Glucans

Abstract

The present invention relates to a glucan having a weight average molar mass of 15,000 to 50,000 g/mol on a single chain basis and a weight average molar mass in aqueous solution on an aggregate basis of 4 to 2010.sup.5 g/mol and existing in gel form in aqueous solution at a concentration 1% at 25 C. and neutral pH and having a melting temperature (gel to sol) of 30 to 44 C. when the glucan is dissolved in water at a concentration of 2%, methods for the production thereof, medical uses thereof, physical supports having the glucan applied thereto or impregnated thereon and in vitro methods of proliferation of skin cells which comprise contacting a population of skin cells with the glucan.

Claims

1. A gel glucan product obtainable by a) heating an aqueous solution of soluble yeast glucan molecules to a temperature of 120 C. to 130 C. and holding the solution at that temperature for 10 to 30 mins; and b) cooling the glucan solution to a temperature of 35 C. to 50 C., over a time period of less than 80 minutes, wherein said glucan is present in said gel glucan product at a concentration of 1 to 6%, has a weight average molar mass of 15,000 to 50,000 g/mol on a single chain basis and a weight average molar mass in aqueous solution on an aggregate basis of 4 to 2010.sup.5 g/mol, and wherein said gel glucan product has a gel to sol melting temperature between 30 C. and 44 C.

2. The gel glucan product of claim 1, wherein said glucan has a weight average molar mass of 20,000 to 40,000 g/mol on a single chain basis.

3. The gel glucan product of claim 1, wherein said glucan has a weight average molar mass of 25,000 to 30,000 g/mol on a single chain basis.

4. The gel glucan product of claim 1, wherein said glucan is in aqueous solution at a concentration of about 2%.

5. The gel glucan product of claim 1, wherein said glucan is derived from Saccharomyces cerevisiae.

6. The gel glucan product of claim 1, wherein said 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.

7. The gel glucan product of claim 1, wherein said glucan is essentially free of repetitive (1,6) linked glucosyl residues.

8. The gel glucan product of claim 1, wherein said gel glucan product has a gel to sol melting temperature of about 33 C.

9. The gel glucan product of claim 1, wherein in step a) the glucan is held at the temperature of 120 C. to 130 C. for about 20 minutes.

10. The gel glucan product of claim 1, wherein in step a) the aqueous solution of glucan molecules is heated to a temperature of 120 C. to 125 C.

11. The gel glucan product of claim 10, wherein in step a) the glucan is held at the temperature of 120 C. to 125 C. for about 20 minutes.

12. The gel glucan product of claim 1, wherein the glucan is cooled over a time period of 50 to 60 minutes.

13. The gel glucan product of claim 1, wherein said method is preceded by a formolysis step wherein a particulate glucan starting material is suspended in formic acid in order to remove -(1,6) linked glucosyl side chains and to solubilise the particulate glucan.

14. The gel glucan product of claim 13, wherein the formolysed product is filtered through a mesh of about 0.2 .

15. The gel glucan product of claim 1, which is present in a pharmaceutical composition also comprising one or more pharmaceutically acceptable diluents or carriers.

16. A physical support having applied thereto or impregnated therein the gel glucan product of claim 1.

17. The physical support of claim 16, wherein said physical support is selected from the group consisting of a woven, non-woven, knitted, foam or adhesive substrate; a patch, dressing, plaster, bandage, film or gauze.

Description

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

(2) FIG. 1 illustrates the SEC-MALS-RI chromatograms of a number of batches of 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 the glucan product analyzed in aqueous buffer (0.1 M 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. The aqueous SEC-MALS-RI results, in combination with the results in DMAc/LiCl, show that the glucan exist as aggregates in the aqueous solution.

(4) FIG. 3 shows storage modulus, G (Pa), plotted against temperature for a glucan gel according to the present invention. The data was obtained by small strain oscillatory measurements using a Stresstech HR rheometer and the following temperature scan: 70 to 10 C. at a rate of C./min, kept at 10 C. for 2 h and then 10 to 70 C. at a rate of C./min. The melting temperature of this gel (gel to sol) is determined to approximately 33 C. based on where the increasing temperature curve levels out (G0 Pa).

(5) FIG. 4 shows the viscosity measurements of a 2% glucan gel according to the present invention using an up-down rate ramp method. The data was obtained at 30 C. with an equilibration time of 3 min at 2 rpm prior to first measurement and 30 sec equilibration before measurement on each consecutive speed after that. Calculated 10 rpm viscosity was 1772 cP from the IPC Paste model in the Rheocalc software of Brookfield Engineering Inc.

(6) FIG. 5 describes the release of TNF- from human myeloid dendritic cells derived from peripheral blood monocytes cultured in the presence of 200 g/ml of the glucan gel of the present invention, curdulan or LPS. The cytokine was measured in culture medium supernatants at 24 h post-stimulation using a commercially available ELISA kit.

(7) FIG. 6 illustrates the release of CXCL10 (IP10) from human myeloid dendritic cells derived from peripheral blood monocytes cultured in the presence of 200 g/ml of the glucan gel of the present invention, curdlan or LPS. The chemokine was measured in culture medium supernatants at 24 h post-stimulation using a commercially available ELISA kit.

(8) FIG. 7 shows the secretion of CXCL-10 from macrophages harvested from db/db mice costimulated in vitro by LPS and the glucan gel of the present invention. Lane 1; LPS alone, lane 2; LPS+20 g/ml of the glucan gel of the present invention, lane 3; LPS+2 g/ml of the glucan gel of the present invention. *p<0.05. *p<0.01.

(9) FIG. 8 shows the results of a wound healing trial using the glucan gel according to the present invention in a 2% and 4% concentration in an aqueous solution compared to a growth factor cocktail with a known efficacy profile. The dressing+water was used as vehicle control. Both 2% and 4% concentrations are effective, with 4% being more effective, although less effective than the GF cocktail.

(10) FIG. 9 shows activation of complement by different batches of beta-glucans. Accumulation of fluid-phase terminal complement complex in human serum was measured. 241-7, 231-0, 411-8, 391-8 are beta-glucans in gel form representing glucans of the present invention. VLMSG represent a non-complement activating formulation of a soluble beta-glucan which is not in gel form. The horizontal dotted line represents the spontaneous complement activation in human serum.

(11) FIG. 10 shows secretion of TNF from a dectin-1 over-expressing RAW cell-line variant. The soluble yeast beta glucan 421-4 represents a glucan of the present invention, while 161194, 30395, xx0995 and 51196 are clear, non-gelling variants.

(12) FIG. 11 shows the biological effect in the dectin-1 over-expressing RAW cell line of soluble glucan (SG) subjected to heating and rapid cooling (HC) as described in Example 1. GC065 rn 9270 is a soluble glucan product with a broken gel, as described in Table 1. Treatment according to Example 1 rescues the biological effect of the broken product.

(13) FIG. 12 shows the biological effect in the dectin-1 over-expressing RAW cell line of soluble glucan (SG) subjected to heating and rapid cooling (HC) as described in Example 1. 421-4 is a SG product corresponding to the product described as a soft gel in Table 1. Treatment according to Example 1 enhances the biological effect of SG batch 421-4.

EXAMPLES

Example 1

Preparation of Gel Glucan Product of the Present Invention

(14) An aqueous solution of 1.5 to 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).

(15) a. Heat Treatment:

(16) 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.

(17) 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.

(18) b. Active Cooling:

(19) 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

In Vivo Wound Healing in Mouse Model

(20) The impact of test glucans and controls 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). Upon acclimation (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 ill monitored on a daily basis throughout the study.

(21) 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 a glucan or control by injection 50 l of a 2% solution 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. For the experimental groups receiving glucan treatments was 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. For the positive control group treatments was reapplied daily until post-wounding day 6wounds in this group received a total of 7 applications of the growth factor combination treatment. On post-wounding days 4, 8 and 12 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 and 8, wounds were re-dressed as above with Bioclusive film dressing. Healing was determined as wound closure relative to the wound size at day 0.

(22) The results are shown in Table 1 and FIG. 8 below.

(23) TABLE-US-00002 TABLE 1 Treatment of wounds in diabetic mice Healing Healing Healing wounds, wounds, wounds, Product Day 8 day 12 day 15 Negative control (dressing only) 0/10 1/10 2/10 Vehicle control (water + dressing) 1/10 3/10 4/10 Broken gel product 3/10 7/10 7/10 Soft gel glucan (2%) 5/10 9/10 10/10 Solid gel glucan (4%) 8/10 10/10 10/10 Positive control (GFcoctail) 10/10 10/10 10/10

(24) The soft gel glucan product and the solid gel glucan product are both yeast derived glucans which have been prepared by the methods of the present invention. Both are yeast derived glucans which have been treated with formic acid to remove (1,6) linked glucosyl units found in the native yeast glucan side chains. The solid and soft gel glucans have been prepared using the new heating and rapid cooling protocol described herein (Example 1). Surprisingly the solid gel (a 4% product) demonstrates enhanced wound healing activity as compared to the soft gel glucan product (a 2% product).

(25) The broken gel product describes a yeast derived glucan product where the (inter-molecular) conformation of the molecules in the gel has been destroyed to a large degree by exposure to an agent which interferes with hydrogen bonding. This glucan is not able to exert a similar beneficial efficacy/healing profile compared to the gel product of the invention. This result clearly shows that the gel structure of the glucan according to the present invention is a surprisingly important property for in vivo efficacy.

(26) The results clearly show that the use of a glucan gel produced according to the invention elicits a more potent response in wound treatment models compared to the delivery vehicle and negative control. The fact that the broken gel product gives inferior results also points to the necessity of the existence of an intact gel structure or specific high order conformation within a glucan gel.

Example 3

Determination of Molar Mass

(27) The molar mass of a series of yeast glucan products was determined using size exclusion chromatography as previously defined in the description. The experiment was performed with the glucan in aqueous solution and thus gives molar mass values for the glucan aggregates within the glucan sample and not on a single chain bases. 5 glucan samples were tested, all were derived from yeast and all had had their (1,6) linked side chains diminished. 4 were in solution and the 5th was a gel glucan in accordance with the present invention (prepared in accordance with Example 1).

(28) The calculated average molar mass for the four glucans in aqueous solution varied from 1.0-3.7410.sup.5 g/mol.

(29) The glucan of the invention had an average molar mass of 810.sup.5 g/mol.

(30) In other experiments, the glucans of the present invention had molar masses which varied from 5-1510.sup.5 g/mol.

Example 4

Determination of Melting Point

(31) Determination of the melting point of a glucan gel produced according to the present invention was performed as described in the description and the results are shown in FIG. 3.

Example 5

Viscosity Measurements

(32) Determination of the viscosity of a glucan gel produced according to the present invention (prepared according to Example 1) was performed as described in the description and the results are shown in FIG. 4.

Example 6

Biological Activity

(33) The effect of the gel glucans of the present invention, which were prepared as different batches, each according to Example 1, on the release of TNF and CXCL-2 and -10 is described herein and shown in the Figures.

(34) In addition, the ability of the beta glucans to activate the complement system was measured. The complement system is composed of a series of serum proteins. The system is a part of the innate immune system, and is activated upon infection or detection of pathogen associated molecular patterns. Activation of the system results in a cascade of cleavage of the complement proteins, which ultimately leads to formation of a terminal complement complex (TCC). Accumulation of TCC can be measured by detection of a neo-epitope using monoclonal antibodies, and can thus serve as an indicator of complement activation.

(35) The glucan was diluted (1:10) in human serum to a volume of 100 The mixture was incubated at 37 C. for 30 min, then diluted 1:5 in PBS before the relative amount of fluid-phase TCC was determined in triplicate using a commercially available ELISA-test kit for human TCC. The results are shown in FIG. 9.

(36) The soluble beta-glucan of the present invention, in gel form, activates the complement system in human serum. However, activation of complement is not a common feature of soluble beta-glucans, as exemplified by VLMSG in FIG. 9. The molecular weight of VLMSG range from 10.sup.3 to approximately 510.sup.6 g/mol, with a mean value 1,310.sup.4 g/mol, and yields a clear solution.

(37) It was demonstrated that gel-forming soluble yeast beta glucans of the present invention prepared according to Example 1 stimulate the release of TNF from dectin-1 over-expressing RAW cells, while non-gelling, clear, variants of soluble yeast beta glucans apparently do so to a much lesser extent (FIG. 10).