Glucan compositions

Abstract

The present invention relates to a gel composition comprising a glucan and a gelling agent, said composition having a melting point (gel to sol) above 37 C., as well as to the uses of this composition in therapy, in particular in wound healing.

Claims

1. A gel composition comprising 0.1%-6% glucan derived from yeast and 0.2%-3% gelling agent, said composition having a melting point (gel to sol) above 37 C., wherein said gelling agent is a cellulose derivative.

2. The composition of claim 1 wherein the glucan and the gelling agent are present as a hydrogel.

3. The composition of claim 1 wherein the gelling agent is selected from the group consisting of carboxymethyl cellulose, methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl methyl cellulose phthalate.

4. The composition of claim 1, wherein the glucan is derived from Saccharomyces cerevisiae.

5. The composition of claim 1 wherein the glucan is a beta glucan comprising a backbone of -(1,3)-linked glucosyl residues and side chains comprising 2 or more -(1,3)-linked glucosyl residues, the sidechains being attached to the backbone via a -(1,6)-linkage.

6. The composition of claim 1 wherein the glucan is essentially free of repetitive -(1,6)-linked glucosyl residues.

7. The composition of claim 1 which comprises 0.2%-4% glucan and 0.25%-2% gelling agent.

8. The composition of claim 1 wherein the gel exists in gel form at 25 C. at pH 4 to 8.

9. The composition of claim 1 which at 25 C. has a viscosity of at least 1000 cP.

10. The composition of claim 1 which comprises about 2% glucan and about 1.5% carboxymethyl cellulose.

11. A method of assisting wound or ulcer healing or treating oral mucositis or cancer in a subject which comprises administering to said subject the composition of claim 1.

12. The method of claim 11 wherein the composition is topically applied to a subject.

13. The method of claim 11 wherein said ulcer is a diabetic ulcer.

14. A physical support having applied thereto or impregnated therein, the composition of claim 1.

15. The physical support of claim 14 selected from the group consisting of a woven, non-woven, knitted, foam or adhesive substrate.

16. A gel composition comprising 0.1%-6% glucan derived from yeast and 0.2%-3% gelling agent, wherein the gelling agent is a cellulose derivative, and wherein said gel composition is obtainable by a method which comprises: a) treating an aqueous solution of glucan molecules derived from yeast, to dissociate the glucan's hydrogen bonds; b) treating the aqueous solution to reform hydrogen bonds within the glucan; wherein the gelling agent is added prior to step a) or after step a) but prior to step b).

17. The method of claim 16, wherein step a) comprises heating the aqueous solution of glucan molecules, with or without gelling agent, and step b) comprises cooling the aqueous solution of glucan and gelling agent.

18. The method of claim 17, wherein the aqueous solution, with or without gelling agent, is heated to at least about 100 C.

19. The method of claim 17, wherein the mixture of glucan and gelling agent is rapidly cooled in step b).

20. The method of claim 17, wherein the mixture of glucan and gelling agent is cooled to below 40 C.

21. The composition of claim 9, which at 25 C. has a viscosity of at least 1500 cP.

22. The physical support of claim 14, selected from a patch, dressing, plaster, bandage, film or gauze.

23. A gel composition comprising a glucan derived from yeast and a gelling agent, said composition having a melting point (gel to sol) above 37 C., wherein said gelling agent is a cellulose derivative and wherein the gel exists in gel form at 25 C. at pH 4 to 8.

24. The composition of claim 23 wherein the glucan and the gelling agent are present as a hydrogel.

25. The composition of claim 23 wherein the gelling agent is selected from the group consisting of carboxymethyl cellulose, methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl methyl cellulose phthalate.

26. The composition of claim 23, wherein the glucan is derived from Saccharomyces cerevisiae.

27. The composition of claim 23 wherein the glucan is a beta glucan comprising a backbone of -(1,3)-linked glucosyl residues and side chains comprising 2 or more -(1,3)-linked glucosyl residues, the sidechains being attached to the backbone via a -(1,6)-linkage.

28. The composition of claim 23 wherein the glucan is essentially free of repetitive -(1,6)-linked glucosyl residues.

29. The composition of claim 23 which comprises 0.2%-4% glucan and 0.25%-2% gelling agent.

30. The composition of claim 23 which at 25 C. has a viscosity of at least 1000 cP.

31. The composition of claim 23 which comprises about 2% glucan and about 1.5% carboxymethyl cellulose.

32. A method of assisting wound or ulcer healing or treating oral mucositis or cancer in a subject which comprises administering to said subject the composition of claim 23.

33. The method of claim 32 wherein the composition is topically applied to a subject.

34. The method of claim 32 wherein said ulcer is a diabetic ulcer.

35. A physical support having applied thereto or impregnated therein, the composition of claim 23.

36. The physical support of claim 35 selected from the group consisting of a woven, non-woven, knitted, foam or adhesive substrate.

37. A gel composition comprising a glucan derived from yeast and a gelling agent, wherein the gelling agent is a cellulose derivative, wherein the gel exists in gel form at 25 C. at pH 4 to 8, and wherein said gel composition is obtainable by a method which comprises: a) treating an aqueous solution of glucan molecules derived from yeast, to dissociate the glucan's hydrogen bonds; b) treating the aqueous solution to reform hydrogen bonds within the glucan; wherein the gelling agent is added prior to step a) or after step a) but prior to step b).

38. The method of claim 37, wherein step a) comprises heating the aqueous solution of glucan molecules, with or without gelling agent, and step b) comprises cooling the aqueous solution of glucan and gelling agent.

39. The method of claim 38, wherein the aqueous solution, with or without gelling agent, is heated to at least about 100 C.

40. The method of claim 38, wherein the mixture of glucan and gelling agent is rapidly cooled in step b).

41. The method of claim 38, wherein the mixture of glucan and gelling agent is cooled to below 40 C.

42. The composition of claim 30, which at 25 C. has a viscosity of at least 1500 cP.

43. The physical support of claim 35, selected from a patch, dressing, plaster, bandage, film or gauze.

Description

(1) The invention will now be further described in the following non-limiting Examples and figures in which:

(2) FIG. 1 illustrates the SEC-MALS-RI chromatograms of a number of batches of preferred yeast glucans, defined as branched (1,3) glucan with <2% repetitive (1,6) linked glucosyl units, analyzed in DMAc/with 0.5% LiCl assuming a dn/dc=0.12. As can be seen the molecular weight distribution is in the range of approx. 10,000 g/mol to approx. 200,000 g/mol on the single chain level.

(3) FIG. 2 shows SEC-MALS-RI chromatograms of a number of batches of preferred yeast glucans, defined as branched (1,3) glucan with <2% repetitive (1,6) linked glucosyl units, analyzed in aqueous buffer (0.1M NaNO.sub.3) assuming a dn/dc=0.15. As can be seen the molecular weight distribution is in the range of approx. 10,000 g/mol to above 10,000,000 g/mol on the single chain level. The aqueous SEC-MALS-RI results, in combination with the results in DMAc/LiCl, show that the preferred yeast glucans exist as aggregates/supramolecular structures in the aqueous solution.

(4) FIG. 3: shows the assumed mechanism of action of the soluble beta glucan used in the present invention. The figure shows that the beta glucan (BG) branches simultaneously bind to receptors on e.g. macrophages and thus activate the innate immune system.

(5) FIG. 4 shows wound closure of full-thickness wounds in a db/db mouse model stimulated by topical administration of SG alone (2%), carboxymethyl cellulose alone (1% CMC), the combination of the two (2% SG, 1% CMC), versus vehicle (water) and positive control (rh-PDGF-BB (10 g)+rh-TGF- (1 g) in 0.5% HPMC and 1% CMC.

(6) FIG. 5 shows the change in mean % wound area remaining with time (sem)Groups: (1) Vehicle control, (4) Methocel, (5) Intrasite, (9) 2% SG (1% CMC), (12) +ve control (HPMC), & (13) +ve control (CMC)

(7) FIG. 6 shows change in mean % of original wound area closed by contraction with time (sem)Groups: (1) Vehicle control, (2) 1% CMC, (6) 2% SG, (9) 2% SG (1% CMC), (12) +ve control (HPMC), & (13) +ve control (CMC)

(8) FIG. 7 shows the change in mean % of original wound area closed by contraction with time (sem)Groups: (1) Vehicle control, (4) Methocel, (5) Intrasite, (9) 2% SG (CMC), (12) +ve control (HPMC), & (13) +ve control (CMC)

(9) FIG. 8 shows wound closure of full-thickness wounds in a db/db mouse model stimulated by topical administration of two different formulations of Biotec Pharmacon's Woulgan Biogel, the hydrogel alone, an oat beta-glucan product, versus vehicle (water) and positive control (rh-PDGF-BB (10 g)+rh-TGF- (1 g) in hydrogel

(10) FIG. 9 shows SG and SG mixed with 1.5% carboxymethylcellulose stored in aluminium containers. T=0 represents appearance at study start, and T=6 indicates samples stored 6 months at ambulating temperatures, changing each week between 4 and 37 C.

EXAMPLES

Example 1: Preparation of Gel Glucan Product (SG)

(11) An aqueous solution of 1.5-2% yeast glucan molecules was treated as described below. This aqueous solution was prepared from a particulate glucan preparation by formolysis to selectively remove -1,6 side chains and subsequent purification and diafiltration to remove particulate matter and low molecular weight components from the formolysis solution. A suitable formolysis step is disclosed in Example 3 of EP 0759089 B1. The particulate glucan was itself prepared from cell walls of Baker's Yeast (S. cerevisiae) by separate extractions with alkali, ethanol and water, each extraction being followed by appropriate drying (spray drying and vacuum drying).

(12) a. Heat Treatment:

(13) Heat treatment takes place after the concentration of the glucan solution has been adjusted, normally giving a product volume of approximately 220 liters at a temperature of approximately 60 C., in a closed and agitated 800 liter tank which is heated by introduction of steam to a jacket surrounding the tank.

(14) The product is heated slowly to approximately 105 C. to ensure an even heating of the whole batch, and then more quickly to 123 C. Normal heating time from 60 to 123 C. is 40-50 minutes. The product is then held at 123-125 C. for 20 minutes.

(15) b. Active Cooling:

(16) Active cooling is then started. It is operated manually, by direct opening and closing of hand operated valves. First the steam is carefully evacuated from the jacket to drain, and the drain valves are left open. Cooling water is then carefully introduced to the jacket, slowly at first to avoid excessive thermal stress to the steel of the tank. As the temperature drops the flow of water is increased. Cooling is normally continued until the product temperature reaches 35-40 C. Normal cooling time from 123 to 40 C. is 50-60 minutes.

Example 2: Preparation of Gel Glucan Product

(17) An aqueous solution of 1.5-2% yeast glucan molecules was treated as described below. This aqueous solution was prepared from a particulate glucan preparation by formolysis to selectively remove -1,6 side chains, as described in Example 1.

(18) a. Disruption of Hydrogen Bonds by Addition of Sodium Hydroxide:

(19) Addition of sodium hydroxide took place after the concentration of the glucan solution had been adjusted, giving a product volume of approximately 185 liters in a closed and agitated 800 liter tank which is heated or cooled by introduction of steam or water to a jacket surrounding the tank.

(20) The product was cooled to 18 C., and 24 moles (960 g) of NaOH, dissolved in approximately 10 liters of purified water, was poured slowly (approximately 1 liter per minute) through a hatch in the tank.

(21) b. Restoration of Hydrogen Bonds by Addition of Hydrochloric Acid:

(22) The restoration process was started immediately after the last of the NaOH had been poured into the tank.

(23) Slightly less than 24 moles of HCl, approx 9 liters of 2.4 M HCl solution in purified water, was poured into the tank relatively quickly (in approximately 2 minutes), the pH of the product was measured, and more acid added in small portions until pH reached approximately 4. Total amount of HCl added was 23.4 moles.

(24) c. Removal of Salt

(25) To remove the ions (Na.sup.+ and Cl.sup.) added during steps a and b, the product was diafiltered over a tangential filter against 4 volumes of purified water.

Example 3: Wound Healing Composition In Vivo

(26) The impact of a gel glucan alone (SG) prepared in accordance with Example 1, vehicle (carboxymethyl cellulose or gellan gum) alone, or a combination of SG and vehicle on wound healing was investigated by analysing the repair of full-thickness excisional skin wounds in the diabetic (db/db) mouse model (i.e. BKS.Cg-m Dock7.sup.m+/+Lepr.sup.db/J mice). The combination product of the invention was also prepared in accordance with heating and rapid cooling method described herein and exemplified in Example 1, in short, the glucan and vehicle were dissolved in aqueous solution and then heated in an autoclave to around 120 C. for about 18 minutes. The product was then cooled quickly to allow gel formation as described in Example 1.

(27) Upon acclimatization (5-7 days without disturbance) the animals were housed in groups of 5 animals according to Home Office regulations and the specific requirements of diabetic animals. After experimental wounding, animals were housed in individual cages (cage dimensions 351515 cm with sawdust bedding, changed twice weekly), in an environment maintained at an ambient temperature of 23 C. with 12-hour light/dark cycles. The mice were provided with food (Standard Rodent Diet) and water ad libitum. Following all anaesthetic events, animals were placed in a warm environment and monitored until they were fully recovered from the procedure. All animals received appropriate analgesia (buprenorphine) after surgery and additional analgesics as required. All animal procedures were carried out in a Home Office licensed establishment under Home Office Licences (PCD: 50/2505; PPL: 40/3300; PIL: 50/3482; PIL: 70/4934). The health of animals was monitored on a daily basis throughout the study.

(28) On day 0, animals were anaesthetised (isofluorane & air) and the dorsum shaved and cleaned with saline-soaked gauze. A single standardised full-thickness wound (10.0 mm10.0 mm) was created in the left dorsal flank skin of each experimental animal. Wounds in all treatment groups were subsequently dressed with a circumferential band of the transparent film dressing Bioclusive (Systagenix Wound Management, UK); after which they received either SG, vehicle, or a combination of SG and vehicle by injection 50 l material dissolved in purified water through the Bioclusive film using a 29-gauge needle. Diabetic animals were randomized to one of the treatment regimes using appropriate software.

(29) Treatments were reapplied on post-wounding days 2, 4 and 6. Wound sites in these animals were closely monitored for excessive build-up of applied agents and excessive wound site hydration; if excessive applied agent accumulation/hydration was apparent, previously applied material was removed by aspiration prior to reapplication.

(30) On post-wounding days 4, 8, 12, 16, 20 and 24 all animals were re-anaesthetised, their film dressings and any free debris removed, and their wounds cleaned using saline-soaked sterile gauze. After photography on days 4, 8, 12, 16, 20 and 24 wounds were re-dressed as above with Bioclusive film dressing. Wound healing was evaluated (not quantitatively) according to the presence of fibrin, granulation tissue, angiogenesis and re-epitelisation. Based on appearance of the above mentioned factors neo-dermal tissue formation (healing) were classified as: Very good, good, slight, no.

(31) Wound closure data were further determined from scaled wound images taken of each wound at each assessment point. The area of a given wound, at a given time point, was expressed as a percentage of the area of that wound immediately after injury (i.e. day 0). The mean percentage wound area remaining (& standard error of mean) was calculated for each group and was displayed graphically. The impact of each glucan preparation was compared to that of wounds in receipt of: i). vehicle; and ii) PDGF-BB+TGF- (positive control).

(32) TABLE-US-00002 TABLE 1 Fraction of healing wounds, day 8. Healing (neo-dermal tissue formation) Treatment Very good Good Slight No 1% Carboxymethyl 0/10 3/10 2/10 5/10 cellulose 2% SG 0/10 5/10 4/10 1/10 4% SG 2/10 4/10 3/10 1/10 1% Carboxymethyl 0/10 5/10 4/10 1/10 cellulose + 1% SG 1% Carboxymethyl 3/10 5/10 2/10 0/5 cellulose + 2% SG 1% Carboxymethyl 1/10 9/10 0/5 0/5 cellulose + 4% SG 0.3% Phytagel 0/10 5/10 3/10 2/10 0.3% Phytagel + 2% SG 0/10 8/10 2/10 0/10

(33) The results in Table 1 show that the frequency of healing wounds in receipt of the glucan alone was higher relative to wounds in receipt of the vehicle alone. This suggests that the glucan alone is a better inducer of neo-dermal tissue formation compared to the gelling agent (the vehicle). In addition, there is a clear concentration-dependent shift from a 2% to a 4% glucan solution showing increase wound healing (good to very good). However, the combination of the glucan and both of the vehicles was superior to the single use of each agent (significant shift from slight to good and very good), suggesting a synergistic effect of the combined products.

Example 4: The Impact of Glucan Preparations According to the Invention on Wound Healing

(34) A study was performed to evaluate glucan-based preparations according to the invention with regard to their ability to promote tissue repair in a recognised in vivo model of delayed wound healing. Patients with diabetes are prone to impaired wound healing, with foot ulceration being particularly prevalent. This delay in wound healing also extends to diabetic animals, including the spontaneously diabetic (db/db) mouse (i.e. BKS.Cg-m Dock7.sup.m+/+Lepr.sup.db/J mice).

(35) In this study, the healing of wounds on diabetic mice in receipt of Biotec glucan SG 131-9 (at various concentrations, with or without various vehicles) was compared to that of similar wounds exposed to the vehicles: (i) purified water [water for injection], (ii) 1.0% carboxy-methyl-cellulose, and (iii) 0.3% Phytagel. The healing of diabetic wounds in receipt of Biotech glucan SG 131-9 was also compared to the comparators: (i) Methocela comparator polysaccharide material, and (ii) Intrasite Gela market leading wound management hydrogel preparation. Recombinant human platelet-derived growth factor-BB (rh-PDGF-BB) in combination with recombinant human Transforming Growth Factor-alpha (rh-TGF-) were used as the positive control in this study. This positive control was applied with two carriers0.5% hydroxy propyl methyl cellulose (HPMC) and 1.0% carboxy methyl cellulose (CMC).

(36) Materials and Methods

(37) Materials Under Test

(38) 1. Water for Injection 2. 1.0% Carboxymethylcellulose (CMC, Sigma C5013, sodium salt) in purified water 3. 0.3% Phytagel+4 mM CaCl.sub.2 4. 2.0% Methocel 5. Intrasite 6. 2.0% SG 7. 4.0% SG 8. 1.0% CMC+1.0% SG 9. 1.0% CMC+2.0% SG 10.1.0% CMC+4.0% SG 11. 0.3% Phytagel+2.0% SG 12. rh-PDGF-BB [10%]+rh-TGF- [1%]in 0.5% HPMC 13. rh-PDGF-BB [10%]+rh-TGF- [1%]in 1.0% CMC

(39) The above materials were prepared in accordance with the methods described in Examples 1 and 3. Phytagel is always used with CaCl.sub.2.

(40) BKS.Cg-m Dock7.sup.m+/+Lepr.sup.db/J Diabetic Mouse Model

(41) Mice were brought into the UK aged approximately 5-6 weeks and maintained in house until aged 12 weeks (1 week)according to Home Office regulations and the specific requirements of diabetic animals.

(42) Briefly, on day 0 mice were anaesthetised using isofluorane and air; and their dorsal flank skin was clipped and cleansed according to protocol. A single standardised full-thickness wound (10 mm10 mm) was created in the skin immediately to the left of the spine. Diabetic animals were randomly allocated to one of 13 experimental groups (as described in the table below). Wounds in all groups were dressed with a circumferential band of the semi-occlusive film dressing Bioclusive (Systagenix Wound Management, UK) and treatments (in 50 l volumes [groups 1-11] and 100 l [groups 12 &13]) applied by hypodermic injection through the Bioclusive film. The condition of dressing materials was examined daily throughout the study and replaced as necessary.

(43) Animals in groups 1 through 11 were restrained and treatments reapplied by hypodermic injection through the Bioclusive film on post-wounding days 2, 4 and 6. Any build-up of hydration/previously applied agent was removed by aspiration prior to re-application. For experimental groups 12 & 13 (positive controls) treatments were reapplied daily until post-wounding day 6.

(44) On day 4 all animals were re-anaesthetised, wounds were photographed, and animals were allowed to recover in a warmed environment (34 C.). As wound boundaries were clearly visible through the Bioclusive dressing, and in order to minimise peri-wound damage through repeated dressing removal, it was decided that the film dressings would be retained at this assessment point.

(45) On days 8 & 12, 16 & 20 all animals were re-anaesthetised, their film dressings and any free debris removed, and their wounds cleaned using sterile saline-soaked sterile gauze. Wounds were then photographed, re-dressed (as above) with Bioclusive film dressingand animals were allowed to recover in a warmed environment (34 C.).

(46) Immediately after wounding, and subsequently on days 4, 8, 12, 16, 20 & 24 all wounds were digitally photographed together with a calibration/identity plate (following film dressing removal and wound cleaningwhere applicable).

(47) Experimental Groups:

(48) TABLE-US-00003 Tx Animal Codes & Group Treatment Group name harvesting n 1 Water for Injection EXP-01 BIOT-02.01 to 10 02.10 2 1.0% Carboxymethylcellulose(CMC) in EXP-02 BIOT-02.11 to 10 purified water (50 l) 02.20 3 0.3% Phytagel + 4 mMCaCl.sub.2(50 l) EXP-03 BIOT-02.21 to 10 02.30 4 2.0% Methocel (50 l) EXP-04 BIOT-02.31 to 10 02.40 5 Intrasite (50 l) EXP-05 BIOT-02.41 to 10 02.50 6 2.0% SG (50 l) EXP-06 BIOT-02.51 to 10 02.60 7 4.0% SG (50 l) EXP-07 BIOT-02.61 to 10 02.70 8 1.0% CMC + 1.0% SG (50 l) EXP-08 BIOT-02.71 to 10 02.80 9 1.0% CMC + 2.0% SG (50 l) EXP-09 BIOT-02.81 to 10 02.90 10 1.0% CMC + 4.0% SG (50 l) EXP-10 BIOT-02.91 to 10 02.100 11 0.3% Phytagel + 2.0% SG (50 l) EXP-11 BIOT-02.101 to 10 02.110 12 rh-PDGF-BB [10 g] + rh-TGF- [1ug]- EXP-12 BIOT-02.111 to 7 (100 ul) in 0.5% HPMC 02.117 13 rh-PDGF-BB [10 g] + rh-TGF- [1ug]- EXP-13 BIOT-02.118 to 7 (100 ul) in 1.0% CMC 02.124
Image Analysis of Wound Closure:

(49) Image Pro Plus image analysis software (version 4.1.0.0, Media Cybernetics, USA) was used to calculate wound closure from scaled wound images taken at each assessment point. As the process of wound closure involves the effects of wound contraction (the inward movement of marginal tissue), this was also determined

(50) The following assessments were made: 1. Percentage wound area remaining with time i.e. the open wound area remaining at a given time pointrelative to the area of the same wound immediately after injury on day 0. 2. Percentage wound contraction with time i.e. the difference between the contracted wound area at a given time point and the original wound area [as a percentage of the original wound area.
Assessment of Initiation of Wound Healing (Neo-Dermal Tissue Generation):

(51) All wounds in the study were visually assessed on a daily basis until day 8and subsequently on days 10, 12, 14, 16, 20 & 24 to establish their healing status. Each wound was scored as to whether it was displaying neo-dermal tissue generation activity or not (i.e. whether the wound had initiated the healing process or not). Each wound was assessed by two independent observers and the average percentage of wounds displaying neo-dermal tissue generation activity was compared between treatment groups at each assessment point.

(52) Neo-dermal tissue formation was considered to have initiated when blood vessels within the fascia of the wound base are concealed by overlying material. This concealment may result from the formation of cloudy exudate, polymerised/semi-polymerised fibrin or granulation tissue. Invariably, the first sign of neo-dermal tissue initiation is the formation of a reddish exudate within the wound void.

(53) Results

(54) Wound Closure:

(55) For a given wound at a given time point, wound closure was expressed as the percentage wound area remaining relative to the initial wound area immediately after injury (i.e. day 0). Mean percentage wound area remaining data for all treatment groups are described in Table 2, below.

(56) TABLE-US-00004 TABLE 2 Percentage Wound Area Remaining Data for all study groups. % wound area remaining with time - open wound area (mean +/ standard error) Days post-wounding 4 8 12 16 20 24 Treatment (1) Vehicle - purified water 96.7 2.8 70.1 2.9 60.6 4.7 41.0 6.3 30.8 5.8 22.9 5.6 (2) Vehicle - 1% CMC 97.5 1.9 66.9 4.2 42.3 4.5 21.9 4.3 12.9 3.4 6.1 1.7 (3) Vehicle - 0.3% Phytagel + 4 mM CaCl.sub.2 95.2 3.4 70.4 4.4 49.2 5.1 34.0 6.2 21.6 5.9 12.9 5.3 (4) 2.0% Methocel 99.0 2.2 58.3 5.5 44.4 6.2 28.2 7.3 16.4 5.8 9.3 4.1 (5) Intrasite 95.2 2.2 74.4 4.4 49.1 5.0 28.0 4.6 15.0 3.9 7.8 2.8 (6) 2.0% SG 93.2 2.8 63.7 3.8 37.5 5.0 19.9 3.4 13.1 4.6 8.7 4.3 (7) 4.0% SG 100.2 4.4 64.5 4.7 39.6 5.7 25.6 5.0 20.3 4.7 15.7 5.2 (8) 1.0% SG 131-9 (in 1% CMC) 97.8 2.7 68.8 2.8 37.4 5.0 19.2 4.0 14.9 4.0 10.2 3.9 (9) 2.0% SG 131-9 (in 1% CMC) 98.1 3.0 60.2 6.5 31.3 6.2 15.0 3.6 7.7 2.3 3.9 1.6 (10) 4.0% SG 131-9 (in 1% CMC) 97.2 2.8 66.6 4.5 53.5 5.5 30.4 3.9 18.9 3.7 11.2 2.5 (11) 2.0% SG 131-9 (in 0.3% Phytagel) 97.0 2.0 67.3 3.0 38.7 3.7 24.5 3.3 11.2 1.9 7.0 1.7 (12) Positive control (in 0.5% HPMC) 93.7 3.2 58.0 6.2 17.9 3.9 4.8 2.2 0.04 0.04 0.0 0.0 (13) Positive control (in 1.0% CMC) 91.2 2.4 57.3 1.9 22.2 3.3 6.2 2.3 1.8 1.2 0.9 0.9

(57) As shown in Table 2, and in FIGS. 4 and 5, wound closure profiles of % wound area remaining with time data, were found to differ noticeably between the different treatment groups. Wounds in receipt of water only demonstrated the slowest wound closure and wounds in receipt of the positive controls exhibited the fastest closure, with all other treatment groups falling between. Wounds in receipt of 2% SG (in CMC) were found to close more rapidly than any other experimental treatment group (excluding positive controls).

(58) Both comparators (Methocel and Intrasite) tended to accelerate wound closure compared to water treatment. The final wound closure levels attained by day 24 were 91% for Methocel and 92% for Intrasite.

(59) Application of SG 131-9 (1, 2 or 4%) in CMC tended to accelerate wound closure compared to water treatment. Treatment with 1% SG 131-9 (in CMC) resulted in significantly elevated closure on post-wounding days 12 through 20. Treatment with 2% SG 131-9 (in CMC) appeared to lead to more substantial and sustained effects and was found to result in a significant acceleration in closure from day 12 onwards. Treatment with 4% SG 131-9 (in CMC) though more effective than water, appeared less effective than both the 1% and 2% treatments. The final wound closure levels reached by day 24 were: 90% for 1% SG 131-9 (in CMC), 96% for 2% SG 131-9 (in CMC) and 89% for 4% SG 131-9 (in CMC).

(60) 2% SG 131-9 applied in 1% CMC tended to elevate wound closure to a greater degree than 2% SG 131-9 applied in water. When the three 2% SG 131-9 treatment regimes are compared, it can be seen that all three promoted closure to a greater level than their respective vehicle controls (i.e. water, 1% CMC & 0.3% Phytagel). In absolute terms, 2% SG in CMC tended to result in the highest level of closure. The closure profile of the 2% SG in water treatment group was similar to that of the 2% SG in Phytagel treatment group, both displayed lower levels of closure than wounds treated with the 2% SG 131-9 in CMC formulation.

(61) Of all the SG 131-9 preparations evaluated, 2% SG 131-9 in 1% CMC appeared to be most effective. 2% SG 131-9 (in CMC) was found to promote wound closure to a greater degree than Intrasite, a comparator polysaccharide material and Methocel, a market leading wound management hydrogel preparation.

(62) Wound Contraction

(63) Contraction is the centripetal movement of the wound marginsdue to the compaction of granulation tissue within the body of the wound. The compactional forces, that drive this process, are thought to reside in cells of the fibroblast lineage. In this study, % contraction was calculated as:

(64) $\%\mathrm{contraction}=\frac{\mathrm{The}\mathrm{area}\mathrm{defined}\mathrm{by}\mathrm{the}\mathrm{boundary}\mathrm{of}\mathrm{normal}}{\mathrm{The}\mathrm{original}\mathrm{wound}\mathrm{area}\left(\mathrm{day}0\right)}100$

(65) The wound contraction results are shown in Table 3 below and FIGS. 6 and 7.

(66) TABLE-US-00005 TABLE 3 % of original wound area closed by contraction (mean +/ standard error) Days post-wounding 4 8 12 16 20 24 Treatment (1) Vehicle - purified water 3.3 2.8 16.9 2.9 25.5 4.2 40.2 4.2 52.6 4.9 60.2 5.5 (2) Vehicle - 1% CMC 2.5 1.9 24.4 2.9 44.7 4.3 60.4 3.9 69.3 3.9 76.9 3.1 (3) Vehicle - 0.3% Phytagel + 4 mM CaCl.sub.2 4.8 3.4 19.1 4.3 38.4 5.3 53.5 6.5 64.6 6.6 72.5 6.5 (4) 2.0% Methocel 1.0 2.2 26.9 5.4 42.1 6.3 56.0 6.7 68.4 5.6 77.2 4.3 (5) Intrasite 4.8 2.2 16.1 4.6 41.3 4.2 60.0 4.0 72.9 3.3 80.0 2.9 (6) 2.0% SG 6.8 2.8 25.4 3.6 48.0 5.9 67.0 4.3 74.0 4.3 80.9 4.4 (7) 4.0% SG 0.2 4.4 23.6 4.0 49.7 4.8 62.9 4.5 72.8 4.3 76.3 5.2 (8) 1.0% SG 131-9 (in 1% CMC) 2.2 2.7 21.9 3.0 45.7 3.4 62.8 4.1 71.4 4.2 76.5 4.0 (9) 2.0% SG 131-9 (in 1% CMC) 1.9 3.0 31.0 6.5 56.7 5.5 71.7 3.2 80.1 3.3 88.8 1.4 (10) 4.0% SG 131-9 (in 1% CMC) 2.8 2.8 23.4 3.4 41.8 5.1 62.0 3.7 73.7 3.3 79.9 2.9 (11) 2.0% SG 131-9 (in 0.3% Phytagel) 3.0 2.0 21.9 3.9 50.1 4.0 66.1 2.9 79.8 2.4 85.4 2.2 (12) Positive control (in 0.5% HPMC) 6.3 3.2 27.3 5.2 60.0 2.4 69.2 1.8 76.2 2.8 80.3 3.4 (13) Positive control (in 1.0% CMC) 8.8 2.4 29.2 2.3 59.1 3.1 70.2 2.6 79.2 2.0 83.7 1.6

(67) Noticeably less contraction was evident in the water only treatment group compared to all other treatment groups. The highest levels of contraction was observed with both positive control regimes, 2% SG (in CMC) and at the later time points (days 20 and 24) with 2% SG (in Phytagel).

(68) Both comparators, Methocel and Intrasite, promoted wound contraction relative to water-treatment. Methocel-treated wounds contracted significantly more than those treated with water on days 8, 20 and 24, while Intrasite treated wounds displayed significantly more contraction from day 12 onwards. Both comparator treatment groups tended to display less wound contraction than positive control-treated wounds.

(69) Treatment with SG 131-9 (1%, 2% or 4%) formulated in 1% CMC promoted wound contraction relative to water-treatment. Treatment with each of the concentrations resulted in significantly greater contraction than water treatment from day 12 onwards. 2% SG 131-9 (in CMC) was found to promote wound contraction compared to CMC alone, with significantly elevated contraction observed on days 16 and 24.2% SG (in CMC) was found to be more effective at promoting contraction than both 1% and 4% SG 131-9 (in CMC). Treatment with 2% SG (in CMC) resulted in similar levels of contraction as the positive control treated wounds up to and including day 20 with no significant differences measured between them; whereas, as previously described, CMC alone resulted in less contraction than the positive control treatments. Interestingly, at the final assessment point (day 24), wounds treated with 2% SG 131-9 (in CMC) were found to have contracted to a greater degree than those treated with both positive control treatment.

(70) 2% SG 131-9 applied in 1% CMC tended to elevate wound contraction to a greater degree than 2% SG 131-9 applied in water. In absolute terms, 2% SG in CMC tended to result in the highest level of contraction. 2% SG 131-9 (in Phytagel) was also found to promote wound contraction compared to water treatment and compared to Phytagel alone.

(71) Of all the SG 131-9 preparations evaluated, 2% SG 131-9 in 1% CMC appeared to be most effective in terms of wound contraction. 2% SG 131-9 (in CMC) was found to promote wound contraction to a greater degree than Intrasite and Methocel.

(72) Initiation of Wound Healing (Neo-Dermal Tissue Generation)

(73) All wounds in the study were visually assessed on a daily basis until day 8 and subsequently on days 10, 12, 14, 16, 20 & 24 to establish their healing status. Each wound was scored as to whether it was displaying neo-dermal tissue generation activity or not (i.e. whether the wound had initiated the healing process or not). Each wound was assessed by two independent observers and the average percentage of wounds displaying neo-dermal tissue generation activity was compared between treatment groups at each assessment point.

(74) Wounds in the different treatment groups were found to demonstrate the first signs of healing at varying times after wounding. According to these data the order in which the different groups were found to respond was, from fastest to slowest:

(75) TABLE-US-00006 Based on median no. days to respond Order Treatment 1 +ve control (CMC), +ve control (HPMC) 3 2% SG 131-9 (1% CMC) 4% SG 131-9 (1% CMC) 2% SG131-9 4% SG131-9 Intrasite 8 Methocel 9 2% SG131-9 (Phyta) 10 1% SG131-9 (1% CMC) 11 Phytagel 12 1% CMC 13 Water

(76) Seven of the ten wounds (70%) randomised to water treatment were found to have initiated neo-dermal tissue formation on conclusion of the study on day 24. All wounds in all other groups were found to have initiated neo-dermal tissue formation by this time point.

(77) On consideration of SG formulated in 1% CMC, wounds in receipt of 2% and 4% SG tended to respond first, followed by wounds in receipt of 1% SG. When compared to water-treatment, a significantly greater number of 1% SG 131-9 treated wounds had responded on days 6 to 14, a significantly greater number in receipt of 2% SG 131-9 had responded on days 3 to 14, and a significantly greater number treated with 4% SG 131-9 had responded on days 4 to 14. No significant differences were noted between these three treatment groups and the two positive control treatment groups after day 4. In terms of the average number of days to respond all three concentrations responded significantly earlier than water-treated wounds.

(78) Wounds in receipt of 2% SG formulated in Phytagel were found to respond earlier than wounds in receipt of Phytagel alone. When compared to control groups, significantly more wounds in receipt of 2% SG (in Phytagel) responded on days 4 to 14 than wounds in receipt of water. In terms of the average number of days to respond, 2% SG (Phytagel) responded significantly earlier than water or Phytagel alone.

Example 5: Glucan Gel Stability

(79) Woulgan was prepared as follows: 2.7% SBG (Biotec's soluble yeast beta glucan in purified water) While stirring, Blanose (7H4XF PH, Kirsch Pharma Gmbh, pharma grade carboxymethyl cellulose) was added to a final conc. of 1.5% (w/v). Stirred until CMC was dissolved Glycerol (99.7%) added to a final conc. of 20%. Sterilized in autoclave at 120 C. for 18 min Cooled quickly and the gel allowed to solidify as described in Example 1.
and was stored in aluminium tubes under conditions which accelerate degradation (shaking with alternating temperatures of 4 C. and 37 C.) for up to six months. The SG alone, i.e. without the carboxymethyl cellulose, was prepared in accordance with Example 1 and stored under identical conditions. The starting material SBG is the same starting material as used in Example 1.

(80) As shown in FIG. 9, degradation of the SG gel is enhanced by these storage conditions. Signs of degradation, i.e syneresis, can be visualised as early as 1 month under these conditions. After 6 months, the SG gel shows clear signs of syneresis and is described as a very soft, thin, heterogenous, lumpy, cracked, granular gel, while the gel consisting of SG with added carboxymethyl cellulose is unaltered compared to its appearance at study start and is retained a homogenous and sticky gel throughout the study, at least until 6 months. The combination products have been demonstrated as having enhanced stability as compared to SG alone.