WO2002064178A1

WO2002064178A1 - Use of green tea extract for wound healing

Info

Publication number: WO2002064178A1
Application number: PCT/GB2002/000627
Authority: WO
Inventors: Keith Helliwell
Original assignee: William Ransom & Son Plc
Priority date: 2001-02-15
Filing date: 2002-02-14
Publication date: 2002-08-22

Abstract

The use of green tea extracts in dressing of wounds can in appropriate concentrations in the range 0.1 to less than 10.0 µg/ml (or g) increase the proliferation of human keratinocytes in order to promote more rapid healing. By exposing the wound to appropriate concentrations of green tea extract the rate of healing can be controlled to reduce the production of scar tissue

Description

US_E OF GREEN TEA EXTRACT FOR WOUND HEALING

Background of the Invention

The present invention relates to the healing of wounds and, more specifically, to a product for topical application to the wound in order to facilitate effective healing. In the context of this specification the term wound is to be construed as a trauma wound that penetrates the skin. Examples are wounds caused in an accident or by a third degree burn or deep ulcers such as bedsores and venous ulcers.

The invention relates to the use of extracts of green tea for the manufacture of a medicament for the treatment of such wounds to facilitate effective healing.

Technical Background

Various factors influence the rate at which a wound heals. For example, in order for the skin to reform over the wound, it is necessary for the keratinocytes from which the epidermis is formed to proliferate. By creating conditions in which there is increased proliferation of the keratinocytes, healing can be accelerated. Promoting migration of keratinocytes across a wound site to restore the epidermal layer will also favourably influence the rate and effectiveness of wound healing.

Fibroblasts are cells that draw the wound together. Controlling the rate of proliferation of the fibroblasts can h ' c an effect on whether a scar is left when the wound heals as well as on the speed of healing.

Since there is much activity and cell production at the site of a wound in order for healing to take place, it is desirable to create conditions in which blood flow to the site is enhanced. Therefore, stimulation of the growth of blood capillary tissue or human dermal microvascular endothelial cells (HDMEC) is useful.

For the purposes of this specification, it will be understood that a wound is healed effectively if it is healed quickly and without leaving a scar. To some extent, compromise is necessary as to speed in order to allow time for a continuous layer of skin to form over the wound. Over rapid healing may result in the unsightly puckering of the wound edges.

Prior Art

It is known to dress wounds with products in liquid or ointment form. It is also known to provide dressings for application to a wound that are impregnated with or otherwise have such products applied to them for release into the wound. Typically these products are primarily concerned with creating antiseptic conditions.

The use of various products and formulations for enhancing the production of human epidermal keratinocyte cells has been proposed to accelerate wound healing. These products typically include various growth factors. See for example US-A-5461030 assigned to Life Medical Science, Inc., which teaches the use of formulations that comprise an effective amount of a serum free cellular nutrient medium in combination with an effective amount of at least one cellular growth stimulating compound such as a natural anabolic hormone or transforming growth factor. These ideas are based on technology for culturing layers of keratinocyte cells for use in skin grafts.

US-A- 6022862 assigned to VimRx Pharmaceuticals, Inc., suggests contacting a wound with an effective wound-healing amount of a composition comprising a pseudopterosin or pseudopterosin derivative. This is believed to promote the growth and proliferation of keratinocytes, fibroblasts and endothelial cells. The pseudopterosins are a class of natural products isolated from the sea whip Pseudopterogorgia.

Solution of the Invention

The present invention is defined in the claims and relies on the recognition that green tea extracts (GTE) in an appropriate concentration can be used in the promotion of effective wound healing by topical application to the wound either in the form of a medicament or by means of impregnation into a wound dressing.

There are existing known pharmaceutical uses of GTE, of which the best known is its well documented antibacterial effect. For this purpose concentrations in excess of lmg/ml are typically employed. Topical use of GTE for treatment of various skin infections has been proposed. For example GB-A-2293 548 describes a treatment for Herpes. DE-A-198 38 918 describes a treatment for eczema and JP 10218784 suggests a treatment for allergic dermatitis. Although such infections result in skin lesions these are excluded from the definition of wound in the present specification. The GTE is primarily proposed for the treatment of the infection as a result of which the skin lesions can be expected to heal naturally.

WO 00/47193 A2 (Karolinska Innovations) discloses the use of GTE to treat a variety of conditions relating to angiogenesis, including wound granulation and scar formation. Therefore these treatments are for cases where there has been excessive blood vessel regeneration such as may occur after a wound has healed ineffectively. The experimental work described by Karolinska demonstrates that EGCG (Epigallocatechin-3 gallate) - one of the major catechins present ion GTE inhibits microvascular endothelial cell proliferation. This suggests that GTE would not be useful in wound healing. However Karolinska does not propose the topical application of GTE to a wound and is therefore directed to a different medical use.

It has now been appreciated that the use of green tea extracts in appropriate concentrations preferably in the range 0.1 to less than 10.0 μg/ml (or g) can increase the proliferation of human keratinocytes in order to promote more rapid healing. The antioxidant and antibacterial effects of catechins found in green tea extracts is documented. However the potential of GTE for enhancing effective wound healing by a direct effect on cell proliferation has not previously been appreciated or exploited.

In certain concentrations an extract of green tea may result in inhibition of proliferation of human fibroblasts. Since it has been established that this inhibition is reversible then controlling the amount of green tea to which the wound is exposed during the healing process can be used to control the possibility of scarring more precisely. For example if fibroblast proliferation is inhibited until a sufficient quantity of keratinocyte cells have been generated to restore the epidermal layer, the risk of scar formation and uneven healing may be reduced.

Brief Description of the Drawings

In order that the invention may be well understood some tests and experiments will now be described, by way of example only, with reference to the accompanying diagrammatic drawings, in which:

Figure 1 shows the results from Experiment 1 in lest A using normal human keratinocytes;

Figure 2 shows the results from Experiment 2 in Test A using normal human keratinocytes; Figure 3 shows the results from Experiment 1 in Test B using normal human dermal fibroblasts;

Figure 4 shows the results from Experiment 2 in Test B using normal human dermal fibroblasts;

Figure 5 shows photographs of the results from Experiment 1 in Test C wherein;

photograph a shows a control sample after four days in a base support medium;

photograph b shows the sample with 10 μg/ml GTE after four days in a base support medium;

photograph c shows the sample treated with GTE after seven days, the medium having been changed to a full support medium on day 4;

photograph d shows the sample treated with GTE after seven days, the medium having been changed for further base support medium on day 4;

photograph e shows the sample treated with GTE after ten days, the medium having been changed to a full support medium on day 4;

photograph f shows the sample treated with GTE after ten days, the medium having been changed for further base support medium on day 4; Figure 6 shows the results from Experiment 1 Section 4 in Test C wherein;

photograph a shows a control sample after twelve days in a base support medium.

photograph b shows the control sample after sixteen days, the medium having been changed to a full support medium on day 12;

photograph c shows the sample with lOμg/ml GTE after twelve days in a base support medium;

photograph d shows the sample treated with GTE after sixteen days the medium having been changed for further base support medium on day 12;

photograph e shows the sample treated with GTE after sixteen days the medium having been changed to a full support medium on day 12.

Figure 7 shows the results from Experiment 2 Section 1 in Test C wherein:

photograph a shows the sample with lOμg/ml TGE after four days in a base support medium;

photograph b shows the sample with 10 μg/ml GTE after seven days in a base support medium;

photograph c shows the sample treated with GTE after seven days, the medium having been changed for further base support medium on day four;

photograph d shows the sample treated with GTE after seven days, the medium having been changed to a full support medium on day four;

photograph e shows the sample with lOμg/ml GTE afer ten days in a base support medium;

photograph f shows the sample treated with GTE after ten days, the 5 medium having been changed for further base support medium on day 4;

photograph g shows the sample treated with GTE after ten days, the medium having been changed to a full support medium on day 4.

Figure 8 shows the results from Experiment 2 Section 2 in Test C wherein:

photograph a shows the sample with lOμg/ml GTE after seven days in a o base support medium;

photograph b shows the sample with lOμg/ml GTE after ten days in a base support medium;

photograph c shows the sample treated with GTE after ten days, the medium having been changed for further base support medium at day 5 seven;

photograph d shows the sample treated with GTE after ten days, the medium having been changed to a full support medium on day seven;

photograph e shows the sample with lOμg/ml GTE after thirteen days in a base support medium;

o photograph f shows the sample treated with GTE after thirteen days, the medium having been changed for further base support medium at day seven;

photograph g shows the sample treated with GTE after thirteen days, the medium having been changed for a full support medium on day seven.

Figure 9 shows the results from Experiment 1 in Test E using human dermal microvascular endothelial cell (HDMEC)

Figure 10 shows the results from Experiment 2 in Test E using human dermal micovascular endothelial cells (HDMEC).

Description of Preferred Embodiment

A product for topical application to a wound is prepared by dissolution or incorporation of an extract of green tea in a suitable base. The product can be prepared for application in a number of dosage forms including, but not restricted to, sterile liquids, ointments or powders or embedded in suitable soluble or non-soluble dressings. Preferably the concentration of the green tea extract is in the range 0.1 to 10 μg/ml (or g).

Various forms of green tea extract are commercially available. Green tea extract will normally be quantified on up to eight different catechins in varying proportions and at a preferred concentration of green tea extract in the range 0.1 μg to 10μg/ml(or g) this will deliver 0.025 to 2.5 μg/ml(or ) of catechins in the dosage form..

Evidence of Efficacy

A number of tests were carried out to establish the effect of GTE on the growth of normal human skin cells. For the purposes of these tests the GTE was dissolved in water and sterilized by filtration through a 0.2μ membrane to make a concentrated stock solution of 5mg/ml. The container was covered with foil and stored at 4°C. Various concentrations of the complex in culture medium were prepared from the stock solution on the first day that the complex was to be added to cells in a given experiment. These working solutions were then stored at 4°C and used for re-feeding the cells during the given experiment.

The following tests were carried out:

Test A to establish the effect of GTE on the growth of normal human keratinocytes;

Test B to establish the effect of GTE on the growth of normal human dermal fibroblasts;

Test C to determine the degree of reversibility of GTE on the effect on the growth of normal human dermal fibroblasts;

Test D to establish the possible antiprotease effect of GTE; and

Test E to establish the effect of GTE on the proliferation of human dermal microvascular endothelial cells (HDMEC).

Test A to establish the effect of GTE on the growth of normal human keratinocytes

Experiment 1

The number of control cells in the basal medium decreased during the course of the experiment, being marginally fewer in number on day 4 (Figure 1) and decreasing markedly over the following four days (p < 0.01). The lower doses of GTE stimulated proliferation, for example, 1 μg/ml at day 6 (p <0.0001). Cell numbers in 10 μg/ml of the complex were fewer than in lower concentrations, but by day 6 were ninefold higher than in the control cells in basal medium. Experiment 2

Although cells were seeded at the same density and the same time schedule was followed, the cells in experiment 2 grew better during the three days after seeding. By the day that the initial dose of GTE was added (Day 0) there were actually twice as many cells as in experiment 1. During the next 8 days, the cells in control medium did decrease in number, but not so markedly as in the first experiment (Figure 2). Concentrations of 0.1 μg/ml and 1.0 μg/ml again stimulated cell growth relevant to controls cells at all time points, although there was a decrease in cell number by day 8 to approximately the same 0 number as at the start of the experiment. In 10.0 μg/ml of GTE the number of cells was lower than in controls cells.

Summary of effect on growth of normal human keratinocytes

In both experiments concentrations of 0.1 μg/ml and 1.0 μg/ml of GTE protected human keratinocytes from the deterioration that occurred in cells maintained in the basal (growth 5 factor deficient medium) and stimulated proliferation compared with day 0. The effects appeared to be more marked in the experiment when the number of keratinocytes was lower.

The basal medium (KBM) contains all the nutrients such as amino acids, vitamins and glucose, but does not contain the growth factors which are essential for keratinocyte o proliferation. It is known that normal human keratinocytes maintained in growth factor- deficient medium go into irreversible growth arrest [Pittlelkow, 1986], but it would appear that GTE contains a component that has either a protective effect or acts as a growth factor for keratinocytes. Reference

Pittelkow MR, Wille JJ, Scott RE. Two functionally distinct classes of growth arrest states in Human Prokeratinocytes that Regulate Clonogenic Potential. J Invest Dermatol 1986; 86: 410-417.

Test B to establish the effect of GTE on the growth of normal human dermal fibroblasts

Experiment 1

By day 4 after adding the GTE there was a dose-related decrease in cell numbers compared to control cells (Figure 3), although the decrease only reached statistical significance in 10.0 μg/ml (pθ.001). However, the numbers of cells in 0.1 μg/ml of

GTE were significantly higher (p<0.05) than when the GTE was first added although they decreased during the following four days as did the number of cells in control medium. The number of cells in 10.0 μg/ml remained approximately the same during the eight days of the experiment.

Experiment 2

Fibroblasts were seeded at a lower density to maintain cells in a non-confluent state throughout the experiment. The trend to decreased growth in GTE was more marked than in the first Experiment 1 (Figure 4). By day 7 the numbers of fibroblasts in 1.0 μg/ml appeared to recover and were not significantly different from those in 0.1 μg/ml. Although numbers were lower than in control medium the difference did not reach statistical significance. The number of fibroblasts in 10.0 μg/ml of GTE decreased to 30% of controls (p<0.001). Summary of effect on growth of normal human dermal fibroblasts

In both experiments it appears that all concentrations of GTE cause some inhibition to the growth of normal human dermal fibroblasts. A series of experiments were commissioned to investigate whether this process is either a continuing regression or reversible.

Test C to determine the degree of reversibility of GTE on the effect on the growth of normal human dermal fibroblasts

Methodology as for experiments in Test [B] 'Effect of GTE on the growth of normal human dermal fibroblasts' .

Experiment 1 - replacing the medium at day 4 and day 12

1. The 10 μg/ml dose was tested because if the inhibition observed in some experiments is shown to be reversible for this concentration it will be reversible for lower doses.

2. By day 4 there was no difference observed relative to the controls, no decrease in cell numbers (See Figure 5 (a) and (b))

On day 4 dishes that had been in 10 μg/ml of GTE were divided into 3 sets.

i) Maintained in 10 μg/ml of GTE

ii) Medium changed to DMEM (Dulbecco's Modified Eagle Medium) only

iii) Medium changed to DMEM/10% FCS (Dulbecco's Modified Eagle Medium/10% Foetal Calf Serum)

3. i) on prolonged exposure to 10 μg/ml GTE morphological changes could be seen under the microscope, with more globular cells.

ii) Cells switched to DMEM only underwent a slight deterioration after further 3 and 6 days, with more globular cells (Figure 5 (d) and (f)).

iii) In DMEM/ 10% FCS recovery of cells from 4 days exposure to GTE 10 μg/ml was excellent and six days after medium change the cells were completely confluent (Figure 5 (c) and (e)).

4. After 12 days exposure to 10 μg/ml GTE (Figure 6 (c) most cells were globular.

When the medium was changed to DMEM only, there was no improvement 4 days later (Figure 6 (d)) when the medium was switched to DMEM/10% FCS there was a slight improvement in the ^"morphology of some cells (Figure 6 (e)).

However, it was not considered that this was totally reversible.

This can be compared with the reversibility shown for cells that had been 12 days in DMEM only. When switched to DMEM/10% FCS there was very good recovery by these cells 4 days later (Figure 6 (a) and (b)).

Experiment 2 - reversing the medium at day 4 and day 7

1. Repeat of Experiment 1 above

After 4 days exposure to 10 μg/ml GTE there was a slight improvement in cells switched to DMEM only, and good improvement in cells switched to DMEM/10% FCS (Figure 7 (a) to (g)).

2. After 7 days exposure to GTE the damage was reversible in DMEM/10% FCS, but there was little improvement in cells switched to DMEM only (Figure 8 (a) to (g))-

Summary

The inhibition of the growth of fibroblasts with GTE appears to be fully reversible up to at least 7 days after treatment, but reversibility appears to become less predictable there afterwards.

Test D to establish the possible antiprotease effect of GTE

It was observed during the work on the reversibility of the inhibition of fibroblast growth that with prolonged exposure to 10 μg/ml of GTE there was great difficulty in trying to trypsinise the cells for counting. Even after 1 hour in trypsin it was not possible to release all cells from the plastic substrate. Therefore, assessment of the effects of the extract and of reversing the medium was continued by microscope observation only.

Yager (1999) has reported that the breakdown of the antiprotease shield puts al risk the ability to deposit de novo extracellular matrix (this may also apply to newly formed epithelium). Yager notes that there is a need to develop therapeutic interventions that can augment and protect the antiprotease shield. It is therefore noteworthy that GTE appears to reduce the activity of trypsin in vitro.

Reference

Yager, D.R. and Mwomeh, B.C. (1999). Wound Rep Reg, 7, 433-441 'The proteolytic environment of chronic wounds'. Test E to establish the effect of GTE on the proliferation of human dermal microvascular endothelial cells (HDMEC).

Effect of GTE on growth of HDMEC in modified EGM-2MV (a proprietary growth medium for microvascular endothelial cells).

Experiment 1

HDMEC cells were grown in modified EGM-2 MV medium containing 1.5% FBS (Foetal Bovine Serum). Numbers of control cells at days 4 and 7 were 155% and 174%> respectively of the numbers on day 0. No toxicity was observed and cell growth was slightly increased in all concentrations of GTE (Figure 9) with statistical significance reached in the 1.0 μg/ml concentration on day 7 (117%o of control, p<0.03).

Experiment 2

In the repeat experiment a similar trend was observed (Figure 10) but the differences were smaller. The cells in all groups grew faster than in Experiment 1 with control cells increasing fourfold by day 4 and more than seven-fold by day 7. In this case the proliferation of cells with GTE in comparison with controls at the same time point did not reach statistical significance.

In both Experiments 1 and 2 no toxicity was observed at any concentration.

Claims

1. Use of extract of green tea in the manufacture of a medicament or dressing adapted for topical application to a skin penetrating wound, wherein the green tea extract is present in a therapeutically effective concentration to produce both an increase in the proliferation of keratinocytes and of human dermal microvascular endothelial cells in the dermis of a human.

2. Use as claimed in claim 1, wherein the green tea extract is used in a concentration in the range of 0.1 to 10.0 μg/ml.

3. A wound dressing impregnated with a therapeutically effective amount of an extract of green tea to promote effective healing of the wound.