WO2006067419A2

WO2006067419A2 - Myrtaceous honey and the use thereof as immunomodulator

Info

Publication number: WO2006067419A2
Application number: PCT/GB2005/004945
Authority: WO
Inventors: Robert James Nash
Original assignee: Mnl Pharma Limited
Priority date: 2004-12-21
Filing date: 2005-12-20
Publication date: 2006-06-29
Also published as: GB0427882D0; WO2006067419A3

Abstract

A process for the production of a composition comprising a pyrrolizidine compound comprises the steps of: (a) providing myrtaceous honey; (b) extracting (e.g. isolating or purifying) casuarine and/or a glycoside thereof from the honey; and/or (c) chemically and/or enzymatically modifying the casuarine and/or glycoside thereof present in the honey.

Description

MYRTACEOUS HONEYAND USES THEREOF

Field of the Invention

The present invention relates to the use of certain honeys in medicine, to processes for the production of casuarine and/or a glycoside thereof from honey and to various industrial processes and pharmaceutical compositions based thereon.

Background to the Invention

Casuarine, (1 R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1 ,2,6,7-tetrahydroxypyrrolizidine (1 ) is a naturally- occurring, highly oxygenated bicyclic pyrrolizidine alkaloid of formula:

Casuarine (1)

Casuarine glycosides also occur in nature. For example, casuarine-6-O-α-glucoside (casuarine-6-O-α-D- glucopyranose, 2) occurs in Eugenia jambolana (Wormald et al. (1996) Carbohydrate Letters (2) 169-174).

Casuarine-6-O-α-D-gIucopyranose (2)

Casuarina equisetifolia wood, bark and leaves have been claimed to be useful against diarrhoea, dysentery and colic (Chopra ef a/. (1956) Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research (India), New Delhi, p. 55) and a sample of bark has recently been prescribed in Western Samoa for the treatment of breast cancer. An African plant containing casuarine (identified as Syzygium guineense) has been reported to be beneficial in the treatment of AIDS patients (see Wormald et al. (1996) Carbohydrate Letters (2) 169-74). Eugenia jambolana is a well-known tree in India for the therapeutic value of its seeds, leaves and fruit against diabetes and bacterial infections. Its fruit have been shown to reduce blood sugar levels in humans and aqueous extracts of the bark are claimed to affect glycogenolysis and glycogen storage in animals (Wormald et al. (1996) Carbohydrate Letters (2) 169-74).

WO2004/064715 describes various immunomodulatory applications for casuarine and casuarine glycosides.

Thus, casuarine and its glycosides have great potential as therapeutic agents and there is presently great interest in processes for purification from natural sources as well as in protocols for organic synthesis.

Isolation from natural sources

Casuarine can be isolated from several botanical sources, including the bark of Casuarina equisetifolia (Casuarinaceae), the leaves and bark of Eugenia jambolana (Myrtaceae) and Syzygium guineense (Myrtaceae) (see e.g. Nash et al. (1994) Tetrahedron Letters (35) 7849-7852), Myrtus communis and Casearia sylvestris (Nash, unpublished).

Casuarine-6-α-glucoside (casuarine-6-α-D-glucopyranose, 2) has been isolated from the bark and leaves of Eugenia jambolana (Wormald et al. (1996) Carbohydrate Letters (2) 169-174) but is also present in bark and leaves of Syzygium guineense and leaves, wood and fruit of Myrtus communis (Nash unpublished).

However, both casuarine and its glycosides are present in only small quantities in natural sources with a range of related water soluble alkaloids and are difficult and expensive to purify in commercial quantities. Moreover, purification from natural sources may result in the co-purification of undesirable (and possibly harmful) contaminants.

Chemical synthesis

As highly hydroxylated pyrrolizidine alkaloids, the synthesis of casuarine and its glycosides is technically challenging: unwanted side reactions arise from the reactive hydroxyl groups.

This problem has been tackled by protecting or differentiating the reactivity of the oxygen functions. Bell et al. (1997) Tetrahedron Letters 38(33): 5869-72 describe the synthesis of four diastereoisomers of casuarine from eight carbon sugar lactones by reduction of open chain azidodimesylates by Suzuki-Takaoka reduction to allow the formation of the pyrrolizidine nucleus by bicyclisation (Bell et al. (1997) Tetrahedron Letters 38(33): 5869- 72). However, the use of heavily protected intermediates limits the flexibility of the scheme: it cannot be readily adapted to other pyrrolizidines because the requirement for selective protection, deprotection and activation leads to cumbersome and lengthy schemes, whilst the use of starting materials possessing the correct hydroxyl configurations is limited by their availability.

Another approach is based on tandem [4+2]/[3+2] nitroalkene cycloadditions. It has been used for the synthesis of several pyrrolizidine and indolizidines alkaloids with up to four contiguous stereogenic centres (see Denmark and Hurd (1999) Organic Letters 1(8): 1311-14). The method was later extended by the same workers to the synthesis of (+)-casuarine by the intermolecular [3+2] cycloaddition of a suitable substituted dipolarophile and a flexible, heavily substituted nitronate. However, the synthesis is not readily adaptable to other stereoisomers, is lengthy and the overall yield is modest (20%).

There is therefore a need for alternative processes for the production and/or isolation of casuarine and its glycosides in order to exploit their therapeutic potential and to permit the systematic comparison and evaluation of structure-function relationships.

Summary of the Invention

The present invention is based, at least in part, on the discovery that certain honeys (hereinafter, myrtaceous honeys) contain casuarine and/or glycosides thereof, often in extremely high concentrations.

Without wishing to be bound by any theory, it is thought that the bees involved in producing myrtaceous honeys in some way concentrate casuarine and/or its glycoside from the environment. This concentration may be attendant on the selective harvesting of nectar and/or other plant components by the bees during foraging. The diverse range of related alkaloids in the plants themselves appear to be removed although they may not be in nectar or pollen collected for the honey.

Thus, in a first embodiment the invention finds application in a process for the production of a composition comprising a pyrrolizidine compound comprising the steps of: (a) providing myrtaceous honey; (b) extracting (e.g. isolating or purifying) casuarine and/or a glycoside thereof from the honey; and/or (c) chemically and/or enzymatically modifying the casuarine and/or glycoside thereof present in the honey.

Thus, the invention contemplates a process for the production of a composition comprising a pyrrolizidine compound comprising the use of myrtaceous honey as a starting or source material. When so used, the myrtaceous honey provides casuarine and/or one or more casuarine glycosides, which can be isolated or purified perse or used as reactants in further chemical and/or enzymatic processing steps to yield a wide range of other pyrrolizidine products.

In a second embodiment, the invention finds application in a method of immunomodulation comprising the step of administering an immunomodulatory amount of honey to an individual in need thereof.

In a third embodiment, the invention finds application in the use of honey for the manufacture of a medicament for use in immunomodulation.

In a fourth embodiment, the invention finds application in a method for monitoring the quality of a honey comprising the step of testing the honey for the presence of casuarine and/or a glycoside thereof. In such embodiments, the invention may find application in a process for the production of honey comprising the step of monitoring the quality of the honey by the method of the invention.

In a fifth embodiment, the invention finds application in a process for the production of a pharmaceutical product comprising the step of incorporating honey into the product. In a sixth embodiment, the invention provides a honey fraction or honey derivative enriched in casuarine and/or a glycoside thereof.

Detailed Description of the Invention

Definitions

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

The term honey is used herein to define the sweet, viscous fluid made by honey bees when the nectar, honeydew, juices or other secretions or deposits from plants are gathered, modified and stored in a honeycomb by the bees. The term therefore covers honey in any form, including in particular comb honey (honey presented in its original comb or portions thereof) and extracted honey (honey removed from the comb and presented in various forms including for example: (1) liquid, (2) crystallized or granulated, or (3) partially crystallized). The term raw honey is a term of art used to define honey as it exists in the beehive or as obtained by extraction, settling or straining without the application of heat.

The term myrtaceous honey is used herein to define any honey containing casuarine and/or a glycoside thereof. Most myrtaceous honeys are produced by bees foraging in an environment which includes plants of the family Myrtaceae, but other plant families (e.g. Flacourtiaceae) may characterize the foraging environment (particularly in some South American countries where plant species of the family Flacourtiaceae (such as Casearia spp.) may be widespread. Thus the term myrtaceous is not used herein to define a honey by reference to floral/botanical source.

The terms honey and myrtaceous honey are also used herein in a broader sense to define various compositions derived from comb or extracted (myrtaceous) honey (as defined above). Thus, the term honey is also used sensu lato to cover a wide variety of honey fractions and honey derivatives and references to honey and myrtaceous honey herein are to be interpreted accordingly (and so understood to embrace inter alia such fractions and derivatives).

As used herein, the term honey fraction is used to define one or more components of honey obtained therefrom by chemical and/or physical fractionation. For example, honey fractions enriched (or depleted) in pollen may be produced by centrifugation or filtration, whilst honey fractions enriched in sugars may be obtained by heating, crystallization or solvent extraction. Honey fractions enriched in casuarine and/or a glycoside thereof may be obtained for example by solvent extraction and/or chromatographic fractionation.

As used herein, the term honey derivative is used to define any composition derived from the processing of honey (for example by physical, enzymatic or chemical processing, including for example heating, filtration, hydrolysis, fermentation, enzymatic biotransformation, chemical modification, mixing, freeze-drying, solvent extraction, chromatography, HPLC etc.). Honey beer is an example of a honey derivative. It is produced by fermentation using honey as a sugar source. Zambian honey beer is based on the use of Zambian honey as a sugar source.

The term Zambian honey is used herein to define honey produced by the foraging activity of African honey bees in Zambia.

The term isolated is used herein to indicate that the isolated material (e.g. the casuarine and/or casuarine glycoside) exists in a physical milieu distinct from that in which it occurs in nature. For example, the isolated material may be substantially isolated (for example purified) with respect to the complex milieu in which it naturally occurs. The term isolating in the context of a process step is to be interpreted accordingly.

In some circumstances, the isolated material forms part of a composition (for example a more or less crude extract containing many other substances, such as a honey fraction), which may contain other components. In other circumstances, the isolated phytochemical may be purified to essential homogeneity, for example as determined spectrophotometrically, by NMR or by column chromatography (for example HPLC).

When purified material of the invention is specified herein the absolute level of purity is not critical and those skilled in the art can readily determine appropriate levels of purity according to the use to which the material is to be put. Preferred, however, are purity levels of 90% w/w, 95% w/w, 99% w/w, 99.9% w/w or higher. The term purifying in the context of a process step is to be interpreted accordingly.

Myrtaceous honey

Myrtaceous honey contains casuarine and/or a glycoside thereof. Preferred myrtaceous honeys contain high concentrations of casuarine and/or casuarine glycoside. High concentrations of casuarine and/or casuarine glycoside are concentrations in excess of 10 μg/ml w/w raw comb honey.

For example, the myrtaceous honeys of the invention may contain:

(a) at least 3.0 mg/g w/w;

(b) at least 2.0 mg/g w/w;

(c) at least 1.0 mg/g w/w;

(d) at least 0.5 mg/g w/w; (e) at least 0.3 mg/g w/w;

(f) at least 0.1 mg/g w/w,

of casuarine.

Alternatively, or in addition, the myrtaceous honeys of the invention may contain:

(a) at least 3.0 mg/g w/w;

(b) at least 2.0 mg/g w/w; (c) at least 1.0 mg/g w/w;

(d) at least 0.5 mg/g w/w;

(e) at least 0.3 mg/g w/w;

(f) at least 0.1 mg/g w/w,

of casuarine glycoside.

Particularly preferred are myrtaceous honeys which contain:

(a) at least 3.0 mg/g w/w;

(b) at least 2.0 mg/g w/w;

(c) at least 1.0 mg/g w/w;

(d) at least 0.5 mg/g w/w;

(e) at least 0.3 mg/g w/w; (f) at least 0.1 mg/g w/w,

of casuarine-6-O-α-glucoside (casuarine-6-O-α-D-glucopyranose).

The myrtaceous honey may also contain mixtures of casuarine and one or more casuarine glycosides (for example, mixtures of casuarine and casuarine-6-O-α-glucoside) wherein the combined mass of casuarine and casuarine glycoside(s) present is as follows:

(a) at least 3.0 mg/g w/w;

(b) at least 2.0 mg/g w/w; (c) at least 1.0 mg/g w/w;

(d) at least 0.5 mg/g w/w;

(e) at least 0.3 mg/g w/w;

(f) at least 0.1 mg/g w/w,

In such myrtaceous honeys the relative proportion of casuarine to casuarine glycoside(s) may vary. Preferred are honeys in which the ratio of casuarine to casuarine glycoside(s) is 1 :1 , 1 :10, 1 :100, 1 :1000, 1 :10000 and 1 :100000.

However, myrtaceous honeys may also have a ratio of casuarine glycoside(s) to casuarine of 1 :1 , 1 :10, 1 :100, 1 :1000, 1 :10000 and 1 :100000.

The concentrations set out above refer to the w/w concentrations as found in the honey when raw and/or in comb form. They may also refer to the w/w concentrations as found in the honey when in extracted form (i.e. liquid, crystallized, granulated or partially crystallized). They may also refer to the w/w concentrations as found in the honey when processed (e.g. by hydrolysis or fermentation). Such hydrolysed/fermented honeys

(including honey beers) typically have relatively high concentrations of casuarine. For example, hydrolysed honeys and honey beers may contain casuarine alone, the casuarine glycoside(s) originally present in the honey having been converted to casuarine during hydrolysis/fermentation. As explained earlier, it is thought that the bees involved in producing myrtaceous honeys may concentrate casuarine and/or its glycoside from the foraged plants and/or from the nectar and/or sweet deposits gathered during foraging. Thus, myrtaceous honeys may be produced by apiculture by providing bees with a foraging environment having an appropriate botanical constitution. By "botanical constitution" it is meant the nature of the vegetative ecosystem within the foraging range of the bees, including the identity and absolute and relative numbers of foragable plants.

Suitable foraging environments therefore include those in which plants containing casuarine and/or a casuarine glycoside are present and available for harvesting by bees. Such plants include species from the family

Myrtaceae, including Eugenia (e.g. Eugenia jambolana), Myrtus (e.g. M. communis) and/or Syzygium spp. (for example S. guineense and S. cordatum). Plants from the family Flacourtiaceae (including for example Casearia spp., e.g. C. sylvestris) are also suitable and when present in a foraging environment may give rise to myrtaceous honeys. Other species from these families and genera (or closely related families and genera) may also characterize suitable foraging environments.

These foraging environments may be naturally-occurring or may be artificially created (e.g. by selectively cultivating plants containing casuarine and/or its glycoside(s) within the foraging range of the bees).

An example of a naturally-occurring foraging environment suitable for the production of myrtaceous honey is that present in the honey-producing regions of Zambia. Other naturally-occurring foraging environments suitable for the production of myrtaceous honey include other regions of central and southern Africa, for example: Uganda, Tanzania, The Gambia and South Africa (where Syzygium species occur).

Other suitable foraging environments can be identified for example by reference to the botanical range of any or all of representatives of the family Flacourtiaceae (including for example Casearia spp., e.g. C. sylvestris) and Myrtaceae, including Eugenia (e.g. Eugenia jambolana), Myrtus (e.g. M. communis) and/or Syzygium spp. (for example S. guineense and S. cordatum) and other species from those families and genera (or closely related families and genera). The availability of any or all of these plant species to foraging bees can permit the production of myrtaceous honey.

Myrtaceous honeys may be readily identified by testing for the presence of casuarine, its glycosides (including casuarine-6-O-α-glucoside) and related compounds. Any of a wide variety of techniques can be employed for this purpose. For example, cation exchange chromatography followed by gas chromatography linked to a mass spectrometer may be employed, where detection of casuarine and casuarine glycosides by gas chromatography requires the production of suitable derivatives (such as trimethylsilyl-derivatives, typically prepared using a mixture of hexamethyldisilazane and trimethylchlorosilane in pyridine in a ratio of 2:1:10). Casuarine gives distinctive major fragments using electron impact mass spectroscopy of 462 and 217 amu whereas casuarine glycosides typically show ions at 476, 418, 361 and 217 amu. Conveniently, a single cation exchange step using a strongly acidic resin also allows the concentration of the alkaloids for subsequent final purification. Exemplary ion exchange methods are described in, for example, WO 03/074147 and WO 03/074146. Thus, the invention provides myrtaceous honeys identified by screening honeys produced by apiculture from various floral sources, geographical sources, foraging environments and/or with various species of bee for the presence of casuarine and/or casuarine glycoside(s).

Particularly preferred for use in the invention is Zambian honey. This honey is commercially available and is produced using traditional methods from smoke-cropped bark hives without the use of fertilized floral foraging environments, artificial bee feeds (e.g. sugars) or antibiotics. The method involves the use of a cylinder of bark (secured with pegs), which is hung in a tree to be occupied by a passing swarm of wild African bees. Once a colony is established, the beekeepers smoke the bees (to subdue them) and harvest the honey.

Zambian honey produced in this way is characterized by distinctive smoky flavour, contributed by relatively large amounts of pollen (which also confers a dark brown colour).

The Zambian honey is traditionally used to brew honey beer. It has been found that this beer contains casuarine, which is derived from the hydrolysis of the casuarine glycoside present in the honey used as a sugar source in the beer-making process.

The myrtaceous honey of the invention will typically contain many components other than casuarine and/or casuarine glycosides that are common to all honeys irrespective of geographical or floral origin.

For example, a compositional breakdown of the ancillary components present in an exemplary batch of myrtaceous honeys is shown below:

Average Range Standard Deviation

Fructose/Glucose Ratio 1.23 0.76 - 1.86 0.126 Fructose, % 38.38 30.91 - 44.26 1.77 Glucose, % 30.31 22.89 - 40.75 3.04 Minerals (Ash), % 0.169 0.020 - 1.028 0.15 Moisture, % 17.2 13.4 - 22.9 1.46 Reducing Sugars, % 76.75 61.39 - 83.72 2.76 Sucrose, % 1.31 0.25 - 7.57 0.87 PH 3.91 3.42 - 6.10 —

Total Acidity, meq/kg. 29.12 8.68 - 59.49 10.33 True Protein, mg/100g. 168.6 57.7 - 567 70.9

Biological activities of the materials of the invention

Without wishing to be bound by any theory, it is thought that the immunomodulatory activity of the various materials described herein (including inter alia the honey-derived pyrrolizidine compositions, the casuarine and casuarine glycosides, honey-based medicaments, honey, honey fractions, honey derivatives, honey products and honey-containing compositions and pharmaceuticals, hereinafter referred to as the "materials of the invention") may arise from the stimulation and/or suppression of cytokine secretion in vivo by the casuarine and/or casuarine glycosides contained therein.

In particular, it is thought that that the immunomodulatory activity of the materials of the invention arises from the stimulation of secretion of one or more cytokines (e.g. one or more Th1 cytokines), including interleukins 2 and/or 12 (IL-2 and/or IL-12) and/or the suppression of secretion of one or more Th2 cytokines (e.g. IL-5).

In particular, it is thought that the immunostimulatory activity of the materials of the invention may arise from the stimulation of 11-12 and IL-2 by dendritic cells. This leads to the stimulation of NK cells to produce lFN-γ and induces the development of CD4⁺ Th1 cells. The induced Th1 cells then produce IFN- v and IL-2. The IL-2 then enhances further proliferation of Th1 cells and the differentiation of pathogen (e.g. tumour and virus) - specific CD8⁺ T cells. The IL-2 also stimulates the cytolytic activity of NK cells of the innate immune system.

IL-12 is the primary mediator of type-1 immunity (the Th1 response). It induces natural killer (NK) cells to produce IFN-γ as part of the innate immune response and promotes the expansion of CD4⁺ Th1 cells and cytotoxic CD8⁺ cells which produce IFN-γ. It therefore increases T-cell invasion of tumours as well as the susceptibility of tumour cells to T-cell invasion.

Thus, the materials of the invention are preferably stimulators of cytokine secretion. Particularly preferred are materials which induce, potentiate, activate or stimulate the release one or more cytokines (for example Th1 cytokines, e.g. IL-12 and/or II-2, optionally together with one or more other cytokines) in vivo.

This primary immunomodulatory activity of the materials of the invention is particularly important in certain medical applications (discussed in detail infra). For example, increased production of IL-12 may overcome the suppression of innate and cellular immunities of HIV-1 -infected individuals and AIDS patients.

The cytokine stimulation exhibited by the materials of the invention may be dependent, in whole or in part, on the presence of co-stimulatory agents. Such co-stimulatory agents may include, for example, agents that stimulate the innate immune system, including Toll-like receptor (TLR) ligands. These ligands include microbial products such as lipopolysaccharide (LPS) and/or monophosphoryl lipid) as well as other molecules associated with microbial infection. In many applications, such co-stimulatory agents will be present in the patient to be treated at the time of administration of the compounds of the invention.

Without wishing to be bound by any theory, it is thought that at least some of the pharmacological activities of the materials of the invention may be based on a secondary glycosidase inhibitory activity.

Such glycosidase inhibition may lead to any or all of the following in vivo:

• Modification of tumour cell glycosylation (e.g. tumour antigen glycosylation);

• Modification of viral protein glycosylation (e.g. virion antigen glycosylation);

• Modification of cell-surface protein glycosylation in infected host cells; • Modification of bacterial cell walls.

This ancillary biological activity may therefore augment the primary immunomodulatory activity in some preferred embodiments of the invention. It may be particularly desirable in certain medical applications, including the treatment of proliferative disorders (such as cancer) or in applications where infection is attendant on immune suppression. For example, selective modification of virion antigen glycosylation may render an infecting virus less (or non-) infective and/or more susceptible to endogenous immune responses. In particular, the materials of the invention may alter the HIV viral envelope glycoprotein gp120 glycosylation patterns, hence inhibiting the entry of HIV into the host cell by interfering with the binding to cell surface receptors.

Thus, the materials of the invention are preferably (but not necessarily) glycosidase inhibitors. Particularly preferred are materials which exhibit specificity of glycosidase inhibition, for example Glucosidase I rather than mannosidases.

Medical applications of the materials of the invention

The invention finds broad application in medicine, for example in methods of therapy and prophylaxis.

These medical applications may be applied to any warm-blooded animal, including humans. The applications include veterinary applications, wherein the materials of the invention are administered to non-human animals, including primates, dogs, cats, horses, cattle and sheep.

The materials of the invention are immunomodulators. Thus, they find general application in the treatment or prophylaxis of conditions in which stimulation, augmentation or induction of the immune system is indicated and/or in which suppression or elimination of part or all of the immune response is indicated.

Particular medical uses of the materials of the invention are described in detail below. References to therapy and/or prophylaxis in the description or claims are to be interpreted accordingly and are intended to encompass inter alia the particular applications described below.

1. Increasing the Th1 :Th2 response ratio

General considerations

When the immune system is challenged by a foreign antigen it responds by launching a protective response.

This response is characterized by the coordinated interaction of both the innate and acquired immune systems.

These systems, once thought to be separate and independent, are now recognized as two interdependent parts that when integrated fulfil two mutually exclusive requirements: speed (contributed by the innate system) and specificity (contributed by the adaptive system).

The innate immune system serves as the first line of defence against invading pathogens, holding the pathogen in check while the adaptive responses are matured. It is triggered within minutes of infection in an antigen-independent fashion, responding to broadly conserved patterns in the pathogens (though it is not nonspecific, and can distinguish between self and pathogens). Crucially, it also generates the inflammatory and co-stimulatory milieu (sometimes referred to as the danger signal) that potentiates the adaptive immune system and steers (or polarizes it) towards the cellular or humoral responses most appropriate for combating the infectious agent (discussed in more detail below).

The adaptive response becomes effective over days or weeks, but ultimately provides the fine antigenic specificity required for complete elimination of the pathogen and the generation of immunologic memory. It is mediated principally by T and B cells that have undergone germline gene rearrangement and are characterized by an exquisite specificity and long-lasting memory. However, it also involves the recruitment of elements of the innate immune system, including professional phagocytes (macrophages, neutrophils etc.) and granulocytes (basophils, eosinophils etc.) that engulf bacteria and even relatively large protozoal parasites. Once an adaptive immune response has matured, subsequent exposure to the pathogen results in its rapid elimination (usually before symptoms of infection become manifest) because highly specific memory cells have been generated that are rapidly activated upon subsequent exposure to their cognate antigen.

Interdependence of innate and adaptive responses

It is now thought that the earliest events following pathogen invasion are effected by cellular components of the innate immune system. The response is initiated when resident tissue macrophages and dendritic cells (DCs) encounter pathogen and become activated by signals generated by interaction between pattern-recognition receptors (PRRs) and the pathogen-associated molecular patterns (PAMPs) shared by large groups of microorganisms. The activated macrophages and DCs are stimulated to release various cytokines (including the chemokines IL-8, MIP-1α and MIP-1 β), which constitute the "danger signal" and triggers an influx of Natural Killer (NK) cells, macrophages, immature dendritic cells into the tissues.

Loaded with antigen, the activated DCs then migrate to lymph nodes. Once there, they activate immune cells of the adaptive response (principally naϊve B- and T-cells) by acting as antigen-presenting cells (APCs). The activated cells then migrate to the sites of infection (guided by the "danger signal") and once there further amplify the response by recruiting cells of the innate immune system (including eosinophils, basophils, monocytes, NK cells and granulocytes). This cellular trafficking is orchestrated by a large array of cytokines (particularly those of the chemokine subgroup) and involves immune cells of many different types and tissue sources (for a review, see Luster (2002), Current Opinion in Immunology 14: 129-135).

Polarization of the adaptive immune response

The adaptive immune response is principally effected via two independent limbs: cell-mediated (type 1 ) immunity and antibody-mediated or humoral (type 2) immunity.

Type 1 immunity involves the activation of T-lymphocytes that either act upon infected cells bearing foreign antigens or stimulate other cells to act upon infected cells. This branch of the immune system therefore effectively contains and kills cells that are cancerous or infected with pathogens (particularly viruses). Type 2 immunity involves the generation of antibodies to foreign antigens by B-lymphocytes. This antibody-mediated branch of the immune system attacks and effectively neutralizes extracellular foreign antigens.

Both limbs of the immune system are important in fighting disease and there is an increasing realization that the type of immune response is just as important as its intensity or its duration. Moreover, since the type 1 and type 2 responses are not necessarily mutually exclusive (in many circumstances an effective immune response requires that both occur in parallel), the balance of the type1/type 2 response (also referred to as the Th1:Th2 response ratio/balance by reference to the distinct cytokine and effector cell subsets involved in the regulation of each response - see below) may also play a role in determining the effectiveness (and repercussions) of the immune defence.

In many circumstances the immune response is skewed heavily towards a type 1 or type 2 response soon after exposure to antigen. The mechanism of this type1/type 2 skewing or polarization is not yet fully understood, but is known to involve a complex system of cell-mediated chemical messengers (cytokines, and particularly chemokines) in which the type1/type 2 polarization (or balance) is determined, at least in part, by the nature of the initial PRR-PAMP interaction when the DCs and macrophages of the innate immune system are first stimulated and subsequently by the cytokine milieu in which antigen priming of naϊve helper T cells occurs.

Two cytokines in particular appear to have early roles in determining the path of the immune response. lnterleukin-12 (IL-12), secreted by macrophages, drives the type 1 response by stimulating the differentiation of Th1 cells, the helper cells that oversee the type 1 response. Another macrophage cytokine, interleukin-10 (IL- 10) inhibits this response, instead driving a type 2 response.

The type 1 and type 2 responses can be distinguished inter alia on the basis of certain phenotypic changes attendant on priming and subsequent polarization of naϊve helper T cells. These phenotypic changes are characterized, at least in part, by the nature of the cytokines secreted by the polarized helper T cells.

Th1 cells produce or are regulated by so-called IM cytokines, which include one or more of TNF, IL-1, IL-2, IFN-gamma, IL-12 and/or IL-18. The Th1 cytokines are involved in macrophage activation and Th1 cells orchestrate Type 1 responses. In contrast, Th2 cells produce so-called Th2 cytokines, which include one or more of IL-4, IL-5, IL-10 and IL-13. The Th2 cytokines promote the production of various antibodies and can suppress the type 1 response.

The involvement of Th1 and Th2 cells and cytokines in type 1 :type 2 immune response polarization has given rise to the terms Th1 response and Th2 response being used to define the type 1 and type 2 immune responses, respectively. Thus, these terms are used interchangeably herein.

There is an increasing realization that the type of immune response is just as important in therapy and prophylaxis as its intensity or its duration. For example, an excess Th1 response can result in autoimmune disease, inappropriate inflammatory responses and transplant rejection. An excess Th2 response can lead to allergies and asthma. Moreover, a perturbation in the Th1:Th2 ratio is symptomatic of many immunological diseases and disorders, and the development of methods for altering the Th1 :Th2 ratio is now a priority .As explained earlier, the immune response comprises two distinct types: the Th1 response (type-1 , cellular or cell mediated immunity) and Th2 response (type-2, humoral or antibody mediated immunity).

These Th1 and Th2 responses are not mutually exclusive and in many circumstances occur in parallel. In such circumstances the balance of the Th1/Th2 response determines the nature (and repercussions) of the immunological defence (as explained herein).

The Th1/Th2 balance (which can be expressed as the Th1:Th2 response ratio) is determined, at least in part, by the nature of the environment (and in particular the cytokine milieu) in which antigen priming of naϊve helper T cells occurs when the immune system is first stimulated.

The Th1 and Th2 responses are distinguished inter alia on the basis of certain phenotypic changes attendant on priming and subsequent polarization of naϊve helper T cells. These phenotypic changes are characterized, at least in part, by the nature of the cytokines secreted by the polarized helper T cells.

Th1 cells produce so-called Th1 cytokines, which include one or more of IL-1, TNF, IL-2, IFN-gamma, IL-12 and/or IL-18. The Th1 cytokines are involved in macrophage activation and Th1 cells orchestrate cell- mediated defences (including cytotoxic T lymphocyte production) that form a key limb of the defence against bacterial and viral attack, as well as malignant cells.

Th2 cells produce so-called Th2 cytokines, which include one or more of IL-4, IL-5, IL-10 and IL-13. The Th2 cytokines promote the production of various antibodies and can suppress the Th1 response.

Accordingly, in the mouse, a cell that makes IFN-gamma and not IL-4 is classified as Th1 , whereas a CD4⁺ cell that expresses IL-4 and not IFN-gamma is classified as Th2. Although this distinction is less clear in humans (T cells that produce only Th1 or Th2 cytokines do not appear to exist in humans), the phenotype of the T cell response (Th1 or Th2) can still be distinguished in humans on the basis of the ratio of Th1 to Th2 cytokines expressed (usually, the ratio of IFN-gamma to IL-4 and/or IL-5).

There is an increasing realization that the type of immune response is just as important in therapy and prophylaxis as its intensity or its duration. For example, an excess Th1 response can result in autoimmune disease, inappropriate inflammatory responses and transplant rejection. An excess Th2 response can lead to allergies and asthma. Moreover, a perturbation in the Th1 :Th2 ratio is symptomatic of many immunological diseases and disorders, and the development of methods for altering the Th1 :Th2 ratio is now a priority.

It has now been discovered that the materials of the invention can increase the Th1 :Th2 response ratio in vivo (for example, by preferentially promoting a Th1 response and/or preferentially suppressing a Th2 response).

Thus, the materials of the invention find application in methods of therapy and/or prophylaxis which comprise increasing the Th1 :Th2 response ratio (for example, by preferentially promoting a Th1 response and/or preferentially suppressing a Th2 response). The medical applications contemplated herein therefore include any diseases, conditions or disorders in which an increase in the Th1 :Th2 response ratio is indicated or desired. For example, the medical applications contemplated include diseases, conditions or disorders in which stimulation of a Th1 response and/or suppression of a Th2 response is indicated or desired.

The mechanism(s) by which the compounds of the invention increase the Th1 :Th2 response ratio are not yet fully understood. It is likely that the activity is based, at least in part, on selective Th1 cytokine induction (since Th1 and Th2 cytokines exhibit mutual inhibition), for example in dendritic cells.

For example, the materials of the invention may induce, potentiate, activate or stimulate (either directly or indirectly) the release and/or activity (in vitro and/or in vivo) of one or more Th1 cytokines (for example one or more cytokines selected from IFN-gamma, IL-12, IL-2 and IL-18). Particularly preferred are compounds which induce, potentiate, activate or stimulate the release and/or activity (in vitro and/or in vivo) of IFN-gamma and/or IL-12 and/or IL-2.

Particularly preferred are materials that stimulate the release of IL-2 and IL-12 in dendritic cells.

The materials of the invention may also suppress or inactivate (either directly or indirectly) the release and/or activity (in vitro and/or in vivo) of one or more Th2 cytokines (for example one or more cytokines selected from IL-4, IL-5, IL-10 and IL-13). Particularly preferred are materials which suppress or inactivate the release and/or activity (in vitro and/or in vivo) of IL-5.

Thus, particularly preferred are materials which exhibit a Th1 cytokine stimulatory activity together with a complementary Th2 cytokine inhibitory activity.

Specific examples of applications falling within the general class of treatments based on increasing the Th1 :Th2 response ratio are described in the following sections.

Th 1 -related diseases

Th1-related diseases are diseases, disorders, syndromes, conditions or infections in which Th1 cells are involved in preventing, curing or alleviating the effects of the disease, disorder, syndrome, condition or infection.

Th1 -related diseases may also include diseases, disorders, syndromes, conditions or infections in which the Th1 component of the immune response is pathologically depressed or diseases, disorders, syndromes, conditions or infections in which stimulation of a Th1 response is indicated.

Such conditions may arise, for example, from certain proliferative disorders (typically cancers) in which the proliferating (e.g. tumour) cells exert a suppressive effect on one or more components of the Th1 response. For example, tumour cells may inhibit dendritic cells, cause the expression of inhibitory receptors on T cells, down regulate MHC class I expression and induce the secretion of anti-inflammatory factors and immunosuppressive cytokines which deactivate or suppress immune cell cytotoxicity.

Thus, the materials of the invention find application in the treatment or prophylaxis of Th1 -related diseases.

Examples of Th1-related diseases include infectious diseases (particularly viral infections) and proliferative disorders (e.g. cancer).

Thus, the Th1 -related diseases include any malignant or pre-malignant condition, proliferative or hyper- proliferative condition or any disease arising or deriving from or associated with a functional or other disturbance or abnormality in the proliferative capacity or behaviour of any cells or tissues of the body.

Thus, the invention finds application in the treatment or prophylaxis of breast cancer, colon cancer, lung cancer and prostate cancer. It also finds application in the treatment or prophylaxis of cancers of the blood and lymphatic systems (including Hodgkin's Disease, leukemias, lymphomas, multiple myeloma, and

Waldenstrom's disease), skin cancers (including malignant melanoma), cancers of the digestive tract (including head and neck cancers, oesophageal cancer, stomach cancer, cancer of the pancreas, liver cancer, colon and rectal cancer, anal cancer), cancers of the genital and urinary systems (including kidney cancer, bladder cancer, testis cancer, prostate cancer), cancers in women (including breast cancer, ovarian cancer, gynecological cancers and choriocarcinoma) as well as in brain, bone carcinoid, nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. It also finds application in the treatment or prophylaxis of cancers of unknown primary site.

The Th1-related infectious diseases include bacterial, prion (e.g. BSE and CJD), viral, fungal, protozoan and metazoan infections. For example, the Th1-related infectious diseases include infection with respiratory syncytial virus (RSV), hepatitis B virus (HBV), Epstein-Barr, hepatitis C virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, hantann virus (hemorrhagic fever), human papilloma virus (HPV), tuberculosis, leprosy and measles.

Particularly preferred Th1-related infectious diseases include those in which the pathogen occupies an intracellular compartment, including HIV/AIDS, leishmaniasis, trypanosomiasis, influenza, tuberculosis and malaria.

The materials of the invention may also find application in the treatment of patients in which the Th1 immune response is defective. Such patients may include neonates, juveniles in which the Th1 response is immature and not fully developed, as well as older patients in which the Th1 response has become senescent or compromised over time. In such patient populations the compounds of the invention may be used prophylactically (as a generalized type 1 immune stimulant to reduce the risks of (e.g. viral) infections. Th2-related diseases and allergy

Th2-related diseases are diseases, disorders, syndromes, conditions or infections in which Th2 cells are implicated in (e.g. support, cause or mediate) the effects of the disease, disorder, syndrome, condition or infection.

Thus, the materials of the invention find application in the treatment or prophylaxis of Th2-related diseases.

One important class of Th2-related diseases treatable with the compounds of the invention is allergic disease.

It is well known that genetically predisposed individuals can become sensitised (allergic) to antigens originating from a variety of environmental sources. The allergic reaction occurs when a previously sensitised individual is re-exposed to the same or to a structurally similar or homologous allergen. Thus, as used herein the term allergy is used to define a state of hypersensitivity induced by exposure to a particular antigen (allergen) resulting in harmful and/or uncomfortable immunologic reactions on subsequent exposures to the allergen.

The harmful, uncomfortable and/or undesirable immunologic reactions present in allergy include a wide range of symptoms. Many different organs and tissues may be affected, including the gastrointestinal tract, the skin, the lungs, the nose and the central nervous system. The symptoms may include abdominal pain, abdominal bloating, disturbance of bowel function, vomiting, rashes, skin irritation, wheezing and shortness of breath, nasal running and nasal blockage, headache and mood changes. In severe cases the cardiovascular and respiratory systems are compromised and anaphylactic shock leads in extreme cases to death.

It is known that the harmful, undesirable and/or uncomfortable immunologic reactions characteristic of allergy have a Th2 response component.

As explained above, the materials of the invention may suppress or inactivate (either directly or indirectly) the release and/or activity (in vitro and/or in vivo) of one or more Th2 cytokines (for example one or more cytokines selected from lL-4, IL-5, IL-10 and IL-13). Thus, the materials of the invention may be used to effect a remedial or palliative modulation of the harmful and/or uncomfortable immunologic reactions characteristic of allergic reactions by inhibiting, suppressing or eliminating the Th2 response to the allergen.

The materials of the invention therefore find application in the treatment or prophylaxis of allergy.

Any allergy may be treated according to the invention, including atopic allergy, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, hypereosinophilia, irritable bowel syndrome, allergen-induced migraine, bacterial allergy, bronchial allergy (asthma), contact allergy (dermatitis), delayed allergy, pollen allergy (hay fever), drug allergy, sting allergy, bite allergy, gastrointestinal or food allergy (including that associated with inflammatory bowel disease, including ulcerative colitis and Crohn's disease) and physical allergy. Physical allergies include cold allergy (cold urticaria or angioedema), heat allergy (cholinergic urticaria) and photosensitivity.

Particularly important is the treatment or prophylaxis of asthma. 2. Haemorestoration

The materials of the invention increase splenic and bone marrow cell proliferation and can act as myeloproliferative agents. They therefore find application as haemorestoratives.

Haemorestoration may be indicated following immunosuppressant therapies (such as cyclosporine A, azathioprine or immunosuppressant radiotherapies), chemotherapy (including treatment with both cycle- specific and non-specific chemotherapeutic agents), steroid administration or other forms of surgical or medical intervention (including radiotherapy). Thus, the use of the materials of the invention as haemorestoratives may be adjunctive to other treatments which tend to depress splenic and bone marrow cell populations. Particularly preferred adjunctive therapies according to the invention include the administration of an immunorestorative dose of the materials of the invention adjunctive to: (a) chemotherapy; and/or (b) radiotherapy; and/or (c) bone marrow transplantation; and/or (d) haemoablative immunotherapy.

3. Alleviation of immunosuppression

The materials of the invention may be used to alleviate, control or modify states in which the immune system is partially or completely suppressed or depressed. Such states may arise from congenital (inherited) conditions, be acquired (e.g. by infection or malignancy) or induced (e.g. deliberately as part of the management of transplants or cancers).

Thus, the materials of the invention may find application as adjunctive immunomodulators (e.g. immunostimulants) in the treatment and/or management of various diseases (including certain cancers) or medical interventions (including radiotherapy, immunosuppressant therapy (such as the administration of cyclosporine A, azathioprine or immunosuppressant radiotherapies), chemotherapy and cytotoxic drug administration (for example the administration of ricin, cyclophosphamide, cortisone acetate, vinblastine, vincristine, adriamycin, 6-mercaptopurine, 5-fluorouracil, mitomycin C, chloramphenicol and other steroid- based therapies). They may therefore be used as chemoprotectants in the management of various cancers and infections (including bacterial and viral infections, e.g. HIV infection) or to induce appropriate and complementary immunotherapeutic activity during conventional immunotherapy.

In particular, the materials of the invention may find application as immunostimulants in the treatment or management of microbial infections which are associated with immune-suppressed states, including many viral infections (including HIV infection in AIDS) and in other situations where a patient has been immunocompromised (e.g. following infection with hepatitis C, or other viruses or infectious agents including bacteria, fungi, and parasites, in patients undergoing bone marrow transplants, and in patients with chemical or tumor-induced immune suppression).

Other diseases or disorders which may give rise to an immunosupressed state treatable according to the invention include: ataxia-telangiectasia; DiGeorge syndrome; Chediak-Higashi syndrome; Job syndrome; leukocyte adhesion defects; panhypogammaglobulinemia (e.g. associated with Bruton disease or congenital agammaglobulinemia); selective deficiency of IgA; combined immunodeficiency disease; Wiscott-Aldrich syndrome and complement deficiencies. It may be associated with organ and/or tissue (e.g. bone marrow) transplantation or grafting, in which applications the materials of the invention may be used adjunctively as part of an overall treatment regimen including surgery and post-operative management of immune status.

4. Cytokine stimulation

The materials of the invention may be used to induce, potentiate or activate various cytokines in vivo, including various interleukins (including IL-2 and/or IL-12).

Accordingly, the materials of the invention find general application in the treatment or prophylaxis of conditions in which the in vivo induction, potentiation or activation of one or more cytokines (e.g. IL-12 and/or II-2) is indicated. Such applications may be employed to stimulate particular elements of the cellular immunity system, including dendritic cells, macrophages (e.g. tissue-specific macrophages), CTL, NK, NKT, B and LAK cells.

In such applications, the materials of the invention may be employed as an adjunct to gene therapies designed to increase the production of endogenous cytokines (for example IL-2).

5. Treatment of proliferative disorders

The invention finds application in the treatment of proliferative disorders, including various cancers and cancer metastasis. For example, the materials of the invention may find particular application in the treatment of leukemias, lymphomas, melanomas, adenomas, sarcomas, carcinomas of solid tissues, melanoma (including melanoma of the eye), pancreatic cancer, cervico-uterine cancer, cancers of the kidney, stomach, lung, ovary, rectum, breast, prostate, bowel, gastric, liver, thyroid, neck, cervix, salivary gland, leg, tongue, lip, bile duct, pelvis, mediastinum, urethra, lung, bladder, esophagus and colon, and Kaposi's Sarcoma (e.g. when associated with AIDS).

In such applications the honey, honey fractions and honey-containing compositions of the invention may exhibit a secondary glycosidase inhibitory activity.

The invention may therefore find application in methods of therapy or prophylaxis which comprise the modification of tumour cell glycosylation (e.g. tumour antigen glycosylation), the modification of viral protein glycosylation (e.g. virion antigen glycosylation), the modification of cell-surface protein glycosylation in infected host cells and/or the modification of bacterial cell walls, hence promoting an increased immune response or inhibiting growth/infectivity directly.

6. Use as adjuvant

The materials of the invention find utility as vaccine adjuvants, in which embodiments they may promote, induce or enhance an immune response to antigens, particularly antigens having low intrinsic immunogenicity. Without wishing to be bound by any theory, the materials of the invention may augment vaccine immunogenicity by stimulating cytokine release, thereby promoting T-cell help for B cell and CTL responses. They may also change glycosylate of cancer or viral antigens and increase vaccine effectiveness.

When used as adjuvant, the materials of the invention may be administered concurrently, separately or sequentially with administration of the vaccine. The invention finds application in any vaccine, but may be particularly as a subunit vaccine, a conjugate vaccine, a DNA vaccine, a recombinant vaccine or a mucosal vaccine. The vaccine may be therapeutic or prophylactic. It may be used immunoprophylactically or immunotherapeutically in both human and non-human subjects. Preferred non-human subjects include mammals and birds. Particularly preferred are veterinary applications. Such applications include the treatment or prophylaxis of infection in domesticated animals (for example dogs and cats) and livestock (e.g. sheep, cows, pigs, horses, chickens and turkeys).

Thus, in some embodiments, the materials of the invention may be present in admixture with other vaccine component(s), or else co-packaged (e.g. as part of an array of unit doses) with the other vaccine components with which it is to be used as adjuvant. In yet other embodiments, the use of the materials of the invention as adjuvant is simply reflected in the content of the information and/or instructions co-packaged with the vaccine components and relating to the vaccination procedure, vaccine formulation and/or posology.

7. Dendritic cell-based applications

As described above, it has now been found that the materials of the invention may induce sustained and pronounced cytokine production (e.g. sustained and pronounced IL-12 and/or IL-2 production) in dendritic cells. Thus, the materials of the invention find application in methods of therapy or prophylaxis comprising the induction of cytokine production in dendritic cells or in which the induction of cytokine production in dendritic cells is indicated or required.

Dendritic cell vaccines

In one dendritic cell-based treatment paradigm, the cells are pulsed (primed or spiked) with a particular antigen or antigens (for example, tumour antigen(s)) and then administered to promote a Th1 immune response. The responding T cells include helper cells, especially Th1 CD4⁺ cells (which produce lFN-γ) and killer cells (especially CD8⁺ cytolytic T lymphocytes). The dendritic cells may also mediate responses by other classes of lymphocytes (B, NK, and NKT cells). They may also elicit T cell memory, a critical goal of vaccination.

With regard to antigen selection for use in the dendritic cell vaccines of the invention, both defined and undefined antigens can be employed. The antigens can be xenoantigens or autoantigens. One or more defined neoantigen(s) may be selected: in the case of cancer treatment, the neoantigen(s) may comprise a tumour-associated antigen.

However, most preferred for use according to the invention are peptides (for example, synthetic 9-11 amino acid peptides) containing defined antigens. Such peptides may comprise natural sequences. Alternatively, they may be synthetic analogues designed for enhanced MHC binding. In other embodiments, the antigens used according to the invention are provided in the form of immune complexes. These are preferably delivered to Fc-receptor-bearing DCs so that both MHC class I and MHC class Il peptide sequences are formed. In this way, dendritic cell vaccines can be used according to the invention for inducing both CTLs and Th cells.

In another approach to antigen selection for use according to the invention, the whole antigenic repertoire of any given tumour (or other target cell, such as a virally-infected cell) is explored. Thus, in another embodiment of the invention there is provided DC-tumour cell hybrids in which the dendritic cells are treated with compound (thereby to induce the expression of IL-2) before or after hybridisation.

In yet other embodiments, necrotic or apoptotic tumour cells or cell lysates (for example lysates of infected cells or tumour cells) are used.

Antigens derived from fresh tumour cells (rather than tumour cell lines or defined antigens) may also be employed.

It is also contemplated that the materials of the invention be incorporated into cellular antigens by introducing them into the cellular membrane or into an intracellular compartment (as described for example in WO96017614, the contents of which are incorporated herein by reference).

Various techniques can be used to deliver the selected antigen(s) to the DCs (variously referred to in the art as antigen loading, pulsing, priming or spiking). Preferred are loading techniques which load the DCs internally: this can be achieved through the use of peptides linked to cell-penetrating moieties.

Antigens can also be loaded by transfecting the DCs with encoding nucleic acid (e.g. by electroporation) such that the antigens are expressed by the DC, processed and presented at the cell surface. This approach avoids the need for expensive GMP proteins and antibodies. RNA is preferred for this purpose, since it produces only transient expression (albeit sufficient for antigen processing) and avoids the potential problems associated with the integration of DNA and attendant long-term expression/mutagenesis. Such transfection techniques also permit exploration of the whole antigenic repertoire of a target cell by use of total or PCR-amplified tumour RNA.

Current strategies for using dendritic cells in this way focus on identifying specific tumour antigens and defining antigenic peptides that bind to the particular MHC alleles expressed by each patient. However, a more general approach would involve the stimulation of the dendritic cells in a manner appropriate for potentiating Th1 responses irrespective of the antigens present and either with or without antigen priming. Cytokine production by activated dendritic cells would then promote the appropriate Th1 response.

The dendritic cell based vaccines of the invention find particular application in the treatment or prophylaxis of various proliferative disorders (including various cancers, as described below). In such applications, the dendritic cells are preferably pulsed (primed or spiked) with one or more tumour antigens ex vivo and the materials of the invention used to potentiate the dendritic cell component of the vaccine by contacting the dendritic cells with the compound either ex vivo (before or after pulsing of the cells) or in vivo (for example by co-administration, either concurrently, separately or sequentially, of the dendritic cells and the compound).

The dendritic cell based vaccines of the invention may be used in the treatment or prophylaxis of any malignant or pre-malignant condition, proliferative or hyper-proliferative condition or any disease arising or deriving from or associated with a functional or other disturbance or abnormality in the proliferative capacity or behaviour of any cells or tissues of the body.

Thus, the invention finds application in the treatment or prophylaxis of breast cancer, colon cancer, lung cancer and prostate cancer. It also finds application in the treatment or prophylaxis of cancers of the blood and lymphatic systems (including Hodgkin's Disease, leukemias, lymphomas, multiple myeloma, and Waldenstrom's disease), skin cancers (including malignant melanoma), cancers of the digestive tract (including head and neck cancers, oesophageal cancer, stomach cancer, cancer of the pancreas, liver cancer, colon and rectal cancer, anal cancer), cancers of the genital and urinary systems (including kidney cancer, bladder cancer, testis cancer, prostate cancer), cancers in women (including breast cancer, ovarian cancer, gynecological cancers and choriocarcinoma) as well as in brain, bone carcinoid, nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. It also finds application in the treatment or prophylaxis of cancers of unknown primary site.

The dendritic cell based vaccines of the invention also find application in the treatment or prophylaxis of various infections, including bacterial, viral, fungal, protozoan and metazoan infections. For example, the vaccines may be used in the treatment or prophylaxis of infection with respiratory syncytial virus (RSV), Epstein-Barr, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, hantann virus (hemorrhagic fever), human papilloma virus (HPV), tuberculosis, leprosy and measles.

Particularly preferred is the treatment or prophylaxis of infections in which the pathogen occupies an intracellular compartment or causes the expression of neoantigens by host cells, including HIV/AIDS, leishmania, trypanosomiasis, influenza, tuberculosis and malaria.

The present invention also contemplates a more general approach to DC cell-based therapy which involves the stimulation of the dendritic cells with the materials of the invention irrespective of the antigens present and either with or without antigen priming.

Thus, the invention finds application in therapies in which dendritic cells exposed to the materials of the invention are targeted to diseased or infected tissue (for example injected directly into a tumour), where the cells can prime endogenous T cells extranodally. In such embodiments, the invention contemplates targeting of DCs to a tumour and their activation in situ to elicit immune responses without the need for ex vivo antigen loading.

In yet another embodiment, the invention contemplates in situ DC vaccination where antigen is targeted to DCs in vivo which are then expanded and induced to mature in situ (by the co-administration of one or more DC maturation stimulants). In such embodiments, antigen is targeted to endogenous DCs by any convenient method, for example through the use of exosomes (as described in Thery et al. (2002) Nat Rev Immunol 2: 569-579).

Any class of dendritic cell may be used according to the invention. Thus, the dendritic cells may be myeloid or lymphoid, or mixtures thereof. The myeloid dendritic cells, if used, may be of the Langerhans cell type or interstitial DCs. Alternatively, mixtures of these myeloid subsets may be used. Especially preferred is the use of monocyte-derived DCs (Mo-DCs).

Helper proteins may be used to potentiate the activity of the dendritic cell vaccines of the invention.

Dendritic cell-based approaches to autoimmune disorders

Dendritic cells are also involved in regulating and maintaining immunological tolerance: in the absence of maturation, the cells induce antigen-specific silencing or tolerance. Thus, in another dendritic cell-based treatment paradigm the cells are administered as part of an immunomodulatory intervention designed to combat autoimmune disorders.

In such applications, the suppressive potential of dendritic cells has been enhanced by in vitro transfection with genes encoding cytokines. However, such gene therapy approaches are inherently dangerous and a more efficient and attractive approach would be to pulse dendritic cells in vitro with biologically active compounds which stimulate an appropriate cytokine secretion pattern in the dendritic cells.

As described above, it has now been discovered that the materials the invention can induce sustained and pronounced cytokine production in dendritic cells. Thus, the materials of the invention find application in the enhancement of the suppressive potential of dendritic cells.

Thus, the invention finds application in the treatment or prophylaxis of autoimmune disorders, including myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren syndrome, scleroderma, polymyositis and dermomyositis, ankylosing spondylitis, and rheumatic fever, insulin-dependent diabetes, thyroid diseases (including Grave's disease and Hashimoto thyroiditis), Addison's disease, multiple sclerosis, . psoriasis, inflammatory bowel disease, ulcerative colitis and autoimmune male and female infertility.

8. Wound healing

The materials of the invention can reverse a Th2 type splenocyte response ex vivo in a normally non-healing infectious disease model. Antigen specific splenocyte IFN-gamma can be significantly increased and IL-5 production significantly reduced in such models, indicative of a healing response.

Thus, the invention finds application in the treatment of wounds. In particular, the invention finds application in the treatment or prophylaxis of wounds and lesions, for example those associated with post-operative healing, burns, infection (e.g. necrotic lesions), malignancy or trauma (e.g. associated with cardiovascular disorders such as stroke or induced as part of a surgical intervention). The wound treatments may involve the selective suppression or elimination of a Th2 response (for example to eliminate or suppress an inappropriate or harmful inflammatory response).

Posology

The materials of the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

The amount of the material administered can vary widely. The principal consideration is the concentration of casuarine and/or casuarine glycoside present in the material to be administered. Other secondary considerations include the particular dosage unit employed, the period of treatment, the age and sex of the patient treated, the nature and extent of the disorder treated, and the particular form of the material selected.

Moreover, the materials of the invention can be used in conjunction with other agents known to be useful in the treatment of diseases, disorders or infections where immunostimulation is indicated (as described infra) and in such embodiments the dose may be adjusted accordingly.

In general, the amount of the material administered daily is sufficient to contain casuarine and/or a glycoside thereof in a range from about 0.01 mg/kg to 500 mg/kg. A unit dosage may contain from 0.05 to 500 mg of the casuarine and/or a glycoside, and can be taken one or more times per day.

The preferred route of administration is oral administration. In general a suitable dose will be sufficient to provide the casuarine and/or a glycoside thereof in the range 0.01 to 500 mg per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 50 mg per kilogram body weight per day and most preferably in the range 1 to 5 mg per kilogram body weight per day.

The desired dose is preferably presented as a single dose for daily administration. However, two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day may also be employed. These sub-doses may be administered in unit dosage forms, for example, containing 0.001 to 100 mg, preferably 0.01 to 10 mg, and most preferably 0.5 to 1.0 mg of the casuarine/glycoside thereof per unit dosage form.

Formulation

In embodiments where the materials of the invention comprise myrtaceous honey no formulation steps beyond those usually applied in the collection and preparation of honeys may be necessary. In such embodiments, the honeys of the invention may be provided as comb honey or extracted honey: where the honey is extracted, it may be removed from the comb and presented in various forms including for example: (1 ) liquid; (2) crystallized or granulated; and/or (3) partially crystallized.

In embodiments where the materials of the invention is formulated together with a pharmaceutically acceptable excipient, any suitable excipient may be used, including for example inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc.

The pharmaceutical compositions may take any suitable form, and include for example tablets, elixirs, capsules, solutions, suspensions, powders, granules and aerosols.

The pharmaceutical composition may take the form of a kit of parts, which kit may comprise the materials of the invention together with instructions for use and/or a plurality of different components in unit dosage form.

Tablets for oral use may include the materials of the invention. The tablets may contain the materials of the invention mixed with pharmaceutically acceptable excipients, such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the materials of the invention are mixed with a solid diluent, and soft gelatin capsules wherein the materials are mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the materials of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity.

Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

The materials of the invention may also be presented as liposome formulations.

For oral administration the materials of the invention can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, granules, solutions, suspensions, dispersions or emulsions (which solutions, suspensions dispersions or emulsions may be aqueous or non-aqueous). The solid unit dosage forms can be a capsule which can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and cornstarch.

In another embodiment, the materials of the invention are tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, lubricants intended to improve the flow of tablet granulations and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium, or zinc stearate, dyes, coloring agents, and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient.

Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptably surfactant, suspending agent or emulsifying agent.

The materials of the invention may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally.

In such embodiments, the materials are provided as injectable doses in a physiologically acceptable diluent together with a pharmaceutical carrier (which can be a sterile liquid or mixture of liquids). Suitable liquids include water, saline, aqueous dextrose and related sugar solutions, an alcohol (such as ethanol, isopropanol, or hexadecyl alcohol), glycols (such as propylene glycol or polyethylene glycol), glycerol ketals (such as 2,2- dimethyl-1 ,3-dioxolane-4-methanol), ethers (such as poly(ethylene-glycol) 400), an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant (such as a soap or a detergent), suspending agent (such as pectin, carhomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose), or emulsifying agent and other pharmaceutically adjuvants. Suitable oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil.

Suitable fatty acids include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate.

Suitable soaps include fatty alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamines acetates; anionic detergents, for example, alkyl, aryl, and olefin sulphonates, alkyl, olefin, ether, and monoglyceride sulphates, and sulphosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for example, alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures. The parenteral compositions of this invention will typically contain from about 0.5 to about 25% by weight of the material of the invention in solution. Preservatives and buffers may also be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB. Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The materials of the invention may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Topical formulations may contain a concentration of the compound from about 0.1 to about 10% w/v (weight per unit volume).

When used adjunctively, the materials of the invention may be formulated for use with one or more other drug(s). In particular, the materials of the invention may be used in combination with antitumor agents, antimicrobial agents, anti-inflammatories, antiproliferative agents and/or other immunomodulatory (e.g. immunostimulatory) agents. For example, the materials of the invention may be used with anti-viral and/or anti-proliferative agents such as cytokines, including interleukins-2 and 12, interferons and inducers thereof, tumor necrosis factor (TNF) and/or transforming growth factor (TGF), as well as with myelosuppressive agents and/or chemotherapeutic agents (such as doxorubicin, 5-fluorouracil, cyclophosphamide and methotrexate), isoniazid (e.g. in the prevention or treatment of peripheral neuropathy) and with analgesics (e.g. NSAIDs) for the prevention and treatment of gastroduodenal ulcers.

Thus, adjunctive use may be reflected in a specific unit dosage designed to be compatible (or to synergize) with the other drug(s), or in formulations in which the materials of the invention are admixed with one or more antitumor agents, antimicrobial agents and/or antiinflammatories (or else physically associated with the other drug(s) within a single unit dose). Adjunctive uses may also be reflected in the composition of the pharmaceutical kits of the invention, in which the material of the invention is co-packaged (e.g. as part of an array of unit doses) with the antitumor agents, antimicrobial agents and/or antiinflammatories. Adjunctive use may also be reflected in information and/or instructions relating to the co-administration of the materials of the invention with antitumor agents, antimicrobial agents and/or antiinflammatories.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention. Example 1 : Analysis of myrtaceous honey

Analysis of honey samples was carried out by GC-MS using a Perkin Elmer Autosystem XL gas chromatograph with a high polarity fused-silica column (Varian 'FactorFour' VF-5ms column, 25 m x 0.25 mm i.d., 0.25 μm phase thickness). The carrier gas (helium) flow rate was 1 ml min^"1. TMS derivatives were separated using a temperature programme that started at 160⁰C for 5 min, followed by a linear increase to 300⁰C at a rate of 10⁰C min^"1. The temperature was held at 300⁰C for an additional 10 min; the total analysis time was 29 min. Electron impact mass spectrometry of the column eluant was carried out using a Perkin Elmer TurboMass Gold mass spectrometer, with a quadrupole ion filter system, which was run at 250⁰C constantly during analysis. The detector mass range was set to 100 to 650 amu. The temperature of the transfer line (GC to MS) was held at 250°C. Samples were injected onto the column via a split vent (split ratio 50:1 ) through a fused silica narrow bore injection liner packed with deactivated quartz wool; the injection port temperature was maintained at 200⁰C. The injection volume was 1 μl. System control, data collection and mass spectral analysis was carried out using Perkin Elmer TurboMass software (TurboMass v. 4.4). A calibration curve using authentic casuarine and casuarine-6-O-α-glucoside allowed quantification.

The ion exchange step used involved the strongly acidic cation exchange resin (IR-120, Merck) converted to the H⁺ form using 1M HCI and then washed to neutrality. Those skilled in the art will recognize that any of a wide range of other strongly acidic cation exchange resin (such as Dowex 50) are also suitable.

The honey is dissolved in any volume of 50% (v/v) aqueous ethanol to allow it to be run through a column and the column is then washed with excess distilled or deionised water. Alternatively, though less effectively, the resin may be simply added to the dissolved honey and then washed with water. The resin binds the alkaloids and amino acids allowing fats, proteins, sugars to be washed out. The alkaloids are displaced using a suitable solvent. In this example 2M ammonium hydroxide was employed. The ammonium hydroxide was removed by rotary evaporation to give an enriched fraction which was then dried for production of trimethylsilyl-derivatives for GC-MS analysis. TMS derivatives were prepared using a mixture of hexamethyldisilazane and trimethylchlorosilane in pyridine (Pierce Tri-Sil' silylation reagent, HMDS:TMCS:pyridine in a ratio of 2:1:10). Samples were heated at 60⁰C for 15 minutes and then left at room temperature for at least 60 min. Insoluble reaction products were sedimented by centrifugation, and the supernatant was transferred to fresh vials using a syringe.

Two typical examples of the GC-MS results obtained are shown below for honey containing casuarine-6-O-α- glucoside (which is shown as the major peak at retention time of 18 minutes). A GC trace of a UK heather honey not containing either casuarine or casuarine-6-O-α-glucoside is also shown for comparison. Also shown are traces for casuarine-6-O-α-glucoside as the tms ether having a characteristic mass fragmentation pattern with major ions at 476, 418, 361 , 272 and 217 amu. Casuarine is even more easily recognized by having an unusual distinctive major fragment at 462 amu and has a retention of around 10.5 minutes. GC-MS: Zambian myrtaceous honey sample 1 showing major casuarine-6-O-α-αlucoside peak

GC-MS: Zambian myrtaceous honey sample 2 showing major casuarine-6-O-α-αlucoside peak

GC-MS of UK heather honey showing absence of casuarine and casuarine-6-O-α-qlucoside

Zambian myrtaceous honey sample 2: Mass spectrum of casuarine-6-Q-α-glucoside (tms)

GC-MS chromatogram of casuarine (tms)

Mass spectrum of casuarine (tms)

Example 2: Analysis of Zambian myrtaceous honey samples

Several commercial bottles of organic Myrtaceous honey derived from North Western Zambia (Mwinilunga) sold in the UK by Tropical Forest Products Limited, Aberystwyth were analysed. Bees in this area visit species of Sy∑ygium (Myrtaceae) to collect nectar/pollen containing casuarine and casuarine-6-O-α-glucoside. The commercial honey samples are blended from batches sourced from several villages, and several of these batches were also analysed. All samples were found to contain casuarine or casuarine-6-O-α-glucoside (see Table below). Unlike the plant extracts which were very complex in alkaloids, they were the only major alkaloids in these honeys and could constitute as much as 1.6% fresh weight. Other classes of alkaloids were not detectable.

Batch 4 15.96 0.27

Batch 5 5.20 0.25

Batch 6 3.47 0.89

Batch 7 0.86 1.41

Batch 8 0.61 0.97

Batch 9 1.12 1.47

Batch 10 0.53 1.04

Batch 11 14.17 1.62

Batch 12 5.98 1.10

Batch 13 2.21 1.96

Batch 14 3.01 3.37

Batch 15 1.49 1.15

Batch 16 2.68 1.31

Batch 17 6.11 3.90

Batch 18 3.52 5.25

Batch 1 (re-extracted) 2.56 3.20

Batch 19 (used for beer) 0.46 0.89

Note: Tropical Forest™ 1 is Organic Forest Honey batch expiry date 11^th November 2005 purchased from Fortnum and Mason. Tropical Forest™ is Organic Forest Honey batch expiry date 8^th December 2005 purchased from Fortnum and Mason.

Comparative example 3: Analysis of non-myrtaceous honey samples

By contrast, other honeys (listed below) did not contain detectable concentrations (<1 ng g^" ) of casuarine or casuarine-6-O-α-glucoside (or any related compounds).

(a) Medibee Manuka honey (New Zealand), lot number 10624, best before Nov '06

(b) Comvita Manuka honey (New Zealand), batch 279463, 9400501.

(c) Marnys Plus 1000 Royal Jelly, two samples both lot number 001-03-02.

(d) Libyan orange blossom, thyme and hannon honeys (no batch numbers).

(e) Gales Pure Set Honey batch 5 000354 900907.

Example 4: Oral availability of myrtaceous honey-derived casuarine in human subjects

Oral dosing experiments with two volunteers using myrtaceous honey batch 1 showed rapid uptake of casuarine into blood. The amount of honey consumed in both subjects was 100g dissolved in water, which equated to 6.9 and 6.2 mg of combined casuarine and casuarine-6-O-α-glucoside per kg body mass, respectively. 7 ml blood samples were taken at 0, 25, 60, 90, 120 minutes and spiked with an internal reference of swainsonine (2.2 μg) for quantification purposes.

Blood was processed using cation exchange column chromatography as described for the honey after mixing with an equal volume of ethanol and centrifugation to remove solid material. Both subjects showed peaks of casuarine at 25 minutes of 55 ng ml^"1 and 98 ng ml^"1 respectively, dropping to 38 and 45 ng ml^"1 at 120 minutes. In subject 2 urine was collected in two-hour batches, up to four hours after ingestion of the honey; the amount of casuarine present in urine after the first two-hour period (409 μg) subsequently fell to 186 μg in the period two to four hours after taking the sample of honey.

This shows that myrtaceous honey-derived casuarine is orally available. Casuarine-6-O-α-glucoside (also present in the batch used for dosing: see Table above) was not detected in the blood and it is possible that it was hydrolysed prior to uptake in the gut. Only low levels of casuarine were seen in urine.

Example 5: Oral administration of myrtaceous honey modulates immunological responses in mice

Batch 1 (see Table above) contains both the pyrrolizidine alkaloid casuarine and casuarine-6-O-α-glucoside. Experiments described below show the oral efficacy of the honey, casuarine and 3,7-diepicasuarine in promoting immune activity.

In a series of five separate experiments, 3,7-diepicasuarine (MNLP24) and casuarine (MNLP462a) in PBS were given orally by gavage to 8 week old female BALB/c mice. Mice received 25 mg kg^"1 or 20 mg kg^"1 of casuarine or alternatively 20 mg kg^"1 of 10 mg kg^"1 3,7-diepicasuarine on three consecutive days. Control groups of BALB/c mice were given PBS alone orally by gavage following an identical schedule to that above. On day four, spleens and mesenteric lymph nodes (MLN) were removed and cell suspensions (2.5 x 10⁶ ml^"1) were either incubated unstimulated for 72 hours or stimulated with 0.5 ug ml^"1 anti-CD3 to polyclonally activate T-cells. Supematants were subsequently analysed for IL-12 and IFN-γ production.

In all experiments using casuarine (MNLP462a) and 3,7-diepicasuarine (MNLP24) at 20 mg or 25 mg kg^"1, splenocyte IFN-γ production was significantly enhanced in treated mice compared with controls stimulated with anti-CD3 (Figs. 1 a and 1b).

Splenocyte IL-12 production was also significantly enhanced following in vivo treatment (Fig. 2a) including at the lower dosage level of 10 mg kg^"1 (Fig. 2b). This was invariably significant over 5 separate experiments, even in the supematants from resting non-stimulated cultures.

The effect of the imino sugars on mesenteric lymph node cells using this dosing regime was much less pronounced, and no increases in IFN-γ were measured following treatment. However, IL-12 lymph node cell levels were consistently higher in imino sugar-treated mice (Fig. 3a) and on occasions, these differences were significant (Fig. 3b).

In a further experiment, BALB/c mice received honey (batch 1 : see Table above) by oral gavage diluted to deliver 10 mg kg^"1 of the casuarine-6-O-α-glucoside (MNLP462b) present in the honey daily for three days. Again, on day four, splenocyte and mesenteric lymph node cells were harvested and stimulated with anti-CD3. Splenocyte IL-12 was significantly increased (Fig. 4) although IFN-γ levels were not significantly increased. A similar result was obtained using the same regime and dose with 3,7-diepicasuarine (Fig. 2b). Thus, these imino sugars can significantly boost the cellular immune response by direct oral delivery at concentrations of 10 mg kg^"1 or greater. Myrtaceous honey containing casuarine and casuarine-6-O-α- glucoside can also boost the immune system when given orally.

Example 6: Oral administration of myrtaceous honey modulates immunological responses in humans

Oral dosing experiments with a human volunteer using myrtaceous honey followed by analysis of LPS- stimulated IFN-γ release in whole blood ex vivo revealed that LPS induced a concentration-dependent increase in IFN-y release in whole blood ex vivo.

Blood was taken from a single individual before (day 0) and at various times after oral administration of the myrtaceous honey. Aliquots of whole blood were then incubated with various concentrations of LPS for 20 h at 37°C, 5% COa and 100% humidity. At the end of this period, plasma was collected by centrifugation and stored at -20°C prior to analysis. IFN-γ levels were measured by ELISA. Values represent the mean of n = 3 + standard deviation. The results are shown in Figure 5.

It can be seen that LPS induced a concentration dependent increase in IFN-γ release ex vivo. Release of this cytokine on induction with LPS was enhanced for up to 21 days after administration of the myrtaceous honey. This enhancement was maximal 2 days after oral dosing.

Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A process for the production of a composition comprising a pyrrolidine compound comprising the steps of:

(a) providing myrtaceous honey; (b) extracting (e.g. isolating or purifying) casuarine and/or a glycoside thereof from the honey; and/or

(c) chemically and/or enzymatically modifying the casuarine and/or glycoside thereof present in the honey.

2. The process of claim 1 which comprises the steps (b) and (c) and further comprises the step (d) of extracting (e.g. isolating or purifying) the chemically and/or enzymatically modified casuarine and/or glycoside thereof.

3. The process of claim 1 which comprises the steps of :

(a) providing myrtaceous honey; (b) chemically and/or enzymatically modifying the casuarine and/or glycoside thereof present in the honey; and (c) extracting (e.g. isolating or purifying) the chemically and/or enzymatically modified casuarine and/or glycoside thereof.

4. The process of any one of the preceding claims wherein the pyrrolizidine compound is a polyhydroxylated pyrrolizidine compound (for example, casuarine and/or a glycoside thereof).

5. The process of any one of the preceding claims wherein the extracting step comprises the production of a polar extract and optionally further comprises fractionating the polar extract to produce a fraction enriched in casuarine and/or a glycoside thereof.

6. The process of any one of the preceding claims wherein the chemical and/or enzymatic modification comprises fermentation and/or hydrolysis (for example to hydrolyse a casuarine glycoside to produce casuarine).

7. A method of immunomodulation comprising the step of administering an immunomodulatory amount of honey to an individual in need thereof.

8. Use of honey for the manufacture of a medicament for use in immunomodulation.

9. The method of claim 7 or the use of claim 8 wherein the immunomodulation comprises:

(a) Increasing the Th1 :Th2 response ratio;

(b) Haemorestoration; (c) Alleviation of immunosuppression;

(d) Cytokine stimulation;

(e) Treatment of proliferative disorders (e.g. cancer);

(f) Vaccination, wherein the compound acts as an adjuvant; (g) Vaccination with a dendritic cell vaccine (e.g. a primed dendritic cell vaccine), wherein the dendritic cells are contacted with the compound; (h) Administration of dendritic cells in the treatment or prophylaxis of autoimmune disorders, wherein the dendritic cells are contacted with the compound; and/or (i) Wound healing;

G) Stimulating the innate immune response; (k) Boosting the activity of endogenous NK cells.

10. The method or use of any one of claims 7 to 9 wherein the immunomodulation comprises:

(a) the treatment of Th1 -related diseases or disorders;

(b) the treatment of Th2-related diseases or disorders (for example allergies, e.g. asthma);

(c) the treatment of bacterial infections;

(d) the treatment of viral infections; (e) the treatment of prion (e.g. BSE and CJD), fungal, protozoan or metazoan infections;

(f) the treatment of diseases associated with intracellular pathogens (e.g. leishmaniasis, trypanosomiasis or malaria).

11. The method or use of claim 10 (d) wherein the viral infection is selected from respiratory syncytial virus (RSV), hepatitis B virus (HBV), Epstein-Barr, hepatitis C virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, hantann virus (hemorrhagic fever), human papilloma virus (HPV) and measles.

12. A method for monitoring the quality of a honey comprising the step of testing the honey for the presence of casuarine and/or a glycoside thereof.

13. A method of apiculture or a process for the production of honey comprising the step of monitoring the quality of the honey by the method of claim 12.

14. An immunomodulatory pharmaceutical composition comprising myrtaceous honey (or a fraction or derivative thereof).

15. Myrtaceous honey (or a fraction or derivative thereof) in a pharmaceutical excipient.

16. A myrtaceous honey fraction or derivative enriched in casuarine and/or a glycoside thereof.

17. A composition comprising myrtaceous honey and an immunomodulatory agent.

18. The invention of any one of the preceding claims wherein the honey is: (a) unprocessed (e.g. comb honey);

(b) processed (for example extracted, e.g. being liquid, crystallized or granulated honey)

(c) a honey fraction (e.g. a polar extract enriched in casuarine and/or casuarine glycoside);

(d) a honey derivative (e.g. honey beer).

19. The invention of any one of the preceding claims wherein the casuarine glycoside is casuarine-6-O-α-D- glucoside.