evaluate the 40 and 80 day 
wounds. The results are shown in Table 4. 

At 40 days, the wounds treated with 250 ng/100 ul inhibin injections had the worst 
dermal architecture, the collagen was densely packed and in parallel alignment, reflected 
in an average score of 7.4. The wounds treated with 1000 ng/100 ul injection had an 
average score of 6.0 and were similar to PBS treated control wounds (6.2). The 
unmanipulated control wounds in the groups treated with 1000 and 1500 ng/100 ul 
injection had the best average scores, possibly indicating a systemic effect. The 
histological scores for the wounds treated with 1500 ng/100 ul injection were in 
agreement with the scores for macroscopic appearance, and reflected the superior dermal 
architecture observed at this stage (4.7). 

At 80 days post-wounding, the histological appearance of the wounds treated with 250 
ng/100 ul injection was better than the wounds treated with 1000 or 1500 ng/100 ul 
injection. The collagen bundles were less densely packed and in a more random 
organisation, particularly at the top of the scar, just below the epidermis. Only one scar 
treated with this dose was wide and of poor quality. The wounds which had received 
1000 ng/100 pi injection treatment had densely packed collagen throughout the wounds 



WO 97/15321 



-21- 



PCT/CBW/02559 



and the wounds treated with 1500 ng/100 ul injection, although one had relatively open 
collagen orientation at the top, were in general very wide at the base where the collagen 
was particularly dense. 

Summary 

These results suggest that the lowest dose of inhibin used in this investigation (250 
ng/100 ul injection) had anti-scarring effects. The pilot experiment suggested that a dose 
of 500 ng/100 ul injection also had slight anti-scarring effects. It appears therefore that 
exogenous addition of inhibin has an antiscarring effect and the data suggest the 
optimum dose of inhibin is between 250 and 500 ng/100 ul injection in this treatment 
regime. 



WO 97/15321 



-22- 



PCT/GB96/02559 



Table 1: The TGF-p Receptor family and their known affinities for TGF-p,, 2tndJ , 
Activin, BMP 2,4 and MIS 



Type I Receptors 


TGF-p 


Activin 


BMP 2,4 


MIS 


TGF-p RI 


✓ 








ActR-Ip 




/ 






Atr-I 




/ 












/ 




RPK-I 






/ 




Act R-I 




/ 






TSR-I 


/ 


✓ 






Brk-43E 






/ 




Brk-25D 






/ 




DAF-I 










Type II Receptors 










Act R-II 
Act R-IIB 
Atrll 
TGF-p RII 
Daf4 
C14 


/ 


/ 
/ 
✓ 


/ 


? 



BMP2,4 = Bone Morphogenetic Proteins 
MIS = Mullerian Inhibiting Substance 



WO 97/15321 



-23- 



PCT/GB96/02559 



j^2l Average Histological Scores (80 days post-wounding) Rat Wounds 
Treated with Activin A 



Group 


Dose (ng/100 


Average Score 






ul injection) 












Treated Wounds 


PBS 


U 


A 


10 


2.625 


3.3 


3.65 


B 


5 


3.13 


5.5 


4.075 


C 


2.5 


5.25 


5.45 


4.25 



Dosages relate solely to Activin A treated wounds 
PBS = PBS treated control wounds 
U = Unmanipulated control wound 



WO 97/15321 



PCT/GB96/02559 



-24- 

Table 3: Average Histological Scores (80 days post-wounding) of Rat Wounds 
Treated with Inhibin. 



Group 


Dose (ng/100 
ul injection) 


Average Score 






Treated Wounds 


PBS 


PBS/BSA 


U 


A 


10 


5.13 


4.75 


5.7 


4.675 


B 


100 


7.58 


7.9 


8.0 


4.9 


C 


500 


4.49 


5.08 




5.6 



Dosages relate solely to Activin A treated wounds 
PBS = PBS treated control wounds 
PBS/BSA = PBS/BSA treated control wounds 
U = Unmanipulated control wound 



WO 97/15321 



-25- 



PCT/GB96/02559 



Table 4: Average Histological Scores (40 days and 80 days post-wounding) of 
Wounds Treated with Inhibin 



Group A: 
Dose 

250 ng/100 ul injection 

PBS 

U 



Average Score 
40 days 80 days 

7.4 4.51 
* 5.15 
7.6 5.25 



Group B: 
Dose 

1000 ng/100 pi injection 
PBS 

U 



Average Score 
40 days 80 days 

6.0 5.225 
6.2 
3.9 



Group C: 
Dose 

1500 ng/100 ul injection 

PBS 

U 



Average Score 
40 days 80 days 

4.7 5.2 
4.6 
2.0 



* this denotes where results are not yet available. 



WO 97/15321 



-26- 



PCT/GB96/02559 



CLAIMS 

1 a stimulator of Activin and/or Inhibin for use in promoting the healing of 
wounds and fibrotic disorders with reduced scarring. 

2 a stimulator of Activin and/or Inhibin according to claim 1, selected from 
any one of the group of Activin, Inhibin, a fragment of activin and/or inhibin, a partially 
modified form of activin and/or inhibin, an inhibitor of metabolism of activin and/or 
inhibin, and a stimulator of synthesis of activin and/or inhibin. 

3 a stimulator of Activin and/or Inhibin according to claim 2, comprising a 
partially modified form of activin and/or inhibin having a longer half-life than its parent 
molecule. 

4. A stimulator of Activin and/or Inhibin according to either one of claims 1 
or 2, comprising an antagonist of an antagonist of Activin and/or Inhibin. 

5. A stimulator of Activin and/or Inhibin according to claim 4, the stimulator 
comprising an antagonist of Follistatin. 

6. A stimulator of Activin and/or Inhibin according to any one of the 
preceding claims for use in conjunction with a pharmaceutical^ acceptable carrier, 
diluent or excipient. 

7 a stimulator of Activin and/or Inhibin according to any one of the 

preceding claims for use in conjunction with a composition for promoting the healing of 
wounds or fibrotic disorders with reduced scarring. 



WO 97/15321 



-27- 



PCT/GB96/02559 



g. A stimulator of Activin and/or Inhibin according to any one ot tl 

preceding claims for use in conjunction with a composition for promoting the healing 
chronic wounds. 

9. The use of a stimulator of Activin and/or Inhibin according to any one 

the preceding claims in the preparation of a medicament for promoting the healing 
wounds and fibrotic disorders with reduced scarring 



10. A method for promoting the healing of wounds or fibrotic disorders with 

reduced scarring comprising stimulating Activin and/or Inhibin. 

n . A method according to claim 10, the stimulation being achieved by 

administering to a site a stimulator of Activin and/or Inhibin. 

12. A method according to claim 11, the stimulator comprising a stimulator 
according to any one of claims 1 -8 . 

13. A method according to any one of claims 10-12, Activin and/or Inhibin 
being stimulated immediately prior to wounding. 

14. A method according to any one of claims 10-13, Activin and/or Inhibin 
being stimulated within 14 days of wounding. 

15. A method according to claim 14, Activin and/or Inhibin being stimulated 
within 7 days of wounding. 

16. A method according to claim 15, Activin and/or Inhibin being stimulated 
within 3 days of wounding. 



WO 97/15321 



PCT/GB96/02559 



-28- 



17. A method according to claim 1 6, Activin and/or Inhibin being stimulated 
immediately prior to or immediately after wounding. 

18. A method according to any one of claims 10-17 for use in conjunction with 
a method for promoting the healing of wounds or fibrotic disorders with reduced 
scarring. 

19. A method according to any one of claims 10-17 for use in conjunction with 
a method for promoting the healing of chronic wounds. 



INTERNATIONAL SEARCH REPORT 



I A. CLASSIFICATION OF JSUBJECT MA. it* 

IPC 6 A61K38/22 



Intr -*onal Application No 

PCi/GB 96/02559 



to International Patent gasification (IPC) or to both na tional daafiofaon and IPC 

>S SEARCHED 
,„„,...._.. documentation searched 

IPC 6 A61K C07K 



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k Minimum documentanon searched (dasnficaoon system followed by dassfication symbols) 



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C. DOCUMENTS CONSIDERED TO BE RELEVANT 

Category ' I Citation of document, with ind. canon, where appropnate, of the relevant passages 



WO 93 19769 A (THE VICTORIA UNIVERSITY OF 
MANCHESTER) 14 October 1993 
cited in the application 
see the whole document 

WO 89 11862 A (BIOTECHNOLOGY AUSTRALIA 
PTY. LTD.) 14 December 1989 
see the whole document 

WO 95 26203 A {THE VICTORIA UNIVERSITY OF 
MANCHESTER) 5 October 1995 
see the whole document 



Relevant to claim No. 



1-19 



1-19 



1-19 



I j j Further dotumcitt i are listed in fee continuation of box C. 

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19 February 1997 

i Name and mailing address of the ISA 

European Patent Office, P.B. 5IIS Pattntlaan 2 
NL - 2280 HV Rijswijk 
Td. < * 31*70) 340.2040, Tx. 31 651 epo nl. 
Fax (-> 31*70) 340-3016 

Form PCT.4SA/3I0 (ttccn* ttmx] U«ty IW2) 



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cannot be considered novd or «nnc4becoradere^dto 
involve an inventive step when the document is taken alone 

'V document of particular relevance; the d ^ d J"^ t n ?°r rtM . 
cannot be considered to involve an tnvennvtft^ when me 
ctocument is combined with one or nw«hersu<h docu- 
ments, such combination being obvious to a person stalled 
in the art 

*&• document member of the same patent family 
Datt of mailing of the international search report 



10.03.97 



Authorized officer 



Moreau, J 



INTERNATIONAL SEARCH REPORT 



.emauonai applicauon No. 

PCT/GB 96/02559 



Box I Observations where certain claim* were found unsearchable (Continuation of item I of first sheet) 



This International Search Report has not been established in respect of certain claims under Article 17(2)(a) fot the following reasons: 



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□ 



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This 



international Searching Authority found multiple inventions in this international applicauon, as follows: 



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INTERNATIONAL SEARCH REPORT 

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Patent document 
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date 



WO-A-9319769 



14-10-93 



WO-A-89 11852 



14-12-89 



Patent family 
member(s) 



Publication 
date 



WO-A-9526203 



05-10-95 



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9402366 


EP-A- 


0646012 


HU-A- 


68905 


JP-T- 


7505378 


NO-A- 


943466 


NZ-A- 


249937 


SK-A- 


116894 


AT-T- 


118351 


AU-B- 


611041 


AU-A- 


3759189 


OE-D- 


68921150 


DE-T- 


68921150 


EP-A- 


0368992 


US-A- 


5196192 


GB-A- 


2288118 


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0754058 



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