GB2288118A - Wound healing composition - Google Patents

Abstract

A healing composition is disclosed comprising at least one neutralising antibody or fragment (eg, FAB) thereof of appropriate affinity to a growth factor in combination with a pharmaceutically acceptable carrier.

Description

WOUND HEALING III This invention concerns a composition for promoting the healing of wounds, particularly, though by no means exclusively, for promoting the healing of chronic wounds.

At present there is a lack of effective treatments for chronic wounds such as venous ulcers, diabetic ulcers and bed sores (decubitus ulcers) especially in the elderly and wheel chair bound patients. Additionally, in elderly patients, the wound healing repair process is slower and these patients tend to have a higher incidence of wounds, for example, burns, diabetic ulcers, venous ulcers and the like, which heal slowly or not at all.

The present invention provides a composition which promotes the healing of chronic wounds.

According to the present invention there is provided a healing composition comprising at least one neutralising antibody or fragment (eg, FAB) thereof of appropriate affinity to a growth factor in combination with a pharmaceutically acceptable carrier.

The antibody may be a monoclonal, phage, polyclonal or genetically engineered antibody eg diabody and may be produced in transgenic mice.

Antibody fragments eg FAB may contain only the active binding sites.

The antibody may be specific for a single epitope on the growth factor. The affinity of the neutralising antibody to the growth factor must be appropriate to cause potentiation of the growth factor and not neutralisation. The increased level of endogenous growth factor so induced promotes the healing of a wound.

This composition is particularly applicable to the healing of chronic wounds as a potentiation of endogenous growth factor may help to "kick start" the healing process.

A potentiation of a growth factor in a healing wound by the addition of neutralising antibody thereto is unexpected but has previously been observed in a different system. Heremans et al (Eur. J. Immunol., 1992, 22, 2395-2401) and Martens at al (Eur. J.

Immunol., 1993, 23, 2026-2029) showed a potentiation of the cytokine IL-6 when endogenous monoclonal antibodies against IL-6 were given in mice injected with endotoxin to induce generalised Shwartzman reaction (endotoxin shock).

The hypotheses put forward by the research teams to explain these results may be applied to the present inventors own findings. For example, they propose that if the affinity of the cytokine receptor for the cytokine is higher than that of the antibody, the cytokine may still be active despite its binding to the antibody. Alternatively, the increased cytokine levels after administration of antibody may be due to delayed elimination or to increased production. Delayed elimination of endogenous cytokine might be brought about by complex formation of the cytokine with circulating antibody. Such antibodies may act as carriers of cytokines in the circulation preventing their elimination from the circulation, urine etc.

Alternatively, or additionally, a cytokine may exert negative control over its own synthesis and/or release in vivo. Thus partial neutralisation or sequestration of the cytokine at the tissue level may depress release, resulting in higher production levels (illustrated in Figure 1).

However, the above hypotheses have not been proven and other~mechanisms may be involved which are as yet unknown.

The growth factor may be any growth factor known to be involved in the promotion of wound healing, for example, TGF-13l, TGF-132, TGF-ss3, PDGF-A, PDGF-B, FGF1 FGF2, FGF7, IGF-I, IGF-II, IL1, IL6, IL8, IL10 and TNF The present inventor found that a neutralising monoclonal antibody of low affinity to TGF-ss2 had no anti-scarring effect on the healing of full thickness wounds in adult rats (see the experimental details below and Figure 4) compared to control wounds. Neutralising monoclonal antibody of low affinity to TGF-13l showed a small decrease in scarring whilst a combination of the two monoclonal antibodies gave wound healing without scarring.

These results are more fully understood for the first time as indicating that when a monoclonal antibody to a single epitope of TGF-ss is given, this is effectively the same as giving the growth factor itself, ie., a low affinity, single epitope recognising antibody potentiates the effect of the growth factor and may in fact increase the half life of the growth factor due to its reduced elimination from the circulation. Thus anti-TGF-ssl or anti-TGF-ss2 alone potentiates the effect of TGF-ssl and TFG-ss2 and heals a wound by the formation of scar tissue. ~However, a combination of anti-TGF-ssl and anti-TGF-ss2 causes neutralisation of the growth factors instead of potentiation, resulting in depression of the action of these fibrotic growth factors and wound healing without scarring.

It is likely that recognition of two or more epitopes by two or more neutralising antibodies or fragments thereof results in the formation of a large enough antibody/growth factor complex for rapid detection and clearing by the body's systems (by kupfer cells of the liver), and hence neutralisation. By contrast the detection of a single epitope by a single antibody, or fragment thereof, may result in a small complex, not easily detected and hence potentiation of the growth factor. Further, as summarised in Figs 2 and 3 the affinity of the antibody (low affinity for potentiation, high affinity for neutralisation) and ratio of antibody to growth factor (high for neutralisation, low for potentiation) etc are likely to influence the end result. It follows that the same antibody may be used to acheive either neutralisation or potentiation according to circumstances and to whether it is coupled with another neutralising antibody recognising a different epitope. It further follows that genetically engineered antibodies or coupled antibodies or fragments thereof eg., diabodies capable of for example binding one or more epitope or fragment larger complexes might also be used for the purposes of neutralisation.

The blocking and potentiating properties of neutralising antibodies to TGF-Bl and TGF-132 are summarised in Figures 2 and 3.

Thus the composition of the present invention may be used in combination with known compositions which heal wounds without scarring. It is envisaged that chronic wounds would first be treated with monoclonal antibodies of low affinity to a single epitope on a growth factor to potentiate its effect and "kick start" the wound healing process, followed shortly thereafter by treatment with a composition known to promote wound healing without scarring (as disclosed in PCT/GB93/00586) or by changing to a combination of antibodies (preferably of high affinity) to fibrotic growth factors to inhibit scarring. Thus it is envisaged that chronic wounds may not only be healed, but healed with improved scarring.

EXPERIMENTAL DETAILS Adult, male Sprague-Dawley rats (Charles River UK Ltd., Kent, UK) weighing 225-250 grammes were anaesthetised by halothane, nitrous oxide and oxygen inhalation. After locally clipping the fur, four full-thickness, linear incisions, lcm in length, down to and including the panniculus carnosus were made on the dorsal skin of the animal. The incisions were placed equidistant from the midline and adjacent to the four limbs (Figure 4).

In each animal one of the wounds (control) was unmanipulated, one (sham-control) was injected with PBS/0.1%BSA/4mM HC1 (carrier used to make up the TGF-ss isoforms), one was injected with TGF-ssl or TGF-R2, and one was injected with neutralising antibody to TGF-ssl alone, neutralising antibody to TGF-ss2 alone or a combination of neutralising antibodies to TGF-ssl and TGF-ss2. The actual site of each treatment was rotated between the four wounds to control for anterior-posterior differences in the healing of rodent wounds. All injections were of 100 ul each and administered by local infiltration of the wound margins (inset in Figure 4). The wounds were treated on days 0, 1 and 2 post-wounding. Animals were allowed to recover and housed in individual cages and fed normal rat chow and water ad libitum. Animals were killed by chloroform overdose on various days after wounding and the wounds harvested. All procedures were carried out according to Home Office regulations and under appropriate licences.

The isoform specific neutralising antibodies and the TGF-ss isoforms used in these experiments were a gift from Dr. A.B.Roberts (Lab. of Chemoprevention, NIH, Bethesda, USA). These antibodies have been well characterised and the specificities established (Danielpour et al, In Growth Factors, 2:61, 1989.) Results and discussion of the results of these experiments are published in the PhD Thesis of Mamta Shah, Cutaneous Scarring : Modulation by Transforming Growth Factor-Beta and its Antagonists, University of Manchester, 1993, Chapter 5.

Claims (14)

1. A healing composition comprising at least one neutralising antibody or fragment (eg, FAB) thereof of appropriate affinity to a growth factor in combination with a pharmaceutically acceptable carrier.

2. A composition according to claim 1, wherein the antibody is a monoclonal, phage, polyclonal or genetically engineered antibody eg diabody.

3. A composition according to claim 1 or claim 2, wherein the antibody is produced in transgenic mice.

4. A composition according to claim 1, wherein the antibody fragments eg FAB contain only the active binding sites.

5. A composition according to any preceding claim, wherein the antibody is specific for a single epitope on the growth factor.

6. A composition accoridng to any preceding claim, wherein the affinity of the neutralising antibody to the growth factor is appropriate to cause potentiation of the growth factor and not neutralisation so that the increased level of endogenous growth factor so induced promotes the healing of a wound.

7. A composition according to any preceding claim, which is designed to be effective in the healing of chronic wounds.

8. A composition according to any preceding claim, wherein the growth factor is chosen from the group of growth factors known to be involved in the promotion of wound healing, for example, TGF-13l, TGF-ss2, TGF-B3, PDGF-A, PDGF-B, FGF1 FGF2, FGF7, IGF-I, IGF-II, IL1, IL6, IL8, IL10 and TNF

9. A composition according to any preceding claim, used in combination with compositions which heal wounds without scarring.

10. A composition according to claim 9, wherein the composition which heals wounds without scarring comprises at least one non-fibrotic growth factor.

11. A composition according to claim 10, wherein the non-fibrotic growth factor comprises TGss-ss3.

12. A composition according to claim 11, wherein the non-fibrotic growth factor comprises FGF.

13. A composition according to any one of claims 9 to 12, wherein the composition which heals wounds without scarring comprises neutralising antibodies to at least one fibrotic growth factor wherein the affinity of said antibodies is appropriate to cause neutralisation of said fibrotic growth factor.

14. A composition according to claim 13, wherein the fibrotic growth factor is chosen from the group comprising, TGF-ssl, TGF-ss2, PDGF.