Novel therapeutic uses of glucan

Abstract

A process for the production of beta-(1,3)(1,6) glucan from a glucan containing cellular source is described, together with compositions and uses / methods of treatment involving glucan. The process of the invention comprises the steps of: (a) extracting glucan containing cells with alkali and heat, in order to remove alkali soluble components; (b) acid extracting the cells of step (a) with an acid and heat to form a suspension; (c) extracting the suspension obtained of step (b) or recovered hydrolyzed cells with an organic solvent which is non-miscible with water and which has a density greater than that of water separating the resultant aqueous phase, solvent containing phase and interface so that substantially only the aqueous phase comprising beta-(1,3)(1,6) glucan particulate material remains; wherein the extraction with said organic solvent provides separation of glucan subgroups comprising branched beta-(1,3)(1,6)-glucan, and essentially unbranched beta-(1,3) glucan which is associated with residual non-glucan contaminents; and (d) drying the glucan material from step (c) to give microparticulate glucan.

Description

[0001] The present invention relates to a process for the extraction of a naturally occurring carbohydrate (glucan) from microorganisms as well as the glucan produced by this process. The invention also relates to novel therapeutic uses of glucan.BACKGROUND TO THE INVENTION[0002] Glucan is a generic term referring to an oligo- or polysaccharide composed predominantly or wholly of the monosaccharide D-glucose. Glucans are widely distributed in nature with many thousands of forms possible as a result of the highly variable manner in which the individual glucose units can be joined (glucosidic linkages) as well as the overall steric shape of the parent molecule.[0003] The glucan referred to in this invention typically is a linear chain of multiple glucopyranose units with a variable number of side-branches of relatively short length. The glucosidic linkages are predominantly (not less than 90%) .beta.-1,3 type with a lower number (not greater than 10%) of .beta.-1,6 type linkages; the ...

AI Technical Summary

Benefits of technology

[0050] The alkali extraction step may be carried out in aqueous hydroxide of from about 2% to about 6% concentration (w / v), such as between 3% and 4% (w / v). Sodium hydroxide or potassium hydroxide find particular application because of their availability and relatively low cost. However, any other strong alkali solution which has suitable solubility characteristics, for example, calcium hydroxide or lithium hydroxide, can be used. The yeast is left in contact with the alkali for a time sufficient to remove alkali soluble non-glucan components. Non-glucan components are removed more rapidly at higher temperatures. The digestion may be carried out at temperatures of from about 50.degree. C. to about 120.degree. C., requiring exposure times to the alkali of between fifteen minutes and sixteen hours. During alkali exposure, the process of cytolysis and dissolution of non-glucan components may be facilitated by vigorous mixing of the yeast suspension using appropriate methods such as by example a stirring apparatus or an emulsifying pump.

[0051] Repeat exposure of the yeast cells to fresh batches of alkali solution assists in removing non-glucan material, particularly protein, from the disrupted yeast cells. The number of alkali treatments is not limiting on the invention. However, the process should be repeated until it is apparent that the cells have been lysed and the majority, of non-glucan alkali soluble components extracted. This can be confirmed by visual or chemical analysis (such as by gas chromatography / mass spectrometry). Treatments using low strengths of hydroxide solution and low temperatures of alkali exposure generally may require increased numbers of separate alkali exposures. By way of example, alkali treatment may be repeated from one to six times.

Problems solved by technology

Prior art methods for the production of microparticulate glucan may be regarded as disadvantageous in one or more respects.

These include poor yield (such as less than about 5% w / w), low purity (such as less than about 90% purity), extended processing time, significant waste production, and high cost.

The first reason is the risk of microembolization associated with the injection of microparticulate glucan by intravenous or other parenteral routes.

The disadvantage of this method is that the enzymic digestion process is difficult to control and can result in excessive hydrolysis of the glucan molecule to monosaccharides or oligosaccharides which lack immunostimulatory activity.

A disadvantage of these methods is that of an additional step of complexity in processing operations, which may add considerably to overall manufacturing cost.

The nature of the acid used in the acid exposure step is generally unimportant.

These approaches suffer from a number of disadvantages which include the production of heterogenous glucan products of wide polydispersity which are unsuitable for pharmaceutical use without size fractionation, relative inconvenience, high cost, and production of waste materials.

It is otherwise not possible to produce soluble glucan having the properties described hereafter.

Whereas the physiology of the healing process is well described for acute surgical wounds, it is ill defined for chronic ulcers.

Ulcers typically show poor to negligible healing because of either constant irritation or pressure (such as decubitus ulcers or pressure sores) or restricted blood supply (such as in individuals with arterial ischaemia or venous thrombosis) or infection (such as `tropical` ulcers) or nerve damage (`neurotrophic` ulcers) or diabetes.

The larger ulcers in particular can be debilitating and restrictive and require intensive and expensive therapy to manage.

However, currently available `best-practice` wound management therapies are not uniformly successful, take considerable lengths of time to produce beneficial results, are associated with poor rates of patient compliance, generally are expensive, and are associated with a high incidence of ulcer recurrence.

A particular difficulty in devising a uniformly successful therapy which may be an improvement on current treatment modalities is the non-unifomity of the different types of ulcers where both the underlying aetiologic disease processes and the pathophvsiology within the wounds show considerable variation.

Confounding this variability, is the general poor understanding of which of the different components of the healing response is dysfunctional and therefore contributing the primary pathology of the dysfunctional healing response.

So that successful antagonism of dysfunction of any particular part of the healing cascade in one ulcer type may not necessarily be successful in another ulcer type.

They are a disastrous complication of immobilization.

They may result from shearing forces on the skin, blunt injury to the skin, drugs and prolonged pressure which robs tissue of its blood supply.

Diminished blood circulation of the skin is also a substantial risk factor.

Treatment is difficult and usually prolonged.

Approximately half of venous ulcers are associated with incompetent perforating veins in the region of the ankle, and constitute a long term problem in many immobile patients.

Scarring and secondary infection all impair healing and make recurrences common if healing does occur.

Disruption at the fracture site remains a problem, even where methods of physical immobilization through such mechanical means as rigid splints (such as casts, bandages, etc.), or implants (such as pins, screws, etc) are used.

Such tissues are typically densely fibrous because they are subjected to relatively high stress loads.

It is known that injuries of this kind in such tissues typically are slow to heal, due in part to the relative difficulty of totally resting the injured tissue because of their load bearing functions, but due largely to the characteristically lower level of activity of all aspects of the tissue healing cascade compared to that which is seen in soft tissues.

An important cause of this comparatively lower level of tissue repair activity in tendons and ligaments is a more limited blood supply compared to most soft tissues.

It is well described that ultraviolet light exposure causes damage to skin, particularly long term exposure to sunlight.

The major detrimental effect of ultraviolet light is damage to proteins, particularly DNA and RNA where it results in dimer formation.

Skin has a rich network of immune cells that are equally sensitive to the detrimental effects of ultraviolet light as are other skin cells and exposure to ultraviolet light leads to temporary dysfunction of these cells.

It is likely that this predisposes to the development of skin cancer through reduced immune surveillance capacity within skin.

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