CA2387247C - Use of neutralizing agents to growth factors for reducing wound scarring - Google Patents
Use of neutralizing agents to growth factors for reducing wound scarring Download PDFInfo
- Publication number
- CA2387247C CA2387247C CA002387247A CA2387247A CA2387247C CA 2387247 C CA2387247 C CA 2387247C CA 002387247 A CA002387247 A CA 002387247A CA 2387247 A CA2387247 A CA 2387247A CA 2387247 C CA2387247 C CA 2387247C
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- growth factor
- tgf
- composition
- wound
- pharmaceutically acceptable
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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.
Description
Use of Neutralizing Agents to Growth Factors for Reducing Wound Scarring This is a divisional application of Canadian Patent Application Serial No. 2,105,652 filed on March 30, 1992.
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.
The subject matter of this divisional application is directed to wound healing involving a growth factor inhibiting agent described more in detail hereinunder. The subject matter of the parent application was restricted to wound healing involving a growth factor inhibiting antibody.
However, it should be understood that the expression "the invention" and the like encompass the subject matter of both the parent and the divisional application.
As is well known, the healing of wounds in tissue such as skin generally involves, at least in adult humans and la other mammals, a process of extracellular matrix (ECM) biosynthesis, turnover and organisation which commonly leads to the production of fibrous, connective tissue scars and consequential loss of normal tissue function.
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.
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.
The subject matter of this divisional application is directed to wound healing involving a growth factor inhibiting agent described more in detail hereinunder. The subject matter of the parent application was restricted to wound healing involving a growth factor inhibiting antibody.
However, it should be understood that the expression "the invention" and the like encompass the subject matter of both the parent and the divisional application.
As is well known, the healing of wounds in tissue such as skin generally involves, at least in adult humans and la other mammals, a process of extracellular matrix (ECM) biosynthesis, turnover and organisation which commonly leads to the production of fibrous, connective tissue scars and consequential loss of normal tissue function.
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.
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 trear.menz c; operations satisfactorily since a certain amount of surgery is generally involved which in itself is ls.keiv to result in wounds producing fresh unsightly scar tissue.
T_z adult humans and other mammalian ve-rtebrates, wound heal.ing 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 ar.d embr_vonic 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 necessar_v intercellular conununication, 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 collaqen 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-~C), transforming growth factor beta (TGF-l31, TGF-132, TGF-83 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.
General reviews of 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 ReDorts in Medicinal Chemistry, 1989, Chap, 24 (published by Academic Press, Inc. ).
The recognition of the importance of the role cf 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.
According to one embodiment of 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.
According to another embodiment of the present invention, there is provided a composition for inhibiting scar tissue formation during wound healing, the composition comprising a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of transforming growth factor-RI (TGF-R1) or transforming growth factor-R2 (TGF-R2), or both, together with a - 4a -pharmaceutically acceptable carrier, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the composition is formulated for administration in a wound area before the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
According to yet another embodiment of the present invention, there is provided a composition for inhibiting scar tissue formation during wound healing, the composition comprising a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-(31 or TGF-(32r or both, together with a pharmaceutically acceptable carrier, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the composition is formulated for administration in a wound area during the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
According to yet another embodiment of the present invention, there is provided use of a growth factor-neutralizing agent comprising an antisense oligonucleotide - 4b -specific to a mRNA of TGF-(31 or TGF-P2r or both, in the manufacture of a medicament, for inhibiting scar tissue formation during wound healing, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the medicament is formulated for administration in a wound area before the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
According to yet another embodiment of the present invention, there is provided use of a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-(31 or TGF-PZ, or both, in the manufacture of a medicament, for inhibiting scar tissue formation during wound healing, wherein the agent neutralises the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the medicament is formulated for administration in a wound area during the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
The TGF-8 growth factor family, for example, is believed to have a particularly important regulating role in wound - 4c -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 WO 92/17206 PCT/GB92/00_5%7U
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 trear.menz c; operations satisfactorily since a certain amount of surgery is generally involved which in itself is ls.keiv to result in wounds producing fresh unsightly scar tissue.
T_z adult humans and other mammalian ve-rtebrates, wound heal.ing 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 ar.d embr_vonic 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 necessar_v intercellular conununication, 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 collaqen 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-~C), transforming growth factor beta (TGF-l31, TGF-132, TGF-83 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.
General reviews of 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 ReDorts in Medicinal Chemistry, 1989, Chap, 24 (published by Academic Press, Inc. ).
The recognition of the importance of the role cf 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.
According to one embodiment of 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.
According to another embodiment of the present invention, there is provided a composition for inhibiting scar tissue formation during wound healing, the composition comprising a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of transforming growth factor-RI (TGF-R1) or transforming growth factor-R2 (TGF-R2), or both, together with a - 4a -pharmaceutically acceptable carrier, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the composition is formulated for administration in a wound area before the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
According to yet another embodiment of the present invention, there is provided a composition for inhibiting scar tissue formation during wound healing, the composition comprising a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-(31 or TGF-(32r or both, together with a pharmaceutically acceptable carrier, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the composition is formulated for administration in a wound area during the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
According to yet another embodiment of the present invention, there is provided use of a growth factor-neutralizing agent comprising an antisense oligonucleotide - 4b -specific to a mRNA of TGF-(31 or TGF-P2r or both, in the manufacture of a medicament, for inhibiting scar tissue formation during wound healing, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the medicament is formulated for administration in a wound area before the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
According to yet another embodiment of the present invention, there is provided use of a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-(31 or TGF-PZ, or both, in the manufacture of a medicament, for inhibiting scar tissue formation during wound healing, wherein the agent neutralises the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the medicament is formulated for administration in a wound area during the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
The TGF-8 growth factor family, for example, is believed to have a particularly important regulating role in wound - 4c -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 WO 92/17206 PCT/GB92/00_5%7U
believed to act in cooperation with one another in the complex overall regulatory process that is involved in wound healing. Indeed, W091/04748 discloses the general use of antibodies to TGF-8 to reduce fibrosis in a rat kidney nephrosis induced model. However, it is , now found that not all TGF-13 growth factors are fibrotic and that supressing the activity of TGF13-3 in particular is counter-productive.
W091/04748 meritions TGFa-1 and TGF8-2 as having the function of increasing extracellular matrix production, but does not suggest that any particular TGFB does not have such an effect.
The growth factor neutralising agent mav be a growth factor neutralising antibodv, for example antibodies to TGF-l31, TGF-32 and PDGF.
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-J31, TGF-!32 or PDGF, for examnle.
The growth factor neutralising agent mav alsc comprise a molecule which binds to the growth factor to inhibit receptor binding, for example where the growth factcr is selected from TGF-81 and TGF-r2, the moiecule may be selected from Decorin and Big!ycan.
11 rI I
W091/04748 meritions TGFa-1 and TGF8-2 as having the function of increasing extracellular matrix production, but does not suggest that any particular TGFB does not have such an effect.
The growth factor neutralising agent mav be a growth factor neutralising antibodv, for example antibodies to TGF-l31, TGF-32 and PDGF.
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-J31, TGF-!32 or PDGF, for examnle.
The growth factor neutralising agent mav alsc comprise a molecule which binds to the growth factor to inhibit receptor binding, for example where the growth factcr is selected from TGF-81 and TGF-r2, the moiecule may be selected from Decorin and Big!ycan.
11 rI I
6 PCf/GB92/00570 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.
The growth factor neutralising agent may also be a soluble form of the receptor or the growth factor binding domain of the receptor.
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.
One method of inactivating the growth factor neutralising agent is encapsulation, wherebv the capsules may be degradable by an externa: stimulus to release the active growth factor neutralising agent when required.
The external stimulus may include W light, in vi=ro enzymes, ultrasound or heat.
A second method of inactivating the growth factor neutralising agent mav be bv the molecular addition of a binding mo'Lecule.
_ 7 _ Again, the binding molecule may be detached from the complex to release active growth factor neutralising agent by an external stimulus including W light, in vivo enzymes, ultrasound or heat.
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 mav 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.
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.
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 cf a tablet or capsule for enteral administration. The = .
.. g _ 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.
The method of preparation of the composition may also include a composition comprising active cvtokines.
The invention also provides a method o`
inhibiting scar tissue formation during the healing cf wounds, said method consisting in adminstering to a host su-zfering from tissue wounding a growth factor neutralising agent or agents in the wound area i: a dosage effective to reduce activity of one or more growth factors involved in the process that leads t-- the formation of scar tissue during healing.
Preferably, the inhibitory agent or mixture o_ agents employed for this purpose comprises a neutralising antibodv or antibodies sDecific to one or more of the crowtz factors concerned, or to precurscrs cf such crowth factors. Advantageously, such antibody or each suc: antibody, is a monoclonal ar.tibodv obtained by . . . . .. i ~.:. I . , I I '.
=
_ 9 -recombinant DNA techniques. However, polyclonal antibodies, purified for example by affinity chromo-tography 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.
In regard to precursors of these growth factors, it is known that in many cases the growth factors are initially present in ar, inactive state as part of, or as a ligand bound tc, 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 pro.teolytic action and release of the active growth factors which wili lead to an overall neutralising effect and inhibition of activity in the same way as the alternative process of a direct bindiag of an inhibitory agent to the active arowta factc: molecules themselves or to ce'_lslar receotor sites cf such Qrowtz factois.
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 easi?y 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 t;, "mimic" this binding portion of the sequence. It is, for instance, known that with TGF-81 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-oC 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 receator binding rpgion is that between amino acids 105 and 115.
WO 92/17206 PC'T/G B92/00570 As a further oossibility, 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-8, as reported by Yamaguchi et al, Nature (1990), 346, 281-284.
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 molecsles may include antisense oligonucleotide(s) synthesised against one or more growth factor mRNA seauences to prevent translation thereof, or the molecuie may be a ribosyme ( s) targetted against specifiC sequences of one or more growth factor mRNA seauences to destroy the mRNA and again prevent its translation.
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-i31 and 2 or ?DGF for example, mav be quite sufficient to prevent anv siQ;.;.f1.caa^.:. amoL`.^.-: c-4 sCL'.- z:.ssue from bein: L'roduced.
= - - ~~._h :;._ a- ~ ~ -WO 92/1; 206 PCT/GB92/O057E1 In some cases combined adminstration 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" with.in a single pharmaceutical formulation.
Although it is believed that a series of these growth factors, including at least those cf the Tsr^-3 family and PDGF, normally act in cooperation with'or.e 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 cn production of scar tissue of reducing or neutralising activity of any one growth factor is likelv 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 activit-r of TGF-8 and/or PDGF can generally be very effective in this respect, inhibition of the activity of certain c`_ the other growth factors may,,at least on :t-S own, be less effective under si:n;lar conditions fo_ reducinc scar 1=i..ssue formation, e=:e^ tnouc'.: such cw:ie= qrJw4...
.. . . .i,I-.,i..,,I I I . .
factors may still be necessary, or may at least have a beneficial effect, in connection with promoting wound healing.
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-A and/or PDGF,.having a major role in the formation of scar tissue in combination with a 4ifferent 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 qua:.ity of healing. At least in some cases, such other additional exogeneous growth factor, for use in combination with TGF-3 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-A and/or PDGF neutralisingagent.(s), a preparation may be obtained which not only prevents scarring of a wound, but also accelerates the whole process of wound healing.
WO 92/17206 PC.'T/GB92/00570 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 expectatiori, 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 eariy stage of healing in order to be effective.
In general, the treatment is best carried out before and/er during the granulation phase whilst f:.brin is st'_i1 present, i.e. before the fibrir, has been -ohol=v rep3aced by granulation tissue. This will usually be within a period of about 1Y days after the i^itia:
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 surgica? wounds the commencement of this treatment, say by topical or parer.tera: application of the growth factor neutralising agent ( s) :.n a pharmaceutical formulation appl_ed to the wound area, mav wel! be incorporated as an '_^tecral par-:
of the surgical procedure and be applied before surgery or immediately the main surgery is completed, before or after suturing.
It has also been found, again somewhat surprisingly, that the treatment does not necessarily need to be repetitive and to be continued throughout a?l 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.
Although it is possible that in some cases the overa'Ll 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 =espect c`_ this latter point, it would see:*
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.
By way of further background explanation a:d descr:.ption of the invention, illustrative examples are hereinafter presented of some of the investigations made and results obtained in the development of the inver.tior., from which the skilled person in the art will :ncre read'_lv be able to aonreciate the nature thereoa and t:, put the invention into practical effect.
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.
The preliminary experimental work in these investiaations was carried out using rats as mode_ exgerimer.;.a'_ animals, but the results are a_ =_icable cenera=l-: _, :':.ata..^.s and cther animals.
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.
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 g_oups of these animals, and according to which group was concerned the injections (100 i- 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.
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 davs 70, 112 and 168. All four wounds were excised (with a 0.5; crr. margir. on all sides ) immediately afte:
;1'~_ 1 i WO 92/17206 PC.'T/GB92/00570 death of each animal and were subjected to tissue analysis by conventional immunohistochemical, histological staining and biochemical techniques.
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, rT, laminin and fibronectin or processed for routine histological examination using a variety of connective tissue stains, or they were immediately freeze-dried for biochemical analyses after microscopic dissection.
In the immunohistochemical analyses, primarj ar.d secondary antibodies for indirect immunostaining were used as showr. in the .4-_'Llowin5 tables.
1 ;i TABLE 1 Primary Antibodies Raised Secondary Ab Against Host Source Dilution (Table 2 refl 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 1:10C 2 COLLAGEN
RAT MOUSE a 1:200 3 & 4 XACROPHAGES
RAT
MONOCYTES & MOUSE a 1:200 3 & 4 MACROPHAGES
HUMAN FACTOR RABB?^_' 1:200 2 V_I.
iC; 1 i I
TA-BLE 2 Secondarv 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 STREP"_'AVIDINE d 1:100 4 Key to Source Codes SEROTEC LTD, Oxford, UK.
b Institut Pasteur de Lyon, France.
c DAROPAT"'S, Copenhagen, Denmark.
AMERSHAM, INTERNATIONAL PIc, Amexsham, UK.
Ncte : Secondarv antibodies 1, 2 and 4 were FITC
conjugated (fluorescein isothiocyanate labelled) for immunofluorometric detection and measurement;
3 was biotynlated.
In carrying out the indirect immunostaining, the i.^.cubation with the primary antibodv was for 1 hour 'ollowed bv three rinses i.^. phosphate buffered sa??ne '?BS _ncubation wit:: _ : rn:-cor.jugated secondary ac-:~-;se_a was for 1 hour f.,_wowed bv a further three . . ~ : 1 h ; , : I i 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 sheeo 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 TM
sections were mounted in a non-fading medium, DABCO
(2,4-diazobicyclo-(2,2,2)-octane), and photographed TM - =rna using a Leitz Dialux microscope and kodak Ektachrome 400 ASA film.
For each primary antibodv and each wound, control sections were stained, substituting P3S for the primary antibody.
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.
For the biochemical analyses, the wounds were microscopica?ly dissected out along with a piece of unwounded dorsa'_ skin from eac^ wound and i.ficned_3te_:
freeze-dried to constant weight. The tissue was homogenised in 1 ml of 1M guanidine hydrochloride, 0.15M sodium acetate, O.O1M
EDTA, pH 5.8, for 24 hours at 4 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 100mM phosphate buffer with 5mM 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 separated using the method of R.
Cappelletti, Mario Del Rosso and Vincenzo Chiarugi: A New Electrophoretic Method for the Complete Separation of all Known Animal Glycosaminoglycans in a Monodimentional Run, Analytical Biochemistry, 1979; vol 99; pp. 311-315.
After washing, the pellets were digested with pepsin 100 g/ml in 0.5M acetic acid at 4 C for 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 & Stadler, Determination of Hydroxyproline, Chimica chimica acta 1987 (18):267-273; 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 B. Sykes, B. Puddle, M. Francis and R. Smith: The estimation of two collagens from human dermis by interrupted gel electrophoresis, Biochem Biophys Res Commun. 1976 Oct 18;72(4):1472-80.
The growth factors used in these experiments were commerically available reagents obtained from R & D Systems (Mineapolis, U.S.A.) or British Biotechnology (U.K.) or Serotec (U.K.) and included:-1. Transforming growth factor beta-1 (TGF-!31) derived from porcine platelets - Dose 10 ng/injection.
2. Platelet derived growth factor (PDGF) from porcine platelets - Dose 10 ng/injection.
3. EAidermal growth factor (EGF) derived from mouse saliva:.-y gland - Dose 10 ng/injection.
4. Basic Fibroblast growth factor (bFGF) derived from bovine brain - Dose 10 ng/injection.
5. Acidic Fibroblast growth factor (aFGF) derived from bovine brain - Dose 10 ng/injection.
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 1. TGF Beta neutralising antibody (raised in rabbit against native porcine platelet TGF-!31 -neutralises both TGF13-1 and TGF13-2) - Dose 50 ug/injection.
' I'- a. . . . . I i . CA 02387247 2002-06-25 r r -WO 92/17206 PC.?/6B92/005711 2. PDGF neutralising antibody (raised in goats against native human PDGF) - Dose 20 g/
injection.
3. EGF neutralising antibody (polyclonal antibody raised in mouse against human EGF) - Dose 10 pg/injection.
4. Basic FGF neutralising antibody (raised ir.
rabbits against native bovine brain basic FGF: -Dose 30 g/injection.
5. Acidic FGF neutralising antibody ;raised in rabbit against native bovine brain ac-;dic FGF) -Dose 30 g/injection.
The irrelevant antibodies used fo, the sham control. wounds were either rabbit IgG or goat IgG
according to the host in which neutralising antibody t--the growth factor was raised. The dose of the irrelevant antibody was similar to that of the neutralising antibody.
SummarY of Results In all the experiments conducted, nc differences were found be z~ween the cc::tro: ar.c sham co.. z_ =_ wcLnds ` , .
at anv 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.
However, at least in the case of the experimental wounds treated with the neutralising antibodies to TGF-B 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 fo= the loss of ectodermal hair ioi'_:c? es ;. T:^.is was in s'r.aro contrast to the ot^e_ . =
WO 92/17206 PCT/GB92/005%t-wounds which showed a distinct scar with verzica?i:-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-3 or aDGF
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 az the dermal/epidermal junction. Treatment with neutralising antibodies to TGF-B and PDGF also decreased the number of blood vessels, monocytes and macrophages within the healing wound. By contrast, the pc_s'uti=re control wounds treated with TGF-li and PDGF showed a marked increase in extraceIlular matrix accumulati:.n, in the density of extraceiiular matrix packing and in the number of blood vessels, monocytes and macrophages.
Scarring was more prominent in these growth factor treated wounds comnared to controls.
These results demonstrate the ability of neturalising antibodies to selected growth fac-~:ors markedly to reduce scar tissue formation in adult dermal wound healing. Most importantly, this advantageoss effect was not accomaanied by attendant prob?ems cf delaved wound hea_i^c ^= d3laved epithe? ialis=_cion ani iow wound strenct^.
WO 92/17206 PCf/GB92/00570 Some improvement in reducing sca-r 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-B
and PDGF growth factors. Interestingly, exogenous acidic or basic FGF itself seemed to improve scarring.
It is, however, believed that similar results to TGF-!i 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.
In the case o` TGF-Ji at least, which appears to be highly active in connection with the production and organisation of coilagen, especially collagen I, leading to the formation cf scar tissue, it is believed tha-, 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-A 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 ir.-~'iammatcry cells, especially macrophages, release TGF-A and exhibit this auto-inductzve effect on TGF-f;
synthesis. TGF-!i also stimulates the synthesis and release of other growth factors, e.g. TGF-4C, PDGF, EGF.
TGF-8, 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 proteolyric turnover and organisation of these extracellular matr?x molecules. As the initial fibrin clot 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 extrace1?:::ar matrix molecules are also deposited in this abno:^nal orientation and this eventually gives rise to the scar.
It can be hypothesised that normal wound heal'_.^._ in adults is phylogenetically optiznised for speed of cl-osure in adverse healing conditions. Consequently, the quantities of growth factor released are generallv excessive, giving the speed of the healing process considerable buffering against external noxious factors but with the long term disadvantage of scarring. Moderr:
methods of wound care, e.g. bandages, and reduction in risks of infection have largely eliminated the necessity cf this growth factor "overdrive" so that treatment t:, diminish the active growt:: factc_ prof_=e -G per::-iss;:,le a^c wi__ minimise subsecuer_-z s;.a=_:. :, ~ ~ .
Thus the autocatalytic cascade of events, described above for TGF-J3, is reduced by treatment at an early stage with a neutralising 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.
Practical Usace 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 prac*_ical use, in general an appropriate amount of:
growth factor antibodv 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 a.ctive and responsible for scar tissue 46ormation in the healing of wounds, will be made up bv any of the methods well-known in the art of pharmacv as a pharmaceutical formulat.ion or prepar-ation for administration in any suitable manner to a oatient in need of wounc treatment. Such pharmaceuticai -fo*_-nulat_onG c, preparatic^s may, moreover, be presented a ,I I
WO 92/17206 PCT/f,'892/00570 not only individually for clinical use but they may also be presented as components of first aid kits for example for non-clinical emergency use.
By way of example in relation to TGF-B and PDGF
grawth factors, in general the antibody or antibodies or other neutralising agent(s) should be administered (at least fbr incisional wounds) so as to effectively neutralise between 1 pg - 1 pg TGF-A (:. and 2; and/cr PDGF (but preferably an amount of between 100 pg - 1C
ng) per cm linear incision per administration. As previously indicated, application early in the wound healing process is essential. Normally this wil; be before, during or before and during, the phase of ' cranulation tissue formation, within about 14 3avs, but preÃerably within 7 or 3 days, or less, f:,,iow?^g, wcunding.
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, especiallv subcutaneous i^jection, may also often be preferred so that the n a::t_alisi.:ig antibodv or antibodies c= other agertr s .14:~, .. , I i 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 ass.ociation or admixture with at least one other ingredient constituting a compatible pharmaceutically acceptable carrier, diluent cr 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, hiodegradable/
absorbable patches or dressings for topical application, or to slow release implant systems with a high initial release decaying to a slow release.
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 i.^.= : ia? lv and more slowly in the longer ter=n when a, , ;I , +~ ~ =
WO 92/17206 PCT/GB92/005?() applied to, or implanted within, the wound or tissue viod.
As will be seen, the invention provides a number of different aspects and, in general, it embraces all nove'L 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 bv the ter:ns and expressions used herein merely in a descriptive or explanatory sense.
The growth factor neutralising agent may also be a soluble form of the receptor or the growth factor binding domain of the receptor.
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.
One method of inactivating the growth factor neutralising agent is encapsulation, wherebv the capsules may be degradable by an externa: stimulus to release the active growth factor neutralising agent when required.
The external stimulus may include W light, in vi=ro enzymes, ultrasound or heat.
A second method of inactivating the growth factor neutralising agent mav be bv the molecular addition of a binding mo'Lecule.
_ 7 _ Again, the binding molecule may be detached from the complex to release active growth factor neutralising agent by an external stimulus including W light, in vivo enzymes, ultrasound or heat.
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 mav 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.
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.
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 cf a tablet or capsule for enteral administration. The = .
.. g _ 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.
The method of preparation of the composition may also include a composition comprising active cvtokines.
The invention also provides a method o`
inhibiting scar tissue formation during the healing cf wounds, said method consisting in adminstering to a host su-zfering from tissue wounding a growth factor neutralising agent or agents in the wound area i: a dosage effective to reduce activity of one or more growth factors involved in the process that leads t-- the formation of scar tissue during healing.
Preferably, the inhibitory agent or mixture o_ agents employed for this purpose comprises a neutralising antibodv or antibodies sDecific to one or more of the crowtz factors concerned, or to precurscrs cf such crowth factors. Advantageously, such antibody or each suc: antibody, is a monoclonal ar.tibodv obtained by . . . . .. i ~.:. I . , I I '.
=
_ 9 -recombinant DNA techniques. However, polyclonal antibodies, purified for example by affinity chromo-tography 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.
In regard to precursors of these growth factors, it is known that in many cases the growth factors are initially present in ar, inactive state as part of, or as a ligand bound tc, 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 pro.teolytic action and release of the active growth factors which wili lead to an overall neutralising effect and inhibition of activity in the same way as the alternative process of a direct bindiag of an inhibitory agent to the active arowta factc: molecules themselves or to ce'_lslar receotor sites cf such Qrowtz factois.
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 easi?y 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 t;, "mimic" this binding portion of the sequence. It is, for instance, known that with TGF-81 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-oC 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 receator binding rpgion is that between amino acids 105 and 115.
WO 92/17206 PC'T/G B92/00570 As a further oossibility, 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-8, as reported by Yamaguchi et al, Nature (1990), 346, 281-284.
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 molecsles may include antisense oligonucleotide(s) synthesised against one or more growth factor mRNA seauences to prevent translation thereof, or the molecuie may be a ribosyme ( s) targetted against specifiC sequences of one or more growth factor mRNA seauences to destroy the mRNA and again prevent its translation.
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-i31 and 2 or ?DGF for example, mav be quite sufficient to prevent anv siQ;.;.f1.caa^.:. amoL`.^.-: c-4 sCL'.- z:.ssue from bein: L'roduced.
= - - ~~._h :;._ a- ~ ~ -WO 92/1; 206 PCT/GB92/O057E1 In some cases combined adminstration 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" with.in a single pharmaceutical formulation.
Although it is believed that a series of these growth factors, including at least those cf the Tsr^-3 family and PDGF, normally act in cooperation with'or.e 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 cn production of scar tissue of reducing or neutralising activity of any one growth factor is likelv 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 activit-r of TGF-8 and/or PDGF can generally be very effective in this respect, inhibition of the activity of certain c`_ the other growth factors may,,at least on :t-S own, be less effective under si:n;lar conditions fo_ reducinc scar 1=i..ssue formation, e=:e^ tnouc'.: such cw:ie= qrJw4...
.. . . .i,I-.,i..,,I I I . .
factors may still be necessary, or may at least have a beneficial effect, in connection with promoting wound healing.
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-A and/or PDGF,.having a major role in the formation of scar tissue in combination with a 4ifferent 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 qua:.ity of healing. At least in some cases, such other additional exogeneous growth factor, for use in combination with TGF-3 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-A and/or PDGF neutralisingagent.(s), a preparation may be obtained which not only prevents scarring of a wound, but also accelerates the whole process of wound healing.
WO 92/17206 PC.'T/GB92/00570 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 expectatiori, 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 eariy stage of healing in order to be effective.
In general, the treatment is best carried out before and/er during the granulation phase whilst f:.brin is st'_i1 present, i.e. before the fibrir, has been -ohol=v rep3aced by granulation tissue. This will usually be within a period of about 1Y days after the i^itia:
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 surgica? wounds the commencement of this treatment, say by topical or parer.tera: application of the growth factor neutralising agent ( s) :.n a pharmaceutical formulation appl_ed to the wound area, mav wel! be incorporated as an '_^tecral par-:
of the surgical procedure and be applied before surgery or immediately the main surgery is completed, before or after suturing.
It has also been found, again somewhat surprisingly, that the treatment does not necessarily need to be repetitive and to be continued throughout a?l 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.
Although it is possible that in some cases the overa'Ll 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 =espect c`_ this latter point, it would see:*
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.
By way of further background explanation a:d descr:.ption of the invention, illustrative examples are hereinafter presented of some of the investigations made and results obtained in the development of the inver.tior., from which the skilled person in the art will :ncre read'_lv be able to aonreciate the nature thereoa and t:, put the invention into practical effect.
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.
The preliminary experimental work in these investiaations was carried out using rats as mode_ exgerimer.;.a'_ animals, but the results are a_ =_icable cenera=l-: _, :':.ata..^.s and cther animals.
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.
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 g_oups of these animals, and according to which group was concerned the injections (100 i- 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.
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 davs 70, 112 and 168. All four wounds were excised (with a 0.5; crr. margir. on all sides ) immediately afte:
;1'~_ 1 i WO 92/17206 PC.'T/GB92/00570 death of each animal and were subjected to tissue analysis by conventional immunohistochemical, histological staining and biochemical techniques.
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, rT, laminin and fibronectin or processed for routine histological examination using a variety of connective tissue stains, or they were immediately freeze-dried for biochemical analyses after microscopic dissection.
In the immunohistochemical analyses, primarj ar.d secondary antibodies for indirect immunostaining were used as showr. in the .4-_'Llowin5 tables.
1 ;i TABLE 1 Primary Antibodies Raised Secondary Ab Against Host Source Dilution (Table 2 refl 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 1:10C 2 COLLAGEN
RAT MOUSE a 1:200 3 & 4 XACROPHAGES
RAT
MONOCYTES & MOUSE a 1:200 3 & 4 MACROPHAGES
HUMAN FACTOR RABB?^_' 1:200 2 V_I.
iC; 1 i I
TA-BLE 2 Secondarv 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 STREP"_'AVIDINE d 1:100 4 Key to Source Codes SEROTEC LTD, Oxford, UK.
b Institut Pasteur de Lyon, France.
c DAROPAT"'S, Copenhagen, Denmark.
AMERSHAM, INTERNATIONAL PIc, Amexsham, UK.
Ncte : Secondarv antibodies 1, 2 and 4 were FITC
conjugated (fluorescein isothiocyanate labelled) for immunofluorometric detection and measurement;
3 was biotynlated.
In carrying out the indirect immunostaining, the i.^.cubation with the primary antibodv was for 1 hour 'ollowed bv three rinses i.^. phosphate buffered sa??ne '?BS _ncubation wit:: _ : rn:-cor.jugated secondary ac-:~-;se_a was for 1 hour f.,_wowed bv a further three . . ~ : 1 h ; , : I i 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 sheeo 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 TM
sections were mounted in a non-fading medium, DABCO
(2,4-diazobicyclo-(2,2,2)-octane), and photographed TM - =rna using a Leitz Dialux microscope and kodak Ektachrome 400 ASA film.
For each primary antibodv and each wound, control sections were stained, substituting P3S for the primary antibody.
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.
For the biochemical analyses, the wounds were microscopica?ly dissected out along with a piece of unwounded dorsa'_ skin from eac^ wound and i.ficned_3te_:
freeze-dried to constant weight. The tissue was homogenised in 1 ml of 1M guanidine hydrochloride, 0.15M sodium acetate, O.O1M
EDTA, pH 5.8, for 24 hours at 4 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 100mM phosphate buffer with 5mM 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 separated using the method of R.
Cappelletti, Mario Del Rosso and Vincenzo Chiarugi: A New Electrophoretic Method for the Complete Separation of all Known Animal Glycosaminoglycans in a Monodimentional Run, Analytical Biochemistry, 1979; vol 99; pp. 311-315.
After washing, the pellets were digested with pepsin 100 g/ml in 0.5M acetic acid at 4 C for 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 & Stadler, Determination of Hydroxyproline, Chimica chimica acta 1987 (18):267-273; 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 B. Sykes, B. Puddle, M. Francis and R. Smith: The estimation of two collagens from human dermis by interrupted gel electrophoresis, Biochem Biophys Res Commun. 1976 Oct 18;72(4):1472-80.
The growth factors used in these experiments were commerically available reagents obtained from R & D Systems (Mineapolis, U.S.A.) or British Biotechnology (U.K.) or Serotec (U.K.) and included:-1. Transforming growth factor beta-1 (TGF-!31) derived from porcine platelets - Dose 10 ng/injection.
2. Platelet derived growth factor (PDGF) from porcine platelets - Dose 10 ng/injection.
3. EAidermal growth factor (EGF) derived from mouse saliva:.-y gland - Dose 10 ng/injection.
4. Basic Fibroblast growth factor (bFGF) derived from bovine brain - Dose 10 ng/injection.
5. Acidic Fibroblast growth factor (aFGF) derived from bovine brain - Dose 10 ng/injection.
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 1. TGF Beta neutralising antibody (raised in rabbit against native porcine platelet TGF-!31 -neutralises both TGF13-1 and TGF13-2) - Dose 50 ug/injection.
' I'- a. . . . . I i . CA 02387247 2002-06-25 r r -WO 92/17206 PC.?/6B92/005711 2. PDGF neutralising antibody (raised in goats against native human PDGF) - Dose 20 g/
injection.
3. EGF neutralising antibody (polyclonal antibody raised in mouse against human EGF) - Dose 10 pg/injection.
4. Basic FGF neutralising antibody (raised ir.
rabbits against native bovine brain basic FGF: -Dose 30 g/injection.
5. Acidic FGF neutralising antibody ;raised in rabbit against native bovine brain ac-;dic FGF) -Dose 30 g/injection.
The irrelevant antibodies used fo, the sham control. wounds were either rabbit IgG or goat IgG
according to the host in which neutralising antibody t--the growth factor was raised. The dose of the irrelevant antibody was similar to that of the neutralising antibody.
SummarY of Results In all the experiments conducted, nc differences were found be z~ween the cc::tro: ar.c sham co.. z_ =_ wcLnds ` , .
at anv 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.
However, at least in the case of the experimental wounds treated with the neutralising antibodies to TGF-B 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 fo= the loss of ectodermal hair ioi'_:c? es ;. T:^.is was in s'r.aro contrast to the ot^e_ . =
WO 92/17206 PCT/GB92/005%t-wounds which showed a distinct scar with verzica?i:-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-3 or aDGF
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 az the dermal/epidermal junction. Treatment with neutralising antibodies to TGF-B and PDGF also decreased the number of blood vessels, monocytes and macrophages within the healing wound. By contrast, the pc_s'uti=re control wounds treated with TGF-li and PDGF showed a marked increase in extraceIlular matrix accumulati:.n, in the density of extraceiiular matrix packing and in the number of blood vessels, monocytes and macrophages.
Scarring was more prominent in these growth factor treated wounds comnared to controls.
These results demonstrate the ability of neturalising antibodies to selected growth fac-~:ors markedly to reduce scar tissue formation in adult dermal wound healing. Most importantly, this advantageoss effect was not accomaanied by attendant prob?ems cf delaved wound hea_i^c ^= d3laved epithe? ialis=_cion ani iow wound strenct^.
WO 92/17206 PCf/GB92/00570 Some improvement in reducing sca-r 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-B
and PDGF growth factors. Interestingly, exogenous acidic or basic FGF itself seemed to improve scarring.
It is, however, believed that similar results to TGF-!i 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.
In the case o` TGF-Ji at least, which appears to be highly active in connection with the production and organisation of coilagen, especially collagen I, leading to the formation cf scar tissue, it is believed tha-, 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-A 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 ir.-~'iammatcry cells, especially macrophages, release TGF-A and exhibit this auto-inductzve effect on TGF-f;
synthesis. TGF-!i also stimulates the synthesis and release of other growth factors, e.g. TGF-4C, PDGF, EGF.
TGF-8, 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 proteolyric turnover and organisation of these extracellular matr?x molecules. As the initial fibrin clot 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 extrace1?:::ar matrix molecules are also deposited in this abno:^nal orientation and this eventually gives rise to the scar.
It can be hypothesised that normal wound heal'_.^._ in adults is phylogenetically optiznised for speed of cl-osure in adverse healing conditions. Consequently, the quantities of growth factor released are generallv excessive, giving the speed of the healing process considerable buffering against external noxious factors but with the long term disadvantage of scarring. Moderr:
methods of wound care, e.g. bandages, and reduction in risks of infection have largely eliminated the necessity cf this growth factor "overdrive" so that treatment t:, diminish the active growt:: factc_ prof_=e -G per::-iss;:,le a^c wi__ minimise subsecuer_-z s;.a=_:. :, ~ ~ .
Thus the autocatalytic cascade of events, described above for TGF-J3, is reduced by treatment at an early stage with a neutralising 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.
Practical Usace 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 prac*_ical use, in general an appropriate amount of:
growth factor antibodv 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 a.ctive and responsible for scar tissue 46ormation in the healing of wounds, will be made up bv any of the methods well-known in the art of pharmacv as a pharmaceutical formulat.ion or prepar-ation for administration in any suitable manner to a oatient in need of wounc treatment. Such pharmaceuticai -fo*_-nulat_onG c, preparatic^s may, moreover, be presented a ,I I
WO 92/17206 PCT/f,'892/00570 not only individually for clinical use but they may also be presented as components of first aid kits for example for non-clinical emergency use.
By way of example in relation to TGF-B and PDGF
grawth factors, in general the antibody or antibodies or other neutralising agent(s) should be administered (at least fbr incisional wounds) so as to effectively neutralise between 1 pg - 1 pg TGF-A (:. and 2; and/cr PDGF (but preferably an amount of between 100 pg - 1C
ng) per cm linear incision per administration. As previously indicated, application early in the wound healing process is essential. Normally this wil; be before, during or before and during, the phase of ' cranulation tissue formation, within about 14 3avs, but preÃerably within 7 or 3 days, or less, f:,,iow?^g, wcunding.
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, especiallv subcutaneous i^jection, may also often be preferred so that the n a::t_alisi.:ig antibodv or antibodies c= other agertr s .14:~, .. , I i 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 ass.ociation or admixture with at least one other ingredient constituting a compatible pharmaceutically acceptable carrier, diluent cr 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, hiodegradable/
absorbable patches or dressings for topical application, or to slow release implant systems with a high initial release decaying to a slow release.
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 i.^.= : ia? lv and more slowly in the longer ter=n when a, , ;I , +~ ~ =
WO 92/17206 PCT/GB92/005?() applied to, or implanted within, the wound or tissue viod.
As will be seen, the invention provides a number of different aspects and, in general, it embraces all nove'L 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 bv the ter:ns and expressions used herein merely in a descriptive or explanatory sense.
Claims (25)
1. A composition for inhibiting scar tissue formation during wound healing, the composition comprising a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of transforming growth factor-.beta.1 (TGF-.beta.1) or transforming growth factor-.beta.2 (TGF-.beta.2), or both, together with a pharmaceutically acceptable carrier, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the composition is formulated for administration in a wound area before the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
2. A composition for inhibiting scar tissue formation during wound healing, the composition comprising a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-.beta.1 or TGF-.beta.2, or both, together with a pharmaceutically acceptable carrier, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the composition is formulated for administration in a wound area during the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
3. The composition according to claim 1 or 2, wherein the growth factor-neutralizing agent is encapsulated.
4. The composition according to claim 3, further comprising a capsule which is degradable by an external stimulus to release the growth factor-neutralizing agent.
5. The composition according to claim 4, wherein the external stimulus is UV light, an in vivo enzyme, ultrasound or heat.
6. The composition according to any one of claims 1 to 5, wherein the growth factor-neutralizing agent is bound to a binding molecule to be maintained in an inactive form.
7. The composition according to claim 6, wherein the binding molecule is detached from the growth factor-neutralizing agent by UV light, an in vivo enzyme, ultrasound or heat.
8. The composition according to any one of claims 1 to 7, wherein the pharmaceutically acceptable carrier is a neutral sterile cream, gel, aerosol or powder for topical application.
9. The composition according to any one of claims 1 to 7, wherein the pharmaceutically acceptable carrier is a sterile solution for injection, irrigation or inhalation.
10. The composition according to any one of claims 1 to 7, wherein the pharmaceutically acceptable carrier comprises a sterile dressing for topically covering a wound.
11. The composition according to any one of claims 1 to 7, wherein the pharmaceutically acceptable carrier is a biopolymer or a polymer for implanting within a wound.
12. The composition according to any one of claims 1 to 11, wherein the composition is formulated for administration with a fibroblast growth factor.
13. Use of a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-.beta.1 or TGF-.beta.2, or both, in the manufacture of a medicament, for inhibiting scar tissue formation during wound healing, wherein the agent neutralizes the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the medicament is formulated for administration in a wound area before the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
14. Use of a growth factor-neutralizing agent comprising an antisense oligonucleotide specific to a mRNA of TGF-.beta.1 or TGF-.beta.2, or both, in the manufacture of a medicament, for inhibiting scar tissue formation during wound healing, wherein the agent neutralises the stimulation of macrophage infiltration, fibroblast migration, extracellular matrix synthesis or deposition by fibroblasts, and wherein the medicament is formulated for administration in a wound area during the granulation phase in a dosage effective to reduce fibrotic activity of the growth factor.
15. The use according to claim 13 or 14, wherein the growth factor-neutralizing agent is encapsulated.
16. The use according to claim 15, wherein the agent is encapsulated with a capsule that is degradable by an external stimulus to release the growth factor-neutralizing agent.
17. The use according to claim 16, wherein the external stimulus is UV light, an in vivo enzyme, ultrasound or heat.
18. The use according to any one of claims 13 to 17, wherein the growth factor-neutralizing agent is bound to a binding molecule to be maintained in an inactive form.
19. The use according to claim 18, wherein the binding molecule is detached from the growth factor-neutralizing agent by UV light, an in vivo enzyme, ultrasound or heat.
20. The use according to any one of claims 13 to 19, wherein the growth factor-neutralizing agent is used in conjunction with a pharmaceutically acceptable carrier.
21. The use according to claim 20, wherein the pharmaceutically acceptable carrier is a neutral sterile cream, gel, aerosol or powder for topical application.
22. The use according to claim 20, wherein the pharmaceutically acceptable carrier is a sterile solution for injection, irrigation or inhalation.
23. The use according to claim 20, wherein the pharmaceutically acceptable carrier comprises a sterile dressing for topically covering a wound.
24. The use according to claim 20, wherein the pharmaceutically acceptable carrier is a biopolymer or a polymer for implanting within the wound.
25. The use according to any one of claims 13 to 24, wherein the medicament is formulated for administration with a fibroblast growth factor.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9106678.7 | 1991-03-28 | ||
| GB919106678A GB9106678D0 (en) | 1991-03-28 | 1991-03-28 | Wound healing |
| CA002105652A CA2105652C (en) | 1991-03-28 | 1992-03-30 | Reducing wound scarring with antibodies to growth factors |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002105652A Division CA2105652C (en) | 1991-03-28 | 1992-03-30 | Reducing wound scarring with antibodies to growth factors |
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| CA2387247A1 CA2387247A1 (en) | 1992-10-15 |
| CA2387247C true CA2387247C (en) | 2009-05-19 |
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| CA002387247A Expired - Lifetime CA2387247C (en) | 1991-03-28 | 1992-03-30 | Use of neutralizing agents to growth factors for reducing wound scarring |
| CA002105652A Expired - Lifetime CA2105652C (en) | 1991-03-28 | 1992-03-30 | Reducing wound scarring with antibodies to growth factors |
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| CA002105652A Expired - Lifetime CA2105652C (en) | 1991-03-28 | 1992-03-30 | Reducing wound scarring with antibodies to growth factors |
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| US (4) | US5662904A (en) |
| EP (1) | EP0585242B2 (en) |
| JP (3) | JP3333507B2 (en) |
| AT (1) | ATE182792T1 (en) |
| AU (1) | AU661840B2 (en) |
| CA (2) | CA2387247C (en) |
| DE (1) | DE69229729T3 (en) |
| DK (1) | DK0585242T4 (en) |
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| GB (1) | GB9106678D0 (en) |
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| US5654270A (en) | 1988-06-28 | 1997-08-05 | La Jolla Cancer Research Foundation | Use of fibromodulin to prevent or reduce dermal scarring |
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1991
- 1991-03-28 GB GB919106678A patent/GB9106678D0/en active Pending
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1992
- 1992-03-30 ES ES92907214T patent/ES2136618T5/en not_active Expired - Lifetime
- 1992-03-30 AT AT92907214T patent/ATE182792T1/en not_active IP Right Cessation
- 1992-03-30 AU AU14368/92A patent/AU661840B2/en not_active Ceased
- 1992-03-30 DE DE69229729T patent/DE69229729T3/en not_active Expired - Lifetime
- 1992-03-30 US US08/122,508 patent/US5662904A/en not_active Expired - Lifetime
- 1992-03-30 WO PCT/GB1992/000570 patent/WO1992017206A1/en active IP Right Grant
- 1992-03-30 DK DK92907214T patent/DK0585242T4/en active
- 1992-03-30 JP JP50694492A patent/JP3333507B2/en not_active Expired - Fee Related
- 1992-03-30 CA CA002387247A patent/CA2387247C/en not_active Expired - Lifetime
- 1992-03-30 EP EP92907214A patent/EP0585242B2/en not_active Expired - Lifetime
- 1992-03-30 IE IE101392A patent/IE921013A1/en not_active IP Right Cessation
- 1992-03-30 CA CA002105652A patent/CA2105652C/en not_active Expired - Lifetime
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1999
- 1999-08-05 GR GR990401862T patent/GR3030924T3/en unknown
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2002
- 2002-02-06 JP JP2002029691A patent/JP2002275094A/en active Pending
- 2002-04-08 US US10/117,351 patent/US20020187149A1/en not_active Abandoned
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2005
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- 2005-09-14 JP JP2005267284A patent/JP2006001949A/en active Pending
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2009
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| US20020187149A1 (en) | 2002-12-12 |
| GR3030924T3 (en) | 1999-11-30 |
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| US5662904A (en) | 1997-09-02 |
| IE921013A1 (en) | 1992-10-07 |
| CA2387247A1 (en) | 1992-10-15 |
| DK0585242T3 (en) | 1999-12-06 |
| EP0585242B2 (en) | 2007-03-07 |
| JPH06506205A (en) | 1994-07-14 |
| JP2002275094A (en) | 2002-09-25 |
| EP0585242B1 (en) | 1999-08-04 |
| ES2136618T5 (en) | 2007-10-16 |
| JP2006001949A (en) | 2006-01-05 |
| US20050287139A1 (en) | 2005-12-29 |
| EP0585242A1 (en) | 1994-03-09 |
| ES2136618T3 (en) | 1999-12-01 |
| DE69229729T2 (en) | 2000-01-13 |
| DK0585242T4 (en) | 2007-07-02 |
| CA2105652C (en) | 2005-09-06 |
| DE69229729T3 (en) | 2007-10-18 |
| US20100152095A1 (en) | 2010-06-17 |
| JP3333507B2 (en) | 2002-10-15 |
| AU661840B2 (en) | 1995-08-10 |
| ATE182792T1 (en) | 1999-08-15 |
| DE69229729D1 (en) | 1999-09-09 |
| AU1436892A (en) | 1992-11-02 |
| CA2105652A1 (en) | 1992-09-29 |
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