WO2011032229A1 - A novel epsilon polymorphic form of inulin and compositions comprising same - Google Patents

Abstract

A novel polymorphic form of inulin, designated epsilon inulin (eIN), is described along with methods for the preparation of eIN, compositions comprising eIN and uses thereof. Amongst the uses described is the stimulation of an immune response in a subject for the purposes of preventing or treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic disease, degenerative or ageing disease.

Description

A NOVEL EPSILON POLYMORPHIC FORM OF INULIN

AND COMPOSITIONS COMPRISING SAME

FIELD OF THE INVENTION

The present invention relates to a novel polymorphic form of inulin, designated epsilon inulin (eIN). In a particular application, the invention provides methods for the preparation of eIN, compositions comprising eIN and uses thereof.

INCORPORATION BY REFERENCE

This patent application claims priority from:

United States Patent Application No 61/243975 titled "Polysaccharide polymorph" filed 18 September 2009.

The entire content of this application is hereby incorporated by reference. The following International Patent Applications are referred to herein:

- PCT/AU86/0031 1 (WO 87/02679) titled "Immunotherapeutic treatment",

- PCT/AU89/00349 (WO 90/01949) titled "Gamma inulin compositions", and

- PCT/AU2005/001328 (WO 2006/024100) titled "New polymorphic form of inulin and uses

thereof.

The entire content of these applications is hereby incorporated by reference. BACKGROUND OF THE INVENTION

Inulin is a simple, inert polysaccharide comprising a family of linear β-ϋ-(2— * l)polyfructofuranosyl a-D- glucoses, in which an unbranched chain of fructose moieties is linked to a single terminal glucose. Inulin preparations therefore comprise neutral polysaccharides of simple, known composition, but which are • molecularly polydisperse, with molecular weights ranging up to 16kD or beyond. Inulin is the storage carbohydrate of the family of flowering plants known as Compositae and is cheaply available from chicory, Jerusalem artichoke or dahlia tubers. It has a relatively hydrophobic, polyoxyethylenerlike backbone, and this unusual structure plus its non-ionised nature allows recrystallization and easy preparation in a very pure state. In nature, inulin can exist as chains of up to a degree of polymerisation (DP) of 60 or more fructose moieties, and in a number of distinct polymorphic forms having different solubility characteristics and other properties.

Although the molecular composition of inulin is well known, the reported determinations of its solubility are conflicting. For example, the Merck Index (Thirteen Edition, 2001) describes inulin as "slightly soluble in cold water and organic solvents, soluble in hot", whereas a quantitative study (Phelps, 1965) suggested that two distinct forms of inulin exist; the first obtained by precipitation from water, the second by precipitation from ethanol; both of which are substantially soluble in water at 37°C. The form obtained by precipitation from water is referred to as alpha-iriulin (aIN), and the form obtained by precipitation from ethanol is known as beta inulin (blN).

It was observed that suspensions of aIN and blN became less soluble in water on standing at room temperature (Cooper, PD and M Carter, 1986a). This led to the discovery of a third polymorphic form of particulate inulin, designated gamma inulin (gIN; also referred to as "Gammulin"), which is disclosed in - WO 87/02679 (see also Cooper, PD and M Carter (1986), and Cooper, PD and EJ Steele (1988)). This third polymorphic form is virtually insoluble in water at 37°C, but becomes soluble in concentrated solution (eg 50 mg/ml) at temperatures over 45°C, as are the alpha and beta forms. It is particularly characterised as having a 50% OD700 thermal transition point (solubilisation phase shift in dilute suspension) as sharp as a melting point of 47±1°C.

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More recently, a fourth polymorphic form of particulate inulin, designated delta inulin (dIN; also referred to as "Deltin"), has been identified. Delta inulin is disclosed in WO 2006/024100, and is virtually insoluble in water at 50°C. Indeed, dIN in a concentrated solution (eg 50 mg/ml) is only soluble when heated to a temperature in the range of 70°C-80°C. dIN is further characterised as having a 50% OD700 thermal transition point in dilute aqueous suspensions of 53-58°C. It is conveniently prepared by heating concentrated aqueous suspensions of gIN at >55°C.

These four, previously known, polymorphic forms in which inulin crystallizes may therefore be characterised by their different solubility rates in aqueous media ranging from one that is rapidly soluble at 23 °C (beta ₂3° inulin) through a form soluble at 37°C with a half-time of 8 minutes (alpha 37^s inulin) to a form virtually insoluble at 37°C (gIN) and another virtually insoluble at 50°C (dIN). These latter two, being insoluble at 37°C, may, if introduced into a biological system at this temperature such as a human, persist in such a system in their respective particulate form. While not wishing to be bound by theory, it is now considered that this particulate nature of these forms, at least in part, explains observations first made in the early 1970s that inulin, while physiologically inert in solution (British Pharmaceutical Codex , 1979) and non-antigenic and non-nephrotoxic, (Verroust, PJ et al. (1974)), nevertheless in particulate form is immunologically active; specifically, acting as an activator of the alternative complement pathway (APC) (Cooper, PD and M Carter (1986)). Accordingly, inulin can exist in a number of distinct polymorphic forms having different solubility characteristics and other properties. This patent application relates to a novel inulin polymorphic form, designated epsilon inulin (el ), possessing interesting and useful properties. SUMMARY OF THE INVENTION

Thus, in a first aspect, the present invention provides a substantially purified preparation of inulin, wherein said inulin is in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C. In a second aspect, the present invention provides a pharmaceutical or immunological composition comprising particles of inulin in an epsilon polymorphic form having a 50% OD₇oo thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C.

The composition of the present invention may be formulated in a pharmaceutically acceptable carrier, diluent or excipient in a form suitable for injection, or a form suitable for oral, rectal, vaginal, topical, nasal or ocular administration. The composition may also comprise an active component such as, for example, a vaccinating antigen (including recombinant antigens), an antigenic peptide sequence, or an immunoglobulin. Further, the composition may comprise one or more immune modulator such as a lymphokine or cytokine, a thymocyte stimulator, a macrophage stimulator, or an endotoxin.

In a third aspect, the present invention provides a method of stimulating an immune response in a subject, for the purposes of, for example, preventing of treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic. disease, degenerative or ageing disease, wherein said method comprises administering to said subject an effective amount of a preparation according to the first aspect or a composition according to the second aspect.

In a related, fourth aspect, the present invention provides a method of enhancing an immune response in a subject, for the purposes of, for example, preventing or treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic disease, or degenerative or ageing disease, wherein said method comprises administering to said subject an effective amount of a preparation according to the first aspect or a composition according to the second aspect.

In a fifth aspect, the present invention provides a method for the preparation of inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C, said method comprising heating a suspension comprising gamma inulin (glN) a d/or delta inulin (dlN) at a temperature of > 55°C.

In a sixth aspect, the present invention provides a. method for the preparation of inulin in an epsilon polymorphic form having a 50% OD700 thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C, said method comprising maintaining a solution of inulin at a concentration of 10 mg/ml or more at a temperature in the range of about 50°C to about 85°C for a period of at least 15 minutes up to about 2 weeks or more. In a further aspect, the present invention provides a method for fractionating a polydisperse inulin mixture, said method comprising precipitating from a suspension comprising dissolved inulin, inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C so as to obtain a high molecular weight inulin fraction, and thereafter separating said epsilon inulin (elN) from residual soluble inulin in the suspension so as to obtain a lower molecular weight inulin fraction.

In a yet further aspect, the present invention provides a substantially purified preparation of inulin prepared from a precursor preparation substantially consisting of inulin in an epsilon polymorphic form having a 50% OD₇₀b thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 5.8°C, wherein said purified preparation of inulin has been prepared by dissolving the epsilon inulin (elN) of said precursor preparation by heating in the form of a slurry in a solvent such as water or ethanol, and thereafter, precipitating the inulin as one or more of alpha inulin (aTN), beta inulin (blN), gamma inulin (glN) or delta inulin (dlN). In a still further aspect, the present invention provides a method for assaying for the presence or concentration in a suspension, of inulin that is in an epsilon polymorphic form, wherein said method comprises measuring the 50% OD700 thermal transition point in a dilute aqueous suspension (<0.5 mg/ml).

In a yet still further aspect, the present invention provides a method of treating or preventing radiation- or chemotherapy-induced immunosuppression in a subject, wherein said method comprises administering to said subject an effective amount of a preparation according to the first aspect or a composition according to the second aspect. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides a graph demonstrating the formation of eIN during slow heating (■) or rapid heating (O) of dlN suspensions;

■

Figure 2 provides optical density thermal transition curves of eIN compared with dlN. Dilutions in 5 ml of phosphate buffered saline (PBS) (0.5 mg/ml) of each preparation contained in glass tubes were heated together in a water bath at increasing temperatures. The OD₇oo of each dilution was measured after equilibration at each temperature;

Figure 3 provides optical density thermal transition curves of aIN, bIN and gIN preparations. Five ml dilutions at 0.5 mg/ml in glass tubes of each were heated together in a water bath at increasing temperatures. The OD₇oo of each dilution was measured after equilibration at each temperature. Symbols: +, four batches of gIN; Δ, A ,□, aINs; and o, bIN (from Cooper, PD and EJ Steele, 1991);

Figure 4 provides optical density thermal transition curves of two preparations of dlN compared with those of the respective gIN preparations from which they were made. The OD₇oo of each dilution was measured after equilibration at each temperature (from WO 2006/024100); Figure 5 shows a graph indicating the optimum temperatures of formation of gIN, dlN and eIN from aIN, gIN and dlN respectively. Values are expressed as a percentage of the maximum OD_70Q found;

Figure 6 provides a graph indicating the rate of conversion of dlN to eIN at its optimum temperature of 66°C. Portions of a dlN suspension (5 ml, 50 mg/ml) were heated in a water bath at 66°C and samples then diluted to 0.5 mg/ml in 5 ml PBS at intervals. All samples were then heated for 10 minutes at 60°C for eIN assay;

Figure 7 provides a graph indicating the rate of conversion of gIN and dlN from aIN and gIN, respectively, at their optimum temperatures (45°C and 55°C). The results were obtained by heating portions of aIN and gIN suspensions (5 ml, 50 mg ml) in water baths either at 45°C or 55°C, followed by dilution of samples to 0.5 mg/ml in 5 ml PBS at intervals. All samples were then heated for 10 minutes either at 37°C or 50°C for the assay of gIN and dlN respectively;

Figure 8 provides a bar graph showing the yields of aIN, dlN, gIN and eIN from a typical sample of un- purified inulin. Raw inulin was dissolved to 130 mg/ml in water and the solution then allowed to crystallize at 5°C over 1 week to form aIN before step-wise conversion to glN (45°C), dIN (55°C) and eIN (66°C). The supernatant refractive index was measured at each step to assess the insoluble fraction, taken as the yield of each polymorphic form at that temperature. The "soluble" inulin shown is the yield that failed to form aIN in the original crystallization;

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Figure 9 provides a graph showing the yields of the various polymorphic forms of particulate inulin deposited upon incubation of replicate inulin solutions at different temperatures. A raw inulin solution was ion exchange- and sterile-filtered but otherwise unfractionated, adjusted to 100 mg/ml and portions stood for 3 weeks in rooms or water-baths maintained at the indicated temperatures. At 7-day intervals, the refractive indices of the clear supernatant fluids were measured and the percentage of inulin precipitated calculated;

Figure 10 provides a graph showing the optical density thermal transition curves of each suspension at 3 weeks. Dilutions in 5 ml of phosphate buffered saline (PBS) (0.5 mg/ml) of each unfractionated suspension contained in glass tubes held in ice, were heated together in a water bath at increasing temperatures. The OD₇₀o of each dilution was measured after equilibration at each temperature and expressed as a percentage of the OD₇₀o at 0°C;

Figure 11 provides a graph showing the reversibility of dIN. Samples of three inulin preparations (dIN dissolved then recrystallized either at 5°C, or at 37°C, or at 37°C then heated at 56°C, respectively), were diluted to 0.5 mg/ml in PBS and progressively heated together in a water bath to equilibrate at the indicated temperatures, when their optical densities were measured (from WO 2006/024100);

Figure 12 provides a graph showing the reversibility of eIN. A sample of eIN was dissolved at 80°C and portions were crystallized at 5°C, 37°C and 50°C, then half of the 50°C sample was heated at 66°C for 2 hr to convert to eESf again;

Figure 13 provides graphical results of the solubilisation of dIN and eIN. Samples (0.5 ml) of dIN (1 12 mg/ml) and eIN (45-74 mg/ml) were heated in sealed 1.5 ml Eppendorf™ centrifuge tubes for 15 minutes fully immersed in a water bath at the indicated temperatures, rapidly centrifuged and the refractive index of the supernatants measured;

Figure 14 provides a graph showing the solubility of three inulin polymorphic forms in dimethyl sulphoxide. Samples of 50μ1 of purified glN, dIN and eIN (50 mg/ml in PBS) were added to 450 μΐ of DMSO dilutions in water to make the indicated final concentrations; and Figure 15 provides bar graphs showing superior vaccine adjuvant activity of elN compared to dIN; thirty- five day old Balb/c mice in groups of 10 were given a single intramuscular immunisation with a 0.5 μg dose of Hepatitis B surface antigen (HBsAg) adjuvanted with either 0.2 mg of dIN or elN. Three weeks after immunisation, the mice were bled and the serum analysed for anti-HBsAg antibody titres by

AUSAB® assay (Abbott Laboratories Inc, Abbott Park, IL, United States of America). (A) Shows that the anti-HBsAg antibody titres were significantly (p<0.05) higher in the mice where the HBsAg was adjuvanted with elN. Using an anti-HBsAg antibody titre of 10 IU/ml as a cutoff to define seroprotection, (B) shows that 100% of the mice immunised with the elN-adjuvanted vaccine achieved a seroprotective HBsAg titre compared to only 60% of mice in the dlN-adjuvanted vaccine group (p<0.05 by one sided Fisher exact test).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified a novel, fifth polymorphic form of inulin, designated epsilon inulin (elN). Similar characteristics to those of aIN, bIN, gIN and dIN are seen again in the formation and properties of elN which, however, also has distinguishing unique properties and utilities.

Epsilon inulin may be defined as having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C thereby making it distinct from dIN, and elN is optimally formed in a concentrated aqueous inulin suspension maintained at > 55°C. It has been found that aIN, bIN, gIN and dIN can be converted into elN by heating at an appropriate temperature. For example, it has been found that when concentrated suspensions of dIN are heated rapidly, the dIN dissolves completely by 73°C. Unexpectedly, when the same preparations were heated slowly between 60-80°C, a thick pasty precipitate developed that did not dissolve at 73°C. When examined, this high temperature inulin precipitate had characteristics distinct from all of aIN, bIN, gIN and dIN, thereby indicating the existence of a novel, fifth polymorphic form of inulin (epsilon insulin) extending the series of inulin polymorphic forms with stepwise higher 50% OD₇oo thermal transition points in dilute aqueous suspension.

Thus, in a first aspect, the present invention provides a substantially purified preparation of inulin, wherein said inulin is in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C.

The term "inulin" as used herein refers not only to inulin, -D-[2— l]-polyfructofuranosyl a-D-glucose, but also to derivatives thereof including β 0-[2-→1] polyfructose which may be obtained by enzymatic removal of the end glucose from inulin, for example using an invertase or inulase enzyme capable of removing this end glucose. Other inulin derivatives included within the scope of the term are derivatives of inulin in which the free hydroxyl groups have been etherified or esterified, for example by chemical substitution with alkyl, aryl or acyl groups by methods well known to persons skilled in the art.

The epsilon inulin (eIN) of the present invention preferably shows a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) in the range of about 58°C to about 80°C.

In one embodiment of the preparation of the present invention, the eIN shows low solubility in aqueous media up to 59^°C, more preferably, a low rate of solution in aqueous media up to 75 ^°C.

The single inulin polymer molecules from which eIN particles are comprised may have a molecular weight in the range of from about 5 to about 50 kD.

The degree of polymerisation (DP) of the single inulin polymer molecules from which eIN particles are comprised will typically be high (eg a weight-average DP > 30 fructose moieties, preferably > 35 fructose moieties).

Preferably, the eIN shows a lower level of solubility in dimethyl sulphoxide (a solvent known to neutralise hydrogen bonding), relative to each of alN, bIN, glN and dlN.

Like the other polymorphic forms of inulin, eIN formation has been found to be fully reversible with the other inulin forms upon dissolving and recrystallizing. Thus, eIN appears to result from a conformational rearrangement of the inulin polymer molecules rather than a chemical or covalent change in the structure of the inulin polymer molecules.

The eIN may be derived from inulin purified from various plant sources including dahlia, chicory, Jerusalem artichoke, onion and garlic, inulin produced synthetically from sucrose, glucose or fructose, or inulin synthetically extended in polymer length by methods well known to persons skilled in the art.

The term "substantially purified preparation" as used herein is to be understood as referring to an inulin preparation that is essentially free of other polysaccharides and/or other exogenous biological materials (eg microbial- or plant-derived materials). Such a preparation will comprise no more than 10% (by weight) of exogenous biological materials, and may be prepared by any of the methods well known to persons skilled in the art including well known hot water extraction and purification processes employed in the commercial production of inulin from chicory (Stephen, AM et al, 2006). Delta inulin and glN have been previously identified as immunologically active, particularly as vaccine adjuvants, either alone or associated with an antigen-binding carrier material such as aluminium hydroxide (Cooper, PD and EJ Steele, 1991 ; Cooper, PD et al, 1991a: Cooper, PD et al, 1991b; WO 90/01949 and WO 2006/024100). In Example 6 provided herein, the present inventors not only show that eIN is also immunologically active but, moreover, possesses a level of immunological activity that exceeds that of glN and dlN, thereby offering the possibility that an elN-based adjuvant will be more potent than any of the inulin-based adjuvants studied and/or developed to date.

Further, eIN is the most temperature stable of all of the five inulin polymorphic forms, a fact that means that suspensions of eIN particles remain insoluble at temperatures that would otherwise dissolve the other inulin forms. This provides major stability advantages when eIN is used in manufacturing applications involving heating up to 85°C. Further, it means that eIN particles are stable at high ambient climatic temperatures, a major advantage if eIN is to be used as an adjuvant where temperature stability is a requirement.

In a second aspect, the present invention provides a pharmaceutical or immunological composition comprising particles of inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C. Preferably, the eIN particles of the composition of the present invention are characterised in that they show a low rate of solution in aqueous media up to 59°C, more preferably, a low rate of solution in aqueous media up to 75°C.

Preferably, the eIN particles of the composition of the present invention have a particle diameter within the size range of about 100 nm to about 10 μηι, more preferably, within the size range of about 1 μπι to about 5 μπι.

The composition of the present invention may be formulated in a pharmaceutically acceptable carrier, diluent or excipient in a form suitable for injection, or a form suitable for oral, rectal, vaginal, topical, nasal or ocular administration. The composition may also comprise an active component such as, for example, a vaccinating antigen (including recombinant antigens), an antigenic peptide sequence, or an immunoglobulin. Alternatively, or additionally, the active component may be a lymphokine or cytokine, a thymocyte stimulator, a macrophage stimulator, an endotoxin, a polynucleotide molecule (eg encoding a vaccinating agent) or recombinant viral vector, a whole microorganism (eg a bacterial lysate), or even a whole virus (eg an inactivated or attenuated virus). Indeed, the composition of the present invention is particularly suitable for use with inactivated/attenuated whole virus as the active component.

Preferred vaccinating antigens that are suitable for inclusion in the composition of the present invention include all or antigenic portions of bacteria, viruses, yeasts, fungi, protozoa and other microorganisms or pathogens of human, animal or plant origin and pollens and other allergens, especially venoms (eg bee and wasp venoms), and asthma-inducing allergens such as house dust mite, cat or dog dander.

Particularly preferred vaccinating antigens are: viral antigens of influenza virus such as haemagglutinin protein (eg seasonal strains of inactivated influenza virus, recombinant HA antigen, and seasonal HI, H3 B or pandemic H5 antigen) and influenza nucleoprotein, antigens of the outer capsid proteins of rotavirus, antigens of human immunodeficiency virus (HIV) such as the gpl20 protein of HIV, the surface proteins of respiratory syncytial virus (RSV), antigen E7 of human papilloma virus (HPV), Herpes simplex, Hepatitis B virus (eg HBsAg), surface proteins of Hepatitis C virus (HCV), inactivated Japanese encephalitis virus, surface proteins of Lyssavirus (causative of rabies); and antigens from microorganisms such as Shigella, Porphyromonas gingivalis (eg the proteinase and adhesin proteins), Helicobacter pylori (eg urease),- Listeria monocytogenes, Mycobacterium tuberculosis (eg BCG), Mycobacterium avium (eg hsp65), Chlamydia trachomatis, Candida albicans (eg the outer membrane proteins of C. albicans), Streptococcus pneumoniae, Neisseria meningitidis (eg class 1 outer protein), Bacillus anthracis (causative of anthrax), Coxiella burnetii (causative of Q fever, but which can also induce long-term protection against autoimmune diabetes- ( ie Type I diabetes)) and malaria-causing protozoa (particularly,

Plasmodium falciparum and Plasmodium vivax).

Other particularly preferred antigens are cancer antigens (ie antigens associated with one or more cancers) such as: carcinoembryonic antigen (CEA), mucin-1 (MUC-1), epithelial tumour antigen (ETA), abnormal products of p53 and ras, and melanoma-associated antigen (MAGE).

Where the composition of the present invention comprises a vaccinating antigen, the composition may preferably comprise an antigen-binding carrier material such as, for example, one or more metal salts or precipitates such as magnesium, calcium or aluminium phosphates, sulphates, hydroxides or hydrates thereof (eg aluminium hydroxide and/or aluminium sulphate) and/or one or more proteins, lipids, organic acids including sulphated or phosphorylated polysaccharides (eg heparin, dextran and cellulose derivatives), organic bases such as chitin (poly N-acetylglucosamine) and deacetylated derivatives thereof or basic cellulose derivatives, and/or other antigens. The antigen-binding carrier material may comprise poorly soluble particles of such materials (eg particles of aluminium hydroxide (alum) gel or a hydrated salt complex thereof). Advantageously, the antigen-binding carrier material does not tend to aggregate or is treated to avoid aggregation. Most preferably, the antigen-binding carrier material is aluminium hydroxide (alum) gel, aluminium phosphate gel or calcium phosphate gel.

Preferably, where an antigen-binding carrier material is present, it is present in a form that is intrinsically associated with the eIN, such as, for example, co-crystals with the eIN. Co-crystals of a particulate form of inulin and an antigen-binding carrier material such as a metal salt may be prepared by, for example, a method comprising:

(a) preparing an inulin solution or partial inulin solution by heating an aqueous suspension of eIN particles;

(b) adding to said solution an amount of one or more metal phosphate compounds;

(c) recrystallizing the inulin from said solution;

(d) transforming the recrystallized inulin back to the eIN form; and

(e) isolating formed co-crystals of the eIN and one or more metal phosphate compounds.

The preparation of the solution or partial solution of the inulin in step (a) may comprise either the application of a temperature of about 55 °C for a period of about 30 minutes or such time sufficient to partially but not fully solubilise the eIN particles. The recrystallization of the inulin in step (c) may comprise applying a temperature of about 4°C. The transformation of the recrystallized inulin back to the eIN form in step (d) may comprise applying a series of temperature steps of about 37°C, 45 °C and >48 °C, respectively. The step of isolating eIN co-crystals in step (e) may comprise, for example, centrifugation and washing away of any remaining soluble inulin.

The diameter of the particles of the eIN in combination with an antigen-binding carrier material such as a metal salt may be in a range of from about 100 nm to 10 μπι, more preferably, within the size range of about 1 μπι to about 5 μπι. The particles of the 'eIN in combination with the antigen-binding carrier material may comprise a relative amount (by weight) of the inulin to the antigen-binding carrier material in the range of 1 :20 to 200: 1.

Further, the composition of the present invention may further comprise one or more immune modulator such as a lymphokine or cytokine, a thymocyte stimulator, a monocyte or macrophage stimulator or an endotoxin.

In a third aspect, the present invention provides a method of stimulating an immune response in a subject, for the purposes of, for example, preventing or treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic disease, degenerative or ageing disease, wherein said method comprises administering to a subject an effective amount of a preparation according to the first aspect or a composition according to the second aspect. In a related, fourth aspect, the present invention provides a method of enhancing an immune response in a subject, for the purposes of, for example, preventing or treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic disease, degenerative or ageing disease, wherein said method comprises administering to a subject an effective amount of a preparation according to the first aspect or a composition according to the second aspect.

The term "effective amount" as used herein refers to a non-toxic but sufficient amount of the

preparation/immunological composition to provide the desired effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular

preparation/immunological composition being administered, and the mode of administration etc. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be routinely determined by persons skilled in the art.

The methods of the third and fourth aspects may stimulate/enhance an immune response by activation or modulation of mononuclear immune cell (eg monocytes, macrophages, and dendritic cells) function and/or the complement pathway in a human or non-human animal subject, for the purposes of inducing or modulating an immune response for, for example: the treatment or prevention of an infection by a bacterium, mycoplasma, fungus, virus, protozoan or other microorganism, or of an infestation by a worm or parasite or to treat or prevent immuno-pathology induced by such an infection; the treatment of an immune disorder such as an allergic or rheumatic disease, an autoimmune disease, an immunodeficiency disease, or neurological, dermatological, renal, respiratory or gastrointestinal disorders relating to dysfunction of the immune system; or the treatment of a tumour or cancer. As such, it is also to be understood that the present invention extends to a method for treating or preventing cancer in a subject, wherein said method comprises administering to said subject an effective amount Of effective amount of a preparation according to the first aspect or a composition according to the second aspect.

In a fifth aspect, the present invention provides a method for the preparation of inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C, said method comprising heating a suspension comprising gamma inulin (gIN) and/or delta inulin (dlN) at a temperature of > 55°C. This method for the preparation of eIN is essentially based on its optimal conversion temperature from dIN in aqueous suspensions at temperatures > 55°C. This is contrasted with the optimal conversion temperatures of gIN from aIN (ie 44-45°C) and dIN from gIN (ie 53-55°C).

Preferably, the method of the fifth aspect, comprises the steps of:

(a) heating an aqueous suspension of gIN or dIN at a temperature of > 58°C for a period of at least 15 minutes up to about 2 weeks or more;

(b) thereafter heating said suspension at a temperature in the range of from about 60°C to about 76°C for up to 5 hours, to cause the formation of eIN; and, optionally,

(c) isolating at least a portion of the formed eIN.

Preferably, in step (a), the aqueous suspension is heated for a period of from about 1.5 to 10 hours, most preferably about 3 hours. Preferably, in step (b), the aqueous suspension is heated for a period of from about.0.5 to 5 hours, most preferably about 3 hours. Particles of eIN may be isolated in step (c) by, for example, subjecting the suspension to centrifugation or some other isolation process such as tangential- flow filtration.

Epsilon inulin can also be prepared by precipitation directly from a solution of mixed molecular weight inulin polymer molecules.

Thus, in an alternative to the method of the fifth aspect, the present invention provides, in a sixth aspect, a method for the preparation of inulin in an epsilon polymorphic form having a 50% OD₇oo thermal transition point in dilute aqueous suspensions (<0.5 mg ml) of > 58°C, said method comprising maintaining a solution of inulin at a concentration of 10 mg/ml or more at a temperature in the range of about 50°C to about 85°C for a period of at least 15 minutes up to about 2 weeks or more.

The present inventors have also found that the formation of eIN selects inulin polymer molecules with a" higher mean degree of polymerisation (DP) than aIN, bIN, gIN or dIN. Thus, the process of preparing eIN provides a convenient method whereby a mixed inulin preparation comprising inulin polymer molecules of varying DP (typical of plant-derived inulin sources) can be conveniently fractionated such that the eIN fraction comprises high DP inulin (eg DP > 30 fructose moieties, preferably > 35 fructose moieties) and the soluble, non-eIN fraction comprises lower DP inulin. Since eIN, when redissolved and recrystallized at the relevant temperatures, produces high yields of gIN and dIN, one use of eIN prepared in this manner (ie such that the process effectively results in a fraction of inulin of high DP) is for the convenient production of high yields of gIN and dlN. By the use of eIN as a feedstock, the present inventors have shown that gIN and dlN can be optimally crystallized from concentrated inulin solutions prepared from solub^'ilised eIN at lower than previously quoted optimal temperatures for formation of gIN and dlN, respectively.

·

Thus, in a further aspect, the present invention provides a method for fractionating a polydisperse inulin mixture, said method comprising precipitating from a suspension comprising dissolved inulin, inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C so as to obtain a high molecular weight inulin fraction, and thereafter separating said epsilon inulin (eIN) from residual soluble inulin in the suspension so as to obtain a lower molecular weight inulin fraction.

In a yet further aspect, the present invention provides a substantially purified preparation of inulin prepared from a precursor preparation substantially consisting of inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of

> 58°C, wherein said purified preparation of inulin has been prepared by dissolving the epsilon inulin (eIN) of said precursor preparation by heating in the form of a slurry in a solvent such as water or ethanol, and thereafter, precipitating the inulin as one or more of alpha inulin (aIN), beta inulin (blN), gamma inulin (gIN) and delta inulin (dlN).

In a still further aspect, the present invention provides a method for assaying for the presence or concentration in a suspension of inulin that is in an epsilon polymorphic form, wherein said method comprises measuring the 50% OD₇oo thermal transition point in a dilute aqueous suspension (<0.5 mg/ml). In a yet still further aspect, the present invention provides a method of treating or preventing radiation- or · chemotherapy-induced immunosuppression in a subject, wherein said method comprises administering to said subject an effective amount of a preparation according to the first aspect or a composition according to the second aspect. The invention is hereinafter described by reference to the following non-limiting examples and accompanying figures. EXAMPLES

Example 1 ' Identification of epsilon Inulin (eIN) Materials and Methods

A glass tube containing 7 ml of dIN suspension in water at 112 mg/ml was continuously heated in a water bath at progressively higher temperatures (slow heating: with an increase in temperature of about 1°C per min, allowing 5 min equilibration at each temperature point) indicated by filled squares in Figure 1. After equilibration, 0.5 ml samples were removed from the glass tube, rapidly centrifuged, and the supernatant solute concentration determined. At selected temperatures, glass tubes containing 1.0 ml of the dIN suspension at 20°C were immersed in the water bath for 5 min (rapid heating) and the same sampling procedure applied.

Results

For the rapidly heated sample, the amount of inulin in solution increased in a linear fashion until, at about 70-72°C, the inulin completely dissolved and the solutions cleared rapidly.

For the slowly heated samples, dIN dissolved into solution at the same rate as when heated rapidly until a temperature of about 65°C was obtained (Figure 1). Surprisingly, a thick pasty precipitate developed between 65-70°C that did not dissolve by 75°C, resulting in a deviation from linearity in the slow heating curve at higher temperatures (Figure 1). This result unexpectedly indicated the formation of a novel inulin polymorphic form following slow heating of a suspension at more than 65°C. This precipitate was designated epsilon inulin (eIN). Subsequent studies confirmed that eIN represented a new polymorphic form of inulin with unique properties enabling it to be easily distinguished from previously described inulin forms.

In appearance, gIN and dIN present as milky white, free-flowing suspensions while eIN suspensions, as originally obtained by heating dIN suspensions, typically had a greyish, paste-like appearance and ^' consistency. Transmission electron microscopy (TEM) of such eIN suspensions showed that whilst the eIN particles were of a similar size to the gIN and dIN precursor particles, the eIN particles were less dense than the former, suggesting that some of the inulin in gIN and dIN is removed or leached out in the process of eIN formation. However, when eEN itself is dissolved and recrystallized (as shown in Figures 9 and 12), the resultant particle suspensions have a milky white appearance much like the other inulin forms. Example 2 Assay of eIN

The alpha, beta, gamma and delta polymorphic forms of inulin can be readily identified and assayed on the basis of their respective temperature-dependent solubility in dilute aqueous suspension (<0.5 inulin mg/ml), as measured by changes in the turbidity of the solution as the inulin particles dissolve. When such a dilute suspension is heated step-wise at progressively higher temperatures, the turbidity (measured as the OD₇oo) remains stable for a period, and then drops abruptly at a critical temperature that is reproducible between batches and is characteristic for the particular polymorphic form when purified. Accordingly, the 50% OD₇₀o thermal transition point is the parameter used to characterise each inulin form. In this example, the 50% OD₇₀o thermal transition point of eIN in aqueous suspension was investigated.

Materials and Methods

One dIN and three different eIN preparations were assayed as follows. Each preparation was diluted in 5 ml PBS to 0.5 mg/ml. The diluted preparations, each contained in a glass tube, were heated together in a water bath step-wise at increasing temperatures from 22°C (with the temperature control adjusted to a predetermined setting, and temperatures allowed to equilibrate (5-10 minutes)), and the OD700 of each dilution was measured. The tubes were then returned to the water bath and the temperature increased to the next setting. Results

■ For eIN, the 50% OD₇₀o thermal transition point was 59-68°C (Figure 2). This distinguishes eIN from glN (ie 50% OD₇₀₀47-48^oC: Figures 3 and 4), dIN (ie 50% OD₇₀₀53 -58°C: Figure 4) or various alpha and beta inulins (ie 50% OD₇₀o 10-40°C: Figure 3). Figures 2-4 also show that a temperature of solubility in dilute suspension that clearly differentiates dIN and eIN is 58-60°C as at this temperature, dIN is soluble whereas eIN remains insoluble (also see Figure 13). Thus, it is possible to estimate the eIN content of a

^• particular dilute aqueous suspension of unknown inulin by heating it to 59°C and measuring the

percentage of inulin that remains insoluble (ie the eIN content) and comparing this to the amount which becomes soluble (ie the non-elN content). Example 3 Preparation of eIN

Materials and Methods

1. Epsilon inulin can be conveniently prepared by heating, at an appropriate temperature, concentrated suspensions of dIN. Figure 5 compares the optimum temperatures of formation of eIN from dIN (60-68°C) with the optimum temperatures of formation of gIN from alN (44-45°C) and of dIN from gIN (53-55°C).

The optimum temperatures shown in Figure 5 were measured by heating 1 ml portions of each inulin preparation (-100 mg/ml) for 20 minutes at various temperatures, when 0.1 ml samples were then diluted in 5 ml PBS contained in glass tubes. These were then heated for 10 minutes in a water bath at 40°C ' (gIN), 49°C (dIN) or 60°C (elN) and the OD₇₀o measured (values are expressed as a percentage of the maximum OD found). 2. Alternatively, elN can be prepared directly from a concentrated solution of inulin by completely dissolving inulin powder with a sufficient quantity of appropriate DP polymer lengths (eg inulin extracted from dahlia or chicory roots) in water by heating to 80°C and then maintaining the inulin solution at a constant temperature of 50-85°C or, more preferably, 50-65°C, for a period of at least 15 minutes to up to about 2 weeks or more, whereupon the elN forms as a copious white precipitate from the solution.

Results

Figure 6 shows the rate of elN preparation using method 1 described above; in particular, the figure shows elN conversion from dIN at 66°C. The formation was found to commence rapidly with a half-time of 10-15 min, and was largely complete after 80-90 min at the optimum temperature. Figure 7 shows that the formation of 'gIN and dIN follow very similar kinetics at the optimum temperatures. The yield of each inulin polymorphic form in a step-wise conversion series comprises 65-80% of the polymorphic form from which it is derived (Figure 8), which indicates that the process of conversion is not selecting a minor component of the total population of molecules present in the preparation but rather, is rearranging a major proportion of the total population of molecules into a more temperature stable form. Figures ? and 10 show that the particular inulin polymorphic form that is crystallized from inulin solutions kept at a given temperature, is dependent on that temperature, and that the yields of each polymorphic form follow a similar pattern to that found after heat conversions (Figure 8). Thus, elN is preferentially deposited at temperatures above 50°C. Example 4 Reversibility of elN

All of the four, previously known, polymorphic forms of inulin can be transformed back into lower forms by completely re-dissolving the inulin suspension in hot water and recry stall izing at a lower temperature, · passing up the series beta→ alpha→ gamma→ delta again by treatment at an appropriate temperature. This is illustrated in Figure 1 1, in which dIN dissolved at 80°C and recrystallized at either 5°C and 37°C, yielded typical temperature solubility curves for alN and gIN, respectively, and further incubation of the gIN-at 56°C yielded the typical temperature solubility curve for dIN. In this example, the reversibility of eIN was investigated.

Materials and Methods

Epsilon inulin was dissolved at 80°C and recrystallized at lower temperature before treating at increasing temperatures.

Results

As shown in Figure 12, the process yielded aIN, glN, dIN and, finally, eIN again. Thus, it was found that eIN formation was indeed reversible on dissolving and recrystallizing. However, in this case, while the material crystallizing at 5°C from a solution of eIN had the typical character of aIN, eIN material redissolved and recrystallized at 37°C was nearer dIN than glN in thermal transition temperature. Thus, although eIN formation is reversible, the formation of eE selects a subfraction of inulin from a polymer mix, such that when eIN is solubilised and recrystallized there is a bias towards the formation of inulin polymorphic forms at lower than the normal optimal formation temperatures when bulk inulin is used. This indicates that the optimal temperature of formation of each inulin polymorphic form is not necessarily a constant but can vary with the DP of the inulin polymers being used in their production. Thus, even higher DP starting material inulin polymer mix than that used in this Example (which had a mean DP of 27), would form glN and dIN at progressively lower temperatures. Hence, one use of eIN is to isolate a subfraction of inulin that can then be used to more conveniently produce high yields of glN and dlN.

Example 5 Distinguishing properties of eEV

The four, previously known, polymorphic forms of inulin are primarily distinguished by their thermal transition temperatures of solubility of weak aqueous suspensions as determined by turbidimetric measures, and this has been shown above to be true also for eIN. In addition, the temperatures of formation, either by heat conversion or direct crystallization, have also been shown above to distinguish eIN from the other polymorphic forms. However, these are only a few of a number of characteristics and properties of the various polymorphic forms. In this example, an investigation was conducted to identify other physical parameters which distinguish eIN from the other inulin polymorphic forms.

Materials and Methods

1. Solubility of eIN in water

The solubilities of dIN and eIN were compared at different temperatures, allowing time for equilibration at each temperature point. Samples (0.5 ml) of dIN (112 mg/ml) and eIN (45-74 mg/ml) in water were heated in sealed 1.5 ml Eppendorf™ centrifuge tubes for 15 minutes fully immersed in a water bath at the indicated temperatures, rapidly centrifuged and the refractive index of the supernatants measured.

2. Solubility of el in dimethyl sulphoxide

Samples of 50 μΐ of purified gIN, dl and elN (50 mg/ml in PBS) were added to 450 μΐ of dimethyl sulphoxide (DMSO) dilutions in water to make the indicated final concentrations. As mixing of water with DMSO is exothermic, the DMSO dilutions were held in glass tubes in an ice-bath. After 5 minutes, the tubes were transferred to a water bath at 25°C for 15 minutes, then returned to ice, when ice-cold PBS (4.5 ml) was added, the tubes returned to 25°C for a further 15. minutes, and OD₇₀o measured.

Results

The dlN and elN forms had different solubilities at a given temperature in water, each relation being linear and little affected by concentration (Figure 13); the lines extrapolate to zero solubility at close to the 50% OD₇oo thermal transition point. The curves were followed close to complete solution. Above 72°C (dlN) and 75°C (elN), both forms dissolved almost completely. Gamma inulin behaves similarly, extrapolating to 47°C and dissolving at 65°C (not shown). These solubilisation characteristics again serve to differentiate the polymorphic forms, such that, in a mixture of polymorphic forms, elN can be "freed" from lower polymorphic forms by repeated washing at 60-65°C without major loss of the elN component. It is known that the water-soluble solvent DMSO increases the dielectric constant of water in a mixture, serving to greatly weaken hydrogen bonds. Inulin suspensions in water were found to be completely soluble in DMSO at temperatures down to 0°C, in which they remain dissolved if diluted with water but can be re-precipitated by ethanol. When the solubilities were compared at closely spaced DMSO concentrations (Figure 14), it was seen that gIN, dlN and elN each have different critical DMSO concentrations at which solubility commenced. In particular, gIN was the most sensitive whilst elN was the most resistant to DMSO solubility, suggesting that there is an increasing strength of hydrogen bonding going from beta→ alpha→ gamma→ delta-→ epsilon inulin.

Example 6 Uses of eIN

gIN and dlN polymorphic forms of inulin have been found to be immunologically active and, in particular, enhance the ability of a co-administered antigen to induce antigen-specific antibodies (ie an "adjuvant effect"). This example investigated whether elN was similarly immunologically active. Materials and Methods

To test whether eIN has adjuvant activity, groups of Balb/c mice were immunised with Hepatitis B surface antigen (HBsAg) either by itself or in combination with eIN or dlN. Results

The results of this investigation are shown in Figure 15. It was found that the addition of eIN to HBsAg significantly enhanced the anti-HBsAg antibody response both in terms of elevation of the geometric mean titres and also in terms of the percentage of animals in the group achieving seroprotection, defined as an anti-HBsAg antibody level of 10 IU/ml. It was also found, that on a weight for weight basis, the adjuvant effect of eIN was more potent than dlN.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. REFERENCES

Cooper, PD et al. The adjuvanticity of Algammulin, a new vaccine adjuvant. Vaccine 9:408-415 ( 1991 a).

Cooper, PD et al., Algammulin (gamma inulin/alum hybrid adjuvant) has greater adjuvanticity than alum for hepatitis B surface antigen in mice. Immunology Letters 27: 131-134 ( 1991b).

Cooper, PD and EJ Steele. The adjuvanticity of gamma inulin. Immunol Cell Biol 66:345-352 (1988).

Cooper, PD and EJ Steele. Algammulin, a new vaccine adjuvant comprising gamma inulin particles containing alum: preparation and in vitro properties. Vaccine 9:351-357 (1991).

Cooper, PD and M Carter. Anti-complementary action of polymorphic "solubility forms" of particulate inulin. Mol Immunol 23(S):S95-90l (1986).

Phelps, CF. The physical properties of inulin solutions. Biochem J 95:41-47 (1965).

Stephen, AM, GO Phillips and PA Williams (eds). Food Polysaccharides and their Applications, second edition. CRC Press, Boca Raton FL (2006).

Verroust, PJ et al. Lack of nephritogenicity of systemic activation of the alternate complement pathway. Kidney Int 6: 157-169 (1974).

Claims

1. A substantially purified preparation of inulin, wherein said inulin is in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C.

2. A pharmaceutical or immunological composition comprising particles of inulin in an epsilon polymorphic form having a 50% OD₇oo thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C.

3. The composition of claim 2, wherein the diameter of the eI particles is within the size range of about 100 nm to about 10 μηι.

4. The composition of claim 2 or 3, further comprising an antigen-binding carrier material selected from the group consisting of aluminium hydroxide (alum) gel, aluminium phosphate gel and calcium phosphate gel.

5. The composition of any one of claims 2 to 4, further comprising an active component selected from the group consisting of vaccinating antigens, antigenic peptide sequences, and immunoglobulins.

6. The composition of any one of claims 2 to 5, further comprising one or more immune modulator selected from the group consisting of lymphokines, cytokines, thymocyte stimulators, monocyte or macrophage stimulators, and endotoxins.

7. A method of stimulating an immune response in a subject, for the purposes of preventing or treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic disease, degenerative or ageing disease, wherein said method comprises administering to said subject an effective amount of a preparation according to claim 1 or a composition according to any one of claims 2 to 6.

8. A method of enhancing an immune response in a subject, for the purposes of preventing or treating an infectious disease, autoimmune disease, immunodeficiency disorder, neoplastic disease, degenerative or ageing disease, wherein said method comprises administering to said subject an effective amount of a preparation according to claim 1 or a composition according to any one of claims 2 to 6.

9. ^* A method for the preparation of inulin in an epsilon polymorphic form having a 50% OD₇oo thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C, said method comprising heating a suspension comprising gamma inulin (glN) and/or delta inulin (dlN) at a temperature of > 55°C.

10. A method for the preparation of inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C, said method comprising maintaining a solution of inulin at a concentration of 10 mg/ml or more at a temperature in the range of about 50°C to about 85°C for a period of at least 15 minutes up to about 2 weeks or more.

1 1. A method for fractionating a polydisperse inulin mixture, said method comprising precipitating from a suspension comprising dissolved inulin, inulin in an epsilon polymorphic form having a 50% OD₇oo thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C so as to obtain a high molecular weight inulin fraction, and thereafter separating said epsilon inulin (eIN) from residual soluble inulin in the suspension so as to obtain a lower molecular weight inulin fraction.

·

12. A substantially purified preparation of inulin prepared from a precursor preparation substantially consisting of inulin in an epsilon polymorphic form having a 50% OD₇₀o thermal transition point in dilute aqueous suspensions (<0.5 mg/ml) of > 58°C, wherein said purified preparation of inulin has been prepared by dissolving the epsilon inulin (eIN) of said precursor preparation by heating in the form of a slurry in a solvent such as water or ethanol, and thereafter, precipitating the inulin as one or more of alpha inulin (alN), beta inulin (blN), gamma inulin (glN) and delta inulin (dlN).

13. A method for assaying for the presence or concentration in a suspension of inulin that is in an epsilon polymorphic form, wherein said method comprises measuring the 50% OD₇₀o thermal transition point in a dilute aqueous suspension (<0.5 mg ml).

14. A method of treating or preventing radiation- or chemotherapy-induced immunosuppression in a subject, wherein said method comprises administering to said subject an effective amount of a preparation according to claim 1 or a composition according to any one of claims 2 to 6.