WO2008009894A2

WO2008009894A2 - Use of imino sugars in immunotherapy

Info

Publication number: WO2008009894A2
Application number: PCT/GB2007/002597
Authority: WO
Inventors: Robert James Nash
Original assignee: Summit Corporation Plc
Priority date: 2006-07-15
Filing date: 2007-07-12
Publication date: 2008-01-24
Also published as: GB0614098D0; WO2008009894A3

Abstract

Described are uses for various imino sugars in PRR-mediated immunotherapy or immunoprophylaxis. In particular, the invention relates to the use of imino sugar PRR (particularly C-type lectin) ligands in immune response variegation and/or the stimulation of immune surveillance (for example by the innate immune system) and/or non-specific immunoprophylaxis. The invention therefore finds application in the treatment of pathological variegated states (for example those arising from pathogen-mediated immune system subversion or immune system dysfunction) and for inhibiting or postponing neutrophil apoptosis.

Description

USE OF IMINO SUGARS IN IMMUNOTHERAPY

Field of the Invention

The present invention relates to the use of various imino sugars in PRR-mediated immunotherapy or immunoprophylaxis. In particular, the invention relates to the use of imino sugar PRR (particularly C-type lectin) ligands in immune response variegation and/or the stimulation of immune surveillance (for example by the innate immune system) and/or non-specific immunoprophylaxis. The invention therefore finds application in methods for the treatment of pathological variegated states (for example those arising from pathogen-mediated immune system subversion or immune system dysfunction).

Background to the Invention

Immune Response Variegation

The mammalian immune system comprises two distinct arms, the adaptive and innate, reflecting its evolution from an ancient innate defence mechanism common to all metazoans. The cellular components of the innate immune system include neutrophils, granulocytes, monocytes, macrophages, dendritic cells (DCs) and natural killer (NK) cells. The adaptive immune system consists of B cells and T cells. 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).

Of profound importance is the fact that the immune system is able to launch qualitatively different immune responses against different threats. This phenomenon has been dubbed immune system variegation (see Pulendran (2005): J. Immunol. 173: 2457-2465), and is characterized by qualitative differences arising from variations in the relative contributions of the various components of the innate and adaptive immune systems (both in absolute terms and in the extent of their temporal and spatial specificity).

Immune system variegation has a profound impact on the efficacy of any given immune response, and is particularly important in circumstances where a strong cellular adaptive response is necessary (for example in viral infections or cancer) and in certain chronic infections where the pathogen has subverted the host immune system to induce immune tolerance. As stated in Pulendran (2005): J. Immunol. 173: 2457-2465, knowledge of the mechanisms of immune system variegation: "... is likely to be of critical importance in the design of novel vaccines and drugs that can generate optimally effective immune responses against a multitude of emerging and re-emerging infections. Thus, understanding the molecular mechanisms and players that regulate decision making in the immune response might be considered a Holy Grail of 21st century immunology and a grand challenge for biology."

While such knowledge is at present incomplete, it has become clear that immune system variegation is determined at least in part by a class of proteins known as pathogen-(or pattern-) recognition receptors (PRRs).

Pathogen-Recognition Receptors (PRRs)

The innate immune response has evolved to recognize a few, highly conserved structures present in diverse groups of microorganisms. These highly conserve structures are known as pathogen- associated molecular patterns (PAMPs). They are recognized by a class of receptors known as pathogen-(or pattern-) recognition receptors (PRRs), which are expressed on various effector cells of the innate immune system, including the professional antigen-presenting cells, macrophages and dendritic cells.

The best-studied class of PRR is the Toll-like receptor class (TLRs). Mammalian TLRs comprise at least 10 members, designated TLR1-10, and may be expressed as homodimers or heterodimers (TLR1 plus TLR2 or TLR6 plus TLR2). It seems that different classes of pathogen are recognized by different TLRs. For example, TLR4 appears to be responsible for the detection of Gram-negative bacteria, its cognate PAMP being lipopolysaccharide (LPS). TLR2 appears to have several ligands, including peptidoglycan of Gram-positive bacteria, lipoproteins from Mycobacterium tuberculosis, and certain components of Saccharomyces cerevisiae zymosan, as well as highly purified Porphyromonas gingivalis LPS. TLR3 recognizes dsRNA, while TLR5 binds flagellin and TLR6 cooperates with TLR2 in detecting a subset of bacterial peptidoglycan. TLR7 can be triggered by imidazoquinolines, as well as ssRNA, and may thus be involved in the detection of viral infection. TLR9 detects bacterial and viral DNA sequences containing unmethylated cytosine-guanosine dinucleotides (CpGs). Other members of the mammalian TLR family may be specific for PAMPs characteristic of other classes of pathogens such as fungi (mannan, glucan and mycobacteria (via lipoarabinomannan and/or muramyldipeptide as cognate PAMPs)).

Another major class of PRR are the C-type lectins (reviewed by Figdor et a/. (2002) Nature Reviews Immunology 2: 77-84). These PRRs share a conserved domain (the carbohydrate recognition domain or CRD) which was first characterized in animal lectins and which appears to function as a calcium-dependent carbohydrate-recognition domain. This consists of about 110 to 130 residues and contains four cysteines which are involved in two disulfide bonds. This domain may be present in multiple copies in some C-type lectin PRRs (for example, the mannose receptor contains eight CRDs).

Examples of C-type lectins include DC-SIGN (Dendritic Cell Specific ICAM-3 Grabbing Nonintegrin, or CD209), which can signal in response to Mycobacterium tuberculosis, synergising with LPS to induce IL-10 production by monocyte-derived DCs. The mannose receptor (MR) is involved in recognition of mycobacteria, fungi and protozoa. Dectin-1 acts as a PRR for β-glucan. Other C- type lectins are expressed in DCs (e.g. blood dendritic cell antigen-2 (BDCA-2), dendritic cell immunoactivating receptor (DCAR) and can also act as signalling receptors, though their role in PAMP recognition has yet to be established.

Immune response variegation by PRRs

The mechanism of immune response variegation is presently understood to be mediated, at least in part, by DCs. These cells act (possibly together with other cells of the innate immune system, including NK cells) to receive signals containing information of the nature of the insult (known as DC instruction), integrate these signals (usually over the course of maturation) and then selectively promote CTL, THI -, TH2- and/or regulatory T-cell responses during and after T-cell activation. These interactions, together with the resultant T cell response (i.e. the relative contribution from CTL, T_H1-, T_H2- and regulatory T-cell components) drives immune response variegation (for reviews, see Pulendran (2005): J. Immunol. 173: 2457-2465; Reis e Sousa (2004): Current Opinion in Immunology 16: 21-25; Kapsenberg (2003): Nature Reviews Immunology 3: 984-993).

Signal reception by DCs occurs via PRR stimulation. The pattern of PRR stimulation on DCs (and possibly on other cells of the innate immune system) determines the spectrum of various signalling molecules (including various cytokines) produced by the DCs during Tπ-cell stimulation. In some circumstances, PRR stimulation may be DC subset-specific: in many circumstances, however, it is clear that PRR stimulation occurs across different DC subsets which then exhibit functional plasticity and convergence. The identity and relative concentrations of these DC-produced signalling molecules constitute Kapsenberg's "third signal" (Kalinski et al. (1999) Immunology Today 20: 561- 567). It is this third "polarizing" signal which is presently understood to drive immune response variegation via differential stimulation of CTL, T_H1-, T_H2- and/or regulatory T-cell components.

Various DC-derived molecules which may contribute to the third, polarizing, signal have been identified. Well documented examples are T_H1 -polarizing factors, such as the interleukin-12 (IL-12) family members IL-12, IL-23 and IL-27, Type 1 Interferons (IFNs) and cell-surface expressed Intercellular Adhesion Molecule 1 (ICAM1). T_H2-polarizing factors include Monocytic Chemotactic Protein 1 (MCP1 , also known as CCL2) and OX40 ligand (OX40L). Regulatory T-cell-polarizing factors include IL-10 and Transforming Growth Factor-β (TGF-β).

The third signal therefore acts to drive selectively the development of the distinct effector T-cell subsets (T_H1-, TH2- and/or regulatory T-cell components), each of which is associated with a network of secondary messengers, some of which can feedback positively and so selectively stimulate maturing DCs to produce further polarizing signals and reinforce the effect. These secondary messengers (or tissue factors) include cytokines, chemokines, co-stimulatory factors, eicosanoids and other molecules which can be classified into T_H1-, T_H2- and/or regulatory T-cell- polarizing factors and which act to consolidate immune system variegation first initiated by DC instruction during T-cell stimulation (see table below).

(see Kapsenberg (2003): Nature Reviews Immunology 3: 984-993).

Factors which influence immune system variegation are of great interest and have broad application in the treatment of a wide range of infectious diseases, proliferative disorders, immune diseases (including autoimmune disorders and allergy) and in vaccine development. The present invention is based, at least in part, on the discovery that certain imino sugars which may act as PRR ligands (for example as C-type lectin ligands) can drive immune response variegation. Without wishing to be bound by any theory, it appears that imino sugars capable of binding to PRRs (e.g. to C-type lectins) can act as PAMP surrogates, driving immune response variegation via PRR-mediating signalling in DCs.

Summary of the Invention

In a first aspect, the invention contemplates the use of an imino sugar (or a pharmaceutically acceptable salt or derivative thereof) for the manufacture of a medicament for use in PRR (for example C-type lectin)-mediated immunotherapy or immunoprophylaxis. The PRR (for example C-type lectin)-mediated immunotherapy or immunoprophylaxis may be immune response variegation, for example in the treatment of pathological variegated states, such as those arising from pathogen-mediated immune system subversion or immune system dysfunction. Alternatively, or in addition, the PRR (for example C-type lectin)-mediated immunotherapy or immunoprophylaxis may be the stimulation of immune surveillance (for example by cells of the innate immune system). Alternatively, or in addition, the PRR (for example C-type lectin)-mediated immunoprophylaxis may be non-specific immunoprophylaxis.

In another aspect, the invention contemplates the use of an imino sugar PRR (for example C-type lectin) ligand for the manufacture of a medicament for use in immune response variegation (for example, in the treatment of pathological variegated states, e.g. arising from immune dysfunction or pathogen-mediated immune system subversion).

Any imino sugar may be used according to the invention. Preferred are imino sugars that mediate PRR (for example C-type lectin) stimulation, for example by acting as PRR (for example C-type lectin) ligands. Particularly preferred are imino sugars which initiate PRR-mediated signalling via PRR (for example C-type lectin) binding (for example to a PRR (e.g. C-type lectin) of DCs).

Structurally, the imino sugar may be a piperidine, pyrroline, pyrrolidine, pyrrolidine, indolizidine, nortropane or a mixture of any two or more of the foregoing structural classes.

The PRR (for example C-type lectin) through which the immunotherapy or immunoprophylaxis of the invention is mediated may be any PRR. Particularly preferred are C-type lectins, for example those selected from a mannose receptor and a rhamnose receptor (see Grillon, Monsigny and Kieda (1990) Glycobiology 1(1): 33-8). Similarly, the PRR ligands of the invention may be ligands for any PRR, though particularly preferred are imino sugars which are ligands for a C-type lectin, preferably those selected from: MMR (CD206, macrophage mannose receptor); and/or DEC-205; and/or Dectin 1; and/or Dectin 2; and/or Langerin; and/or DC-SIGN; and/or BDCA-2; and/or DCIR; and/or DLEC; and/or CLEC; and/or a rhamnose-binding C-type lectin.

The PRR (for example C-type lectin) through which the immunotherapy or immunoprophylaxis of the invention is mediated may reside on any cell type (or may be a secreted form). However, particularly preferred are C-type lectins, preferably those which are displayed by (or secreted by) cells of the innate immune system, for example DCs.

The invention finds broad application in all forms of immunotherapy and immunoprophylaxis, including for example: the treatment or prevention of viral infection; the treatment or prevention of bacterial infection; the treatment or prevention of protozoal infection; the treatment or prevention of fungal infection; the treatment or prevention of prion infection; the treatment or prevention of metazoan infection; the treatment or prevention of proliferative disorders (for example cancer); the treatment or prevention of autoimmune disorders; the treatment or prevention of pathogen-mediated immune system subversion; the treatment or prevention of allergy and the treatment or prevention of pathological variegated immune system states.

In another aspect, the invention provides a method for PRR (for example C-type lectin)-mediated immunotherapy or immunoprophylaxis comprising administering an imino sugar (for example, an imino sugar PRR (for example C-type lectin) ligand) to a patient in need thereof.

In another aspect, the invention provides a method for immune response variegation (for example in the treatment of pathological variegated states, such as those arising from pathogen-mediated immune system subversion or immune system dysfunction), comprising administering an imino sugar (for example, an imino sugar PRR (for example C-type lectin) ligand) to a patient in need 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 imino sugar defines a saccharide analogue in which the ring oxygen is replaced by a nitrogen.

The term ligand as used herein in relation to the imino sugars of the invention is intended to define those sugars which can act as binding partners for a PRR. Such ligands therefore include those which bind (or directly physically interact) with PRR in vivo irrespective of the physiological consequences of that binding. Thus, the ligands of the invention may bind PRR as part of a cellular signalling cascade in which the PRR forms a part. Alternatively, they may bind PRR in the context of some other aspect of cellular physiology. In the latter case, the ligands may for example bind PRR at the cell surface without triggering a signalling cascade, in which case the binding may effect other aspects of cell function. Thus, the ligands of the invention may bind PRRs and thereby effect an increase in the concentration of functional PRR at the cell surface (for example mediated via an increase in PRR stability, absolute receptor numbers and/or PRR activity). Alternatively, the imino sugar ligands may bind PRR (or PRR precursors) intracellular^, in which case they may act as molecular chaperones to increase the expression of active PRR. As used herein, the term "mediated", as used herein (and applied for example to various immunotherapies and immunoprophylactic methods, therapies, treatments or interventions) is intended to operate limitatively so that the various methods, therapies, treatments and interventions to which the term is applied are those in which the PRR plays a biological role. In cases where the term is applied to treatment, prophylaxis or intervention (e.g. in the "PRR-mediated treatments" and "PRR-mediated prophylaxis" of the invention), the role played by the PRR may be direct or indirect and may be necessary and/or sufficient for the operation of the treatment, prophylaxis or outcome of the intervention.

The term immune surveillance is a term of art and is used herein to define the monitoring function by which the immune system protects against pathological cellular changes (including those brought about by infection or cancer). As used herein, immune surveillance specifically includes PRR- mediated immune surveillance.

The term non-specific immunoprophylaxis is used herein to define prophylactic methods which are mediated by components of the immune system (for example, by one or more components and/or cells of the innate immune system) and which are not targeted (or limited in efficacy) to any particular infection, disease, syndrome, condition or pathological process. Preferred non-specific immunoprophylactic methods according to the invention are those which are effected by a stimulation of immune surveillance in the patient undergoing treatment. Non-specific immunoprophylaxis may therefore be effected according to the invention by stimulating the endogenous immune system of the patient undergoing treatment whereby immune surveillance by the immune system of the patient is potentiated or stimulated. Such stimulation may be PRR- mediated.

The term adjunctive (as applied to the use of the imino sugars of the invention in therapy) defines uses in which the imino sugar is administered together with one or more other drugs, interventions, regimens or treatments (such as surgery and/or irradiation). Such adjunctive therapies may comprise the concurrent, separate or sequential administration/application of the sugars of the invention and the other treatment(s). Thus, in some embodiments, adjunctive use of the imino sugar of the invention is reflected in the formulation of the pharmaceutical compositions of the invention. For example, adjunctive use may be reflected in a specific unit dosage, or in formulations in which the imino sugar of the invention is present in admixture with the other drug(s) with which it is to be used adjunctively (or else physically associated with the other drug(s) within a single unit dose). In other embodiments, adjunctive use of the imino sugar of the invention may be reflected in the composition of the pharmaceutical kits of the invention, wherein the imino sugar of the invention is co-packaged (e.g. as part of an array of unit doses) with the other drug(s) with which it is to be used adjunctively. In yet other embodiments, adjunctive use of the imino sugar of the invention may be reflected in the content of the information and/or instructions co-packaged with the sugar relating to formulation and/or posology.

The term alkaloid is used herein sensu stricto to define any basic, organic, nitrogenous compound which occurs naturally in an organism. However, it should be noted that the term alkaloid is also used herein sensu lato to define a broader grouping of compounds which include not only the naturally-occurring alkaloids, but also their synthetic and semi-synthetic analogues and derivatives. Thus, as used herein, the term alkaloid covers not only naturally-occuring basic, organic, nitrogenous compounds but also derivatives and analogues thereof which are not naturally occurring (and which may not be basic).

Most known alkaloids are phytochemicals, present as secondary metabolites in plant tissues (where they may play a role in defence), but some occur as secondary metabolites in the tissues of animals, microorganisms and fungi. There is growing evidence that the standard techniques for screening microbial cultures are inappropriate for detecting many classes of alkaloids (particularly highly polar alkaloids, see below) and that microbes (including bacteria and fungi, particularly the filamentous representatives) will prove to be an important source of alkaloids as screening techniques become more sophisticated. Structurally, alkaloids exhibit great diversity. Many alkaloids are small molecules, with molecular weights below 250 Daltons. The skeletons may be derived from amino acids, though some are derived from other groups (such as steroids). Others can be considered as sugar analogues. It is becoming apparent (see Watson et al. (2001) Phytochemistry 56: 265-295) that the water soluble fractions of medicinal plants and microbial cultures contain many interesting novel polar alkaloids, including many carbohydrate analogues. Such analogues include a rapidly growing number of polyhydroxylated alkaloids.

The term isolated as applied to the imino sugars of the invention is used herein to indicate that the sugar exists in a physical milieu distinct from that in which it occurs in nature or in a purified form. For example, the isolated material may be substantially isolated (for example purified) with respect to the complex cellular milieu in which it naturally occurs. When the imino sugar is purified, 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 or higher. In some circumstances, the isolated sugar may form part of a composition (for example a more or less crude extract containing many other substances) or buffer system, which may for example contain other components. In other circumstances, the isolated imino sugar may be purified to essential homogeneity, for example as determined spectrophotometrically, by NMR or by chromatography (for example GC-MS). The term neutropenia is used herein to define a condition characterized by a circulating neutrophil count of less than about 2000 per mm³ (for example less than about 1500 per mm³). Grade 1 neutropenia in humans is the lower limit of normal to 1500 neutrophils per mm³, less than 1500 to 1000 neutrophils per mm³ is grade 2 neutropenia, about 1000-500 neutrophils per mm³ is grade 3 neutropenia and less than about 500 neutrophils per mm³ is considered to be grade 4 neutropenia. The term is also used herein to indicate a condition characterized by loss (or reduction) of neutrophil function with no attendant reduction in circulating neutrophil count.

The term pharmaceutically acceptable derivative as applied to the imino sugars of the invention define sugars which are obtained (or obtainable) by chemical derivatization of the parent imino sugars of the invention. The pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with mammalian tissues without undue toxicity, irritation or allergic response (i.e. commensurate with a reasonable benefit/risk ratio). Preferred derivatives are those obtained (or obtainable) by alkylation, esterification or acylation of the parent imino sugars of the invention. The derivatives may be active per se, or may be inactive until processed in vivo. In the latter case, the derivatives of the invention act as pro-drugs. Particularly preferred pro-drugs are ester derivatives which are esterified at one or more of the free hydroxyls and which are activated by hydrolysis in vivo. The pharmaceutically acceptable derivatives of the invention retain some or all of the activity of the parent sugar. In some cases, the activity is increased by derivatization. Derivatization may also augment other biological activities of the sugar, for example bioavailability.

The term pharmaceutically acceptable salt as applied to the imino sugars of the invention defines any non-toxic organic or inorganic acid addition salt of the free base sugar which are suitable for use in contact with mammalian tissues without undue toxicity, irritation, allergic response and which are commensurate with a reasonable benefit/risk ratio. Suitable pharmaceutically acceptable salts are well known in the art. Examples are the salts with inorganic acids (for example hydrochloric, hydrobromic, sulphuric and phosphoric acids), organic carboxylic acids (for example acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and mandelic acid) and organic sulfonic acids (for example methanesulfonic acid and p-toluenesulfonic acid). The imino sugars of the invention may also be converted into salts by reaction with an alkali metal halide, for example sodium chloride, sodium iodide or lithium iodide. Preferably, the imino sugars of the invention are converted into their salts by reaction with a stoichiometric amount of sodium chloride in the presence of a solvent such as acetone.

The term indicated is a term of art used herein in relation to a disease, condition, subject or patient population to convey the clinical desirability or necessity of a particular intervention in relation to that disease, condition, subject or patient population. This might be the case, for example, where intervention would be palliative, preventative or (at least partially) curative.

These salts and the free base sugars can exist in either a hydrated or a substantially anhydrous form. Crystalline forms of the sugars of the invention are also contemplated and in general the acid addition salts of the imino sugars of the invention are crystalline materials which are soluble in water and various hydrophilic organic solvents and which in comparison to their free base forms, demonstrate higher melting points and an increased solubility.

In its broadest aspect, the present invention contemplates all optical isomers, racemic forms and diastereomers of the imino sugars of the invention. Those skilled in the art will appreciate that, owing to the asymmetrically substituted carbon atoms present in the sugars of the invention, they may exist and be synthesised and/or isolated in optically active and racemic forms. Thus, references to the imino sugars of the present invention encompass the imino sugars as a mixture of diastereomers, as individual diastereomers, as a mixture of enantiomers as well as in the form of individual enantiomers.

Therefore, the present invention contemplates all optical isomers and racemic forms thereof of the sugars of the invention, and unless indicated otherwise (e.g. by use of dash-wedge structural formulae) the sugars shown herein are intended to encompass all possible optical isomers of the sugars so depicted. In cases where the stereochemical form of the sugar is important for pharmaceutical utility, the invention contemplates use of an isolated eutomer.

Imino sugars for use according to the invention

Any imino sugar may be used according to the invention, providing that it can mediate PRR (for example C-type lectin) immunotherapy or immunoprophylaxis.

Imino sugars for use according to the invention may be readily identified by screening assays which detect: (a) binding to a PRR (for example C-type lectin); and/or (b) the stimulation of PRR (for example C-type lectin) signalling. In the latter case, assays for PRR (for example C-type lectin) signalling activity may involve the use of PRR (for example C-type lectin)-bearing immune cells (typically DCs) as test reagent. Those skilled in the art will readily be able to identify appropriate conditions and formats for such assays, including inter alia the nature and number of the dendritic cells, the relative concentrations of imino sugar and cells, the duration of stimulation with imino sugar and the methods used to detect signalling (for example by immunoassay for cytokine release). The imino sugar may be an alkaloid. Particularly preferred are polyhydroxylated alkaloids. Preferred are imino sugars having a small molecular weight, since these may exhibit desirable pharmacokinetics. Thus, the imino sugar may have a molecular weight of 100 to 400 Daltons, preferably 150 to 300 Daltons and most preferably 200 to 250 Daltons. Also preferred are non- metabolizable imino sugars. Such sugars may exhibit extended tissue residence durations, and so exhibit favourable pharmacokinetics.

In a preferred embodiment, the imino sugar has the formula:

wherein R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof.

Particularly preferred are imino sugars having the formula:

In a particularly preferred embodiment the imino sugar is 1 R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)- 1 ,2,6,7-tetrahydroxypyrrolizidine (casuarine), wherein R is hydrogen and having the formula:

or a pharmaceutically acceptable salt or derivative thereof.

The imino sugar may also be a casuarine glycoside, or a pharmaceutically acceptable salt or derivative thereof. In such embodiments, the imino sugar is preferably casuarine-6-α-D-glucoside of the formula:

or a pharmaceutically acceptable salt or derivative thereof.

Other suitable imino sugars for use according to the invention are selected from:

(a) 3,7-diepi-casuarine;

(b) 7-epi-casuarine;

(c) 3,6,7-triepi-casuarine;

(d) 6,7-diepi-casuarine;

(e) 3-epi-casuarine;

(f) 3,7-diepi-casuarine-6-α-D-glucoside;

(g) 7-epi-casuarine-6-α-D-glucoside;

(h) 3,6,7-triepi-casuarine-6-α-D-glucoside; (i) 6,7-diepi-casuarine-6-α-D-glucoside; and (j) S-epi-casuarine-δ-α-D-glucoside, or a pharmaceutically acceptable salt or derivative thereof.

In another preferred embodiment the imino sugar has the formula:

In such embodiments, most preferred are imino sugars having the formula:

Examples of such preferred imino sugars include N-hydroxyethylDMDP having the formula:

or a pharmaceutically acceptable salt or derivative thereof.

In another embodiment, the imino sugar has the formula:

wherein R¹ is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups and R² is selected from hydrogen, hydroxy and alkoxy, or a pharmaceutically acceptable salt or derivative thereof.

In such embodiments, the imino sugar preferably has the formula:

In such embodiments, R¹ may be a saccharide moiety (for example a glucoside or arabinoside moiety).

In another embodiment, the imino sugar has the formula:

or a pharmaceutically acceptable salt or derivative thereof.

In such embodiments, the imino sugar is preferably 2-hydroxy-1 ,2-cis-castanospermine having the formula:

or a pharmaceutically acceptable salt or derivative thereof.

Alternatively, the imino sugar may be 2-hydroxy-1,2-trans-castanospermine having the formula:

or a pharmaceutically acceptable salt or derivative thereof.

Particularly preferred are imino sugars selected from the table below (or stereochemical variants thereof):

Other suitable imino sugars for use according to the invention include the "ring contracted swainsonines" (see US5075457, the content of which relating to ring-contracted swainsonines is incorporated herein by reference).

For example, the imino sugar may be a ring-contracted swainsonine of the formula:

1S, 2R, 7R, 7aR)-1,2,7-trihydroxy(7)

or of the formula:

7S-epimer (8)

The L-forms of swainsonine and its ring-contracted derivatives may be particularly preferred.

In many embodiments of the invention, the imino sugar is isolated or purified. However, in some embodiments the use of an isolated or purified imino sugar is not required, and crude extracts suffice.

The imino sugars need not be naturally occurring, and may be synthetic analogues or derivatives of naturally occurring counterparts. Such analogues or derivatives are preferably pharmaceutically acceptable analogues, salts, isomers or derivatives as herein defined. However, preferred imino sugars are phytochemicals. Such phytochemicals may be isolated from natural sources or synthesised in vitro. Particularly preferred are imino sugars selected from the following classes:

(a) piperidine imino sugars;

(b) pyrroline imino sugars; (c) pyrrolidine imino sugars;

(d) pyrrolidine imino sugars;

(e) indolizidine imino sugars;

(f) nortropane imino sugars.

However, imino sugar mixtures containing two or more different imino sugars representative of one or more of the classes listed above may also be used.

PRR-ligands Preferred imino sugars are PRR (for example C-type lectin) ligands (as defined herein). Such ligands may be identified by assays for PRR (for example C-type lectin) binding. These may involve competitive binding assays using an isolated PRR (for example C-type lectin) and a known cognate PAMP ligand as test reagents. Such competitive binding assays are routine in the art, and those skilled in the art will readily be able to identify appropriate conditions and formats for such assays.

The ligands of the invention may bind any PRR (for example C-type lectin), including any of the lectins described in Figdor et al. (2002) Nature Reviews Immunology 2: 77-84 (the disclosure of which relating to the identification of various C-type ligands being incorporated herein by reference). Preferably, the imino sugars of the invention bind to PRRs/lectins displayed on DCs, though they may bind to PRRs/lectins on other cells including other cells of the innate immune system as well as to PRRs/lectins on, for example, macrophages and T-cells.

Thus, the imino sugars of the invention may be ligands for type I and/or type Il C-type lectins. In particular, the imino sugars of the invention may be ligands for C-type lectins selected from: (a) MMR (CD206, macrophage mannose receptor); and/or

(b) DEC-205; and/or

(c) Dectin 1 ; and/or

(d) Dectin 2; and/or

(e) Langerin; and/or (f) DC-SIGN; and/or

(g) BDCA-2; and/or (h) DCIR; and/or

(i) DLEC; and/or 0) CLEC; and/or (k) a rhamnose-binding C-type lectin. Mannose and/or rhamnose analogues

The imino sugars may also act as sugar (for example mannose and/or rhamnose) analogues. Such analogues are imino sugars which share some or all of the binding properties of mannose and/or rhamnose in vivo (without necessarily sharing all of the attendant functional properties thereof).

Such sugar analogues may be identified by assays for saccharase (e.g. mannosidase and/or rhamnosidase) inhibitory activity. Such enzyme assays are routine in the art, and those skilled in the art will readily be able to identify appropriate conditions and formats for such assays.

Thus, preferred rhamnose analogues for use according to the invention are imino sugars which exhibit inhibitory activity against one or more rhamnosidase enzyme(s). Similarly, preferred mannose analogues for use according to the invention are imino sugars which exhibit inhibitory activity against one or more mannosidase enzyme(s).

In yet other embodiments, preferred imino sugars may be rhamnose analogues which bind to the rhamnose receptor PRR (see Grillon, Monsigny and Kieda (1990) Glycobiology 1(1): 33-8). Such binding perse need not necessarily trigger the rhamnose receptor-mediated signalling pathway (i.e. initiate the cellular signalling cascade in which the rhamnose receptor forms a part): other co- stimulatory events may be required. Moreover, the binding may occur in the context of some other aspect of cellular physiology. In the latter case, the imino sugars may act as ligands as hereinbefore defined and may for example bind rhamnose receptor at the cell surface without triggering a signalling cascade, in which case the binding may effect other aspects of cell function. Thus, the rhamnose analogues of the invention may bind to the rhamnose receptor and thereby effect an increase in the concentration of functional rhamnose receptor at the cell surface (for example mediated via an increase in receptor stability, absolute receptor numbers and/or receptor activity). Alternatively, the rhamnose analogues may bind rhamnose receptors (or rhamnose receptor precursors) intracellularly, in which case they may act as molecular chaperones to increase the expression of active PRR.

Similarly, other preferred imino sugars may be mannose analogues which bind to the mannose receptor PRR (as described infra). Again, such binding perse need not necessarily trigger the mannose receptor-mediated signalling pathway (i.e. initiate the cellular signalling cascade in which the mannose receptor forms a part): other co-stimulatory events may be required. Moreover, the binding may occur in the context of some other aspect of cellular physiology. In the latter case, the imino sugars may act as ligands as hereinbefore defined and may for example bind mannose receptor at the cell surface without triggering a signalling cascade, in which case the binding may effect other aspects of cell function. Thus, the mannose analogues of the invention may bind to the mannose receptor and thereby effect an increase in the concentration of functional mannose receptor at the cell surface (for example mediated via an increase in receptor stability, absolute receptor numbers and/or receptor activity). Alternatively, the mannose analogues may bind mannose receptors (or mannose receptor precursors) intracellularly, in which case they may act as molecular chaperones to increase the expression of active PRR.

Medical applications of the invention

The invention finds application in various forms of immunotherapy and immunoprophylaxis, and may therefore be embodied in various methods of treatment and in various pharmaceutical compositions.

The invention finds particular application in immune response variegation. The term "immune response variegation" is a term of art, as explained in the Background to the Invention (above). The invention may therefore find application in the modulation of the relative contributions of the various components of the innate and adaptive immune systems (either in absolute terms or in the extent of their temporal and spatial activity). In particular, the invention finds application in immune response variegation characterized by the modulation of the relative activities of the T_H1 -, T_H2- and/or regulatory T-cell compartments.

Thus, the invention finds application in the treatment of pathological variegated states. Such states may arise from immune dysfunction or pathogen-mediated immune system subversion (discussed below). Immune response variegation effected according to the invention is often indicated in chronic diseases states, particularly chronic infections (which typically reflect an inappropriate, undesirable or pathological immune response variegation).

The invention therefore finds broad application in the treatment or prevention of viral infection; the treatment or prevention of bacterial infection; the treatment or prevention of protozoal infection; the treatment or prevention of fungal infection; the treatment or prevention of prion infection; the treatment or prevention of metazoan (e.g. helminth) infection or infestation ; the treatment or prevention of proliferative disorders (for example cancer) and the treatment or prevention of autoimmune disorders. Particularly preferred is the treatment or prevention of chronic viral, bacterial, protozoal, fungal, prion or metazoan (e.g. helminth) infections or infestations.

In particular, the invention contemplates the treatment of states, diseases or disorders characterized by the presence of a pathological (or undesirable or inappropriate) immune response variegation. Specific examples of such states, diseases or disorders are discussed below:

Pathogen-mediated immune system subversion

Certain microbial compounds produced during infection can arrest DCs in their immature state and/or instruct (or prime) them on maturation to initiate an immune response that is polarized towards a T_REG type. The resultant immunological tolerance benefits the pathogen and can permit chronic infection. Many eukaryotic pathogens (including protozoa, helminths, fungi and ectoparasites) can act in this way.

Examples of pathogens which subvert the host immune response by arresting DCs in the immature state or otherwise inhibiting DC activation or function include Plasmodium falciparum (by binding to CD36 and CD52), Mycobacterium spp. (which can bind to DC-SIGN via mannosylated lipoarabinomannan), hepatitis C virus (HCV), herpes simplex virus (HSV), cytomegalovirus, Bacillus anthracis, lymphocytic choriomeningitis virus (LCMV), lymphocytic choriomeningitis virus (LCMV) and lymphocytic choriomeningitis virus (LCMV). Examples of pathogens which instruct or prime DCs to initiate an immune response that is polarized towards a T_REG type include Bordatella pertussis and Schistosoma mansoni.

The invention may therefore find application in the treatment of chronic infection or infestation caused by any of the foregoing pathogens.

Certain pathogens may also induce neutropenia. Neutrophils are short lived, professional phagocytic cells which are important in host resistance to microbial invasion. They play a protective role in challenges by Candida albicans, Salmonella enteric subspecies, Tyhipimurium, Yersinia enterocolitica, Chlymadia trachomatis and Toxoplasma gondii. Some of these are known to have rhamnose as a key cell wall component. Neutropenia - depleted levels of circulating neutrophils - is a risk associated with the above mentioned infections. Evidence has shown that impaired protective acquired immunity is correlated to neutropenia, hence the importance of neutrophil presence during immune responses. During infection with Toxoplasma gondii, for example, neutrophil depletion leads to impaired immunity and lethal systemic pathology (Bennouna et al., 2003).

Thus, the invention finds application in the treatment or prophylaxis of pathogen-mediated neutropenia, for example in the treatment or prophylaxis of the clinical sequelae of infection with a pathogen selected from Candida albicans, Salmonella enteric subspecies, Tyhipimurium, Yersinia enterocolitica, Chlymadia trachomatis and Toxoplasma gondii.

Allergy

Allergic diseases (including allergic rhinitis, asthma and atopic dermatitis) have diverse clinical features but exhibit common immunological characteristics. Patients have an undesirable immune response variegation characterized by elevated allergen-specific T_H2 cells and allergen-specific IgE.

The invention finds application in the treatment of such allergic diseases and disorders by effecting a rebalancing or remodelling of the undesirable immune response variegation associated with allergy by dampening the T_H2 component of the immune response (for example via stimulation of the T_H1 and/or T_REG components). Autoimmunity lntrathymic deletion of developing T cells with high avidity for self-antigens restricts the repertoire of peripheral autoreactive T cells, but this process does not proceed to completion. This results in the presence of lymphocytes with avidity for self-antigens in the periphery. While it is thought that the resulting overlap in the recognition of self and non-self is necessary for effective immunity, the phenomenon creates the potential for autoimmune disease. In such diseases, lymphocytes with avidity for self-antigens mediate pathological immune responses (typically inflammation and tissue damage).

Autoimmune disease, which affects approximately 5% of the population (a disproportionate number being women), comprises a heterogeneous group of poorly understood disorders including multiple sclerosis (MS), rheumatoid arthritis, early-onset diabetes, thyroid diseases, Grave's disease, psoriasis, systemic lupus erythematosus, scleroderma, ankylosing spondilitis and myasthenia gravis.

The invention finds broad application in the treatment of autoimmune diseases and disorders by effecting a rebalancing or remodelling of the undesirable immune response variegation associated with autoimmunity by stimulating an autoreactive T_REG component.

The invention also finds application in the remodelling of the immune response variegation in patients undergoing immunosuppressive therapies (or therapies in which immunosuppression is a side effect), in patients suffering from Graft-versus-host disease and as an adjunctive therapy in transplantation. It may also find application in polarizing vaccine-induced immune responses, either as an adjunct to vaccination or as part of an adjuvant composition.

Inhibition of neutrophil apoptosis

The invention may be used for inhibiting or postponing neutrophil apoptosis, the treatment or prophylaxis of a disease or disorder in which the inhibition or postponement of neutrophil apoptosis is indicated or the treatment or prophylaxis of a disease or disorder characterized by excessive neutrophil apoptosis.

Neutrophils (PMN) are key components of the inflammatory response and as such contribute to the killing of bacteria, fungi and parasites. In addition, recent evidence suggests their involvement in the development of the immune response. The cells secrete cytokines (IL-12) and chemokines (CCL3, CCL4, CCL5 (Rantes), and CCL20 (MIP-3α) that display potent chemotactic activity for immature bone marrow-derived dendritic cells (DC) (Bennouna et a/., 2003). Parasite (Toxoplasma gondii) - stimulated PMN also release soluble factors that trigger DC activation, as measured by IL-12 (p40) and TNF-α production as well as up-regulation of co-stimulatory molecules CD40 and CD86. This recruitment and activation of DCs leads in turn to Th1 cell activation and effective immune responses to infection including stimulation of IFN-g. Thus, neutrophils are now recognized as important decision shapers during the early phases of immune responses. Neutrophil depletion in vivo leads to defective splenic DC cytokine responses to infection. DC activation is driven at least in part by PMN-derived TNF-α. The receptors on neutrophils responding to T. gondii and causing IL-12 and cytokine release are not proven. It is notable that casuarines also promote production of IL-12, increased CD40 and CD86 expression and resulting in increased IFN-g by splenocytes (McGowan, 2006).

α-L-Rhamnosyl- receptors have so far only been reported on human neutrophils (Grillon et a/., 1990). They appear by far the major C-type lectin receptors on these cells. These receptors are numerous with 55,000 per cell and their affinity reached 2 x 10⁸. Their number increased in response to GM-CSF, T cells and B cells. Approximately 60% of leukocytes produced in bone- marrow are neutrophils and 100 billion granulocytes are produced and enter the blood daily. Physiological cell death in neutrophils is caused by apoptosis. Studies have shown that neutrophil apoptosis can be delayed by over expression of GM-CSF and G-CSF (Simon, 2001). It is notable that GM-CSF is a lectin-like molecule. This suggests that lectin-sugar recognitions may be involved in the ability of cells to achieve proper defence responses. The amplification of the α-L-rhamnose receptor expression on granulocytes by mononuclear cells with GM-CSF suggests these receptors might be involved in one of the immunological adhesion functions of these cells. Rhamnose is a key component of the cell wall of many bacteria and as such rhamnosyl-fragments would indicate a bacterial infection. Rhamnose is not a mammalian sugar and as such only rhamnosyl- fragments from bacteria would be found in the blood.

Haemorestoration and haematopoetic recovery

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

Haemorestoration may be indicated following 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 imino sugars 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 imino sugar adjunctive to: (a) chemotherapy; and/or (b) radiotherapy; and/or (c) bone marrow transplantation; and/or (d) haemoablative immunotherapy. The therapies of the invention may also include bone-marrow reconstitution, haematopoietic recovery and/or engraftment. Such therapies may be adjunctive to cytotoxic/cytoablative therapy (e.g. radio- or chemotherapy) during cancer management.

Posology

The imino sugars 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 imino sugar administered can vary widely according to 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 imino sugar selected. Moreover, the imino sugars 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 effective amount of the imino sugar administered will generally range from about 0.01 mg/kg to 500 mg/kg daily. A unit dosage may contain from 0.05 to 500 mg of the imino sugar, and can be taken one or more times per day. The imino sugar can be administered with a pharmaceutical carrier using conventional dosage unit forms either orally, parenterally, or topically, as described below. The preferred route of administration is oral administration. In general a suitable dose will be in the range of 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 active ingredient per unit dosage form.

Formulation

The compositions of the invention comprise the imino sugar of the invention, optionally together with a pharmaceutically acceptable excipient. The imino sugar of the invention may take any form. It may be synthetic, purified or isolated from natural sources (for example from Casuarina equisetifolia or Eugenia jambolana), using techniques described in the art (and referenced infra). When isolated from a natural source, the imino sugar of the invention may be purified. However, the compositions of the invention may take the form of herbal medicines. Such herbal medicines preferably are analysed to determine whether they meet a standard specification prior to use. The herbal medicines for use according to the invention may be dried plant material. Alternatively, the herbal medicine may be processed plant material, the processing involving physical or chemical pre- processing, for example powdering, grinding, freezing, evaporation, filtration, pressing, spray drying, extrusion, supercritical solvent extraction and tincture production. In cases where the herbalmedicine is administered or sold in the form of a whole plant (or part thereof), the plant material may be dried prior to use. Any convenient form of drying may be used, including freeze- drying, spray drying or air-drying.

In embodiments where the imino sugar 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 cornstarch 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 composition 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 imino sugar of the invention, either alone or together with other plant material associated with the botanical source(s) (in the case of herbal medicine embodiments). The tablets may contain the imino sugar 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 imino sugar of the invention is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is 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 sugars 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 sugars of the invention may also be presented as liposome formulations. For oral administration the imino sugar 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 imino sugars 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 imino sugars of the invention may also be administered parenteral^, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally. In such embodiments, the imino sugar is 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 imino sugar 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 imino sugars 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 sugar from about 0.1 to about 10% w/v (weight per unit volume).

When used adjunctively, the imino sugars of the invention may be formulated for use with one or more other drug(s). In particular, the imino sugars of the invention may be used in combination with antitumor agents, antimicrobial agents, antiinflammatories, antiproliferative agents and/or other immunomodulatory (e.g. immunostimulatory) agents. For example, the imino sugars of the invention may be used with anti-viral and/or antiproliferative 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 imino sugar is 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 imino sugar 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 imino sugar with antitumour 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 : Binding of casuarine to the mannose receptor

The ability of casuarine to bind to the mannose receptor was determined using a competitive ELISA assay using mannose immobilized at 20 mol % on an ELISA plate with po!y[N-(2 hydroxyethyl) acrylamide] as target.

The ability of the test compounds to compete for binding to the target with a (CRD4-7)-Fc conjugate (the CRD4-7 moiety derived from the native macrophage mannose receptor) was determined using anti-Fc-HRP (horseradish peroxidase) as the detection reagent. Using mannose as a positive control, casuarine was shown to have a higher affinity than mannose for this mannose receptor complex. These results show that casuarine may bind to the mannose receptor in vivo. Thus, casuarine may effect PRR-mediated immunotherapy or immunoprophylaxis via binding to the mannose receptor (though it should be noted that PRR-mediated immunotherapy or immunoprophylaxis by casuarine may arise from its binding to other PRRs in vivo and such a mechanism is not excluded by this experiment).

Example 2: Binding of 3,7-diepicasuarine to the mannose receptor

The assay format described in Example 1 was also used to assess the ability of 3,7-diepicasuarine to bind to the mannose receptor. Again, using mannose as a positive control, 3,7-diepicasuarine was shown to have a higher affinity than mannose for this mannose receptor complex. These results show that 3,7-diepicasuarine may bind to the mannose receptor in vivo. Thus, 3,7- diepicasuarine may effect PRR-mediated immunotherapy or immunoprophylaxis via binding to the mannose receptor (though again it should be noted that PRR-mediated immunotherapy or immunoprophylaxis by 3,7-diepicasuarine may arise from its binding to other PRRs in vivo and such a mechanism is not excluded by this experiment).

Example 3: Rhamnose Receptors on Neutrophils

We show below that casuarine and 3,7-diepicasuarine inhibit apoptosis in murine splenocyte cultures. These compounds also up-regulate CD40 (macrophages and DCs) and CD86 (DCs) expression with activity comparable to that of LPS, TNF-α and parasite products (schistosome) (McGowan, 2006). Like GM-CSF and G-CSF, the imino sugars casuarine and 3,7-diepicasuarine postpone neutrophil apoptosis via interactions with the α-L-rhamnose receptor. Interestingly the mannose analogue D- swainsonine has also been shown to stimulate bone marrow proliferation (Olden, et al, 1991). Mannose and rhamnose analogues have been shown to be enantiomeric in that D-swainsonine potently inhibits mannosidases and L-swainsonine inhibits rhamnosidase (Davis et al., 1996). It has also been shown that the two enantiomers of imino sugars can also bind to the same enzymes but with different binding characteristics (Bleriot ef a/., 2006). D-casuarine and the D-3,7-diepicasuarine closely resemble the rhamnose analogue L-swainsonine. The former 2 compounds promote IL-12 production by murine DCs and IFN-g production by murine splenocytes but L-swainsonine does not (McGowan, 2006). Without wishing to be bound by any theory, it is thought that the reason for this could be that the casuarines resemble rhamnosyl-fragments whereas L-swainsonine resembles L- rhamnose itself. Although rhamnose is not a mammalian sugar it will enter the blood via digestion of plant and microbial foods and hence should not promote an immune response by itself. Rhamnosyl- fragments should trigger an immune response because they can only enter the blood and other tissues via infections (cell wall fragments).

D-swainsonine (mannose analogue)

L-swainsonine (rhamnose analogue, Davis et al., 1996)

3,7-diepicasuarine (rhamnose analogue by analogy)

Here we confirm the presence of high affinity α-L-rhamnose receptors on human granulocytes and the inhibition of neutrophil apoptosis by casuarine and 3,7-diepicasuarine in splenocyte cultures. Methods

Granulocyte Isolation

Buffy coats from the blood of two healthy donors were obtained at the Medical School, University of Aberdeen. Granulocytes were separated from mononuclear cells and erthyrocytes according to the method of Grillon et al. (1990). The highly purified cells were determined by a fluorescence- activated cell sorter (FACS) analysis to be 95% neutrophils and 5% eosinophils.

Fluorescent neoglycoprotein cell labelling

The neoglycoproteins D-mannose-BSA; D-galactose-BSA; L-Rhamnose-BSA were bought from Dextra Limited, Reading, UK. They were labelled with AlexaFI488 (green FACS label) and Alexa FI647 (red FACS label) using protein labelling kits from Molecular Probes (bought from Invitrogen catalogue number A10235 and A20173). Labelling of the neoglycoconjugates was successful with an average of 4 Alexafluor molecules attached per sugar-BSA.

Flow Cytometry analysis

The granulocytes were incubated follwing the method of Grillon et al., 1990 with and without GM-

CSF (to increase rhamnose receptor levels) at 37oC for 30 mins. Then each of the three BSA-sugar-Alexa FI647 were added at 20 ul of

200 ug/ml. The concentration was the same as used by Grillon but Alexa Fl is a much brighter dye. The cells were incubated at 37⁰C for 40 mins and then a fresh aliquot of neoglycan was added and the cells incubated at 4⁰C for a further 30 mins.

Murine Splenocyte Cultures

The spleen from an 8-10 week old BALB/c mouse was removed aseptically and placed in a Petri dish containing 5ml of medium. Cell suspensions were prepared by grinding the spleen against a sterile wire mesh (size 0.5mm). The cells were then centrifuged for 5 mins at lOOOrpm. Boyles Solution was added to remove the erthyrocytes and centrifuged for 5 mins. A further two washes in complete medium were carried out and the pellet re-suspended and a cell count performed and incubated for 24-72 hours at 37⁰C at 5% CO₂. Splenocytes were stimulated using LPS (Salmonella abortus) and anti-CD3 (clone C363, Southern Biotech. Associates, USA). At 1ug and 0.5ug/ml respectively. All experiments were in 12 well plates with 1 x 10⁶ cells/well and incubated with agents for 15 hours. Neutrophil antibody 1A8 (551460 BDBiosciences) and CD45R (553088 BDBiosciences) staining of spleen cells used the protocol described by McGowan (2006). Cells were then prepared for FACS analysis using standard methods of 2ml phorbol myristate 50ng/ml, ionomycin 500ng/ml and Brefeldin A at 1 ug/ml. After 4 hours the cells were cooled on ice and washed with 0.5% BSA/2mM EDTA/PBS before resuspending in 100ul anti-FcR. FACS analysis was conducted on a FACSCanto^tm Flowcytometer using FACSDiva^tm software. A neutrophil specific marker (ly-6G0 was used to measure neutrophil numbers. Results

Each neoglycoconjugate was tested in duplicate and FACS results were relative to each other. GaI- BSA served as a "negative control"- showing background non-specific binding. In this case, rhamnose-BSA labelling was much more intense (with a good shift to the right of the fluorescent log scale - by 2 logs). The presence of casuarine and 3,7-diepicasuarine greatly increased the numbers of neutrophils present in the splenocyte cultures both with and without anti-CD3 stimulation. The results are shown below. Data are representative of three independent experiments.

Figure 1 shows percentage increase in neutrophil expression from Spleen cells in the presence of casuarine (C) and 3,7-diepicasuarine (DEC) at both 5 and 1μg/ml.

Figure 2 shows percentage increase in the neutrophil population from spleen cells in the presence of anti-CD3 and anti-CD3 with either casuarine (C) (5 & 1μg/ml) or 3,7-diepicasuarine (DEC) (5 & 1μg/ml).

Discussion

We have confirmed the presence of α-L-rhamnose receptors on human neutrophils as the major C- type lectin receptor. We have also shown that imino sugar α-L-rhamnose analogues can increase the numbers of neutrophils present in murine splenocyte cultures. We have previously shown that an increase in anti-CD3 stimulated IFN-g is shown by splenocytes both in vitro and ex vivo in the presence of the imino sugars casuarine and 3,7-diepicasuarine. The conclusion is that an inhibition of neutrophil apoptosis (shown in vitro here) has occurred as a result of activation of the cells by the imino sugars casuarine and 3,7-diepicasuarine. The boosting of T cell IFN-g response has also been shown to occur as a result of interactions between neutrophils and Toxoplasma gondii by an unknown neutrophil receptor (Bennouna ef a/., 2003). Without wishing to be bound by any theory, the imino sugars may bind to the α-L-rhamnose receptor and cause activation of the neutrophils. This activation is shown by inhibition of apoptosis and the priming of other immune cells as has been shown to also occur as a result of neutrophil activation by parasites.

Neutrophils are short lived, professional phagocytic cells which are important in host resistance to microbial invasion. They play a protective role in challenges by Candida albicans, Salmonella enteric subspecies, Tyhipimurium, Yersinia enterocolitica, Chlymadia trachomatis and Toxoplasma gondii. Some of these are known to have rhamnose as a key cell wall component. Neutropenia - depleted levels of circulating neutrophils - is a risk associated with the above mentioned infections. Evidence has shown that impaired protective acquired immunity is correlated to neutropenia, hence the importance of neutrophil presence during immune responses. During infection with Toxoplasma gondii, for example, neutrophil depletion leads to impaired immunity and lethal systemic pathology (Bennouna ef a/., 2003).

Severe neutropenia occurs following high-dose chemotherapy in cancer patients, and G-CSF is commonly used to decrease the period of severe neutropenia. Imino sugars such as casuarine and 3,7-diepicasuarine could have therapeutic potential by promoting neutrophil survival to reduce severe neutropenia in chemotherapy-treated cancer patients.

Example 4: Micro-Calorimetric Responses of Murine Macrophages to Imino Sugar Analogues of Rhamnose and Mannose

Micro-calorimetry provides a sensitive method for studying the metabolic effects of chemicals added to the medium of cells in culture (Loike et a/., 1981). Application of the method here will allow us to determine the speed of responses to sugar analogues and whether surface receptors are involved. J774.E macrophages are activated (as measured by micro-calorimetry) within seconds by the sugar analogues D-casuarine, D-swainsonine and D-3,7-diepicasuarine. Subsequent addition of compounds gives immediate stepped increases in heat production. Subsequent addition of 6ug/ml of LPS gave no further heat response over an hour. The speed of the response suggests that a surface sugar receptor is involved: glycan changes by glycosidase inhibition affecting the receptors cannot happen in seconds. Culturing J774.E cells with D-swainsonine (50ug/ml), D-casuarine (50 and 1ug/ml) and D-3,7-diepicasuarine (1ug/ml) for 24 hours increases the binding of mannose receptor antibody (FITC) as measured by flow cytometry.

D-Swainsonine, a mannose analogue, has been widely reported to have anti-cancer activity through immune modulation (Olden et al., 1991). The immune modulation, however, has always been ascribed to inhibition of Golgi Mannosidase Il and alterations this causes to surface glycoproteins activating immune cells (Watson et al., 2001). The potent inhibition of mannosidase is also the cause of the toxicity of D-swainsonine through accumulation in the lysosome and inhibition of lysosomal acidic mannosidase. D-Swainsonine and D-castanospermine, a glucose analogue, have also been reported to block mannose receptors through their endogenous production of high mannose terminating glycans (Chung et al., 1984).

D-Swainsonine has been reported to activate peritoneal macrophages and to increase tumouricidal activity (Das ef al., 1995). Using a micro-calorimeter we were able to carry out assays on murine macrophage cell line cultures to determine if D-swainsonine activate the cells before glycan changes could occur. If activation was immediate then glycan changes due to mannosidase inhibition are unlikely to be involved; such glycan changes would require at least one hour or longer. We also wanted to determine if D-casuarine (Nash et al., 1984) and D-3,7-diepicasuarine (both analogous to ring contracted L-swainsonine, a rhamnose analogue) could also activate murine macrophages immediately. D-Casuarine and D-3,7-diepicasuarine have been shown to be promoters of IL-12 (in vitro with sub-optimal LPS in murine macrophages and dendritic cell cultures) (PCT/GB2004/000198).

Studies have shown that the extent of activation by D-swainsonine of peritoneal macrophages to cytotoxicity against tumour cells is comparable to that obtained with known macrophage-activating agents such as interferon and bacterial lipopolysaccharide (Das ef al. 1995; Grzegorzewski ef a/., 1989). D-Swainsonine can also induce tumouricidal activity in resident tissue-specific macrophages of both the lung and spleen, with activation being both time- and dose-dependent (Mohla et al., 1990). This is relevant to the clinical management of metastatic diseases since visceral organs are common sites for metastasis formation.

The purpose of this experiment was to show immediate responses by murine macrophages to D- swainsonine, D-casuarine and D-3,7-diepicasuarine. The three compounds are sugar analogues (and as such are likely to interact with sugar receptors known to occur on the surface of cells of the immune system. We hypothesized that mannose receptors should bind D-swainsonine and rhamnose receptors should bind D-casuarine and D-3,7-diepicasuarine. Mannose receptors are widely distributed but rhamnosyl- receptors have only been reported on neutrophils (Grillon ef a/., 1990). We have recently confirmed their presence on human neutrophils following the Grillon method (Nash, R.J., Wong, S. et al. in preparation). Rhamnose is a key component of the cell wall of many bacteria and as such rhamnosyl-fragments would indicate a bacterial infection; neutrophils and macrophages being the first line of defense. Rhamnose is not a mammalian sugar and as such only rhamnosyl- fragments from bacteria would be found in the blood. It is probable that macrophages will also have rhamnose receptors. Casuarine only differs in hydroxyls from the rhamnose analogue L-swainsonine at C-7. Since both casuarine and 3,7-diepicasuarine are potent immune modulators (stimulating IL-12 production by macrophages and dendritic cells) the orientation of the 7-hydroxyl seems unimportant. Changing the orientation of the hydroxyl at C-6, or removing this hydroxyl, removes promotion of IL-12.

D-swainsonine (mannose analogue)

L-swainsonine (rhamnose analogue, Davis et al., 1996)

3,7-diepicasuarine (rhamnose analogue by analogy)

We chose to conduct the micro-calorimetry experiment using the macrophage cell line J774.E that is known to express C-type lectin receptors. This cell line has been shown to expresses the mannose receptor that we predicted should respond to D-swainsonine. Nothing is known about other C-type lectins expressed on this cell line but it is likely to express other receptors as well. A follow-on experiment measured the binding of a mannose receptor anti-body following incubation of cells in vessels with the compounds for 24 hours. The expectation was that activation of the cells would increase expression of several receptors including the mannose receptor.

Methods

Micro-calorimetry The microcalorimeter was an LKB model 10700-1. The J774.E cell line was provided by Professor Siamon Gordon of the Pathology Department, University of Oxford. Cells were grown in GIBCO IMDWl medium with L-glutamine and 25mM HEPES. The medium contained 10% GIBCO heat inactivated fetal bovine serum and 2% GIBCO Penecillin-streptomycin (10,000 units/ml). Stock cell cultures were maintained in vent capped treated/non-pyrogenic 75ml culture flasks (Corning 430641). Cell counts were conducted using Marienfeld Fuchs Rosenthal slides (0.0625mm²). Trypan blue (0.4% in PBS) was used to determine viability. PBS was GIBCO phosphate buffered saline pH 7.2. Data from the micro-calorimeter was captured and analysed using BioXpert software. The sugar analogues were pumped into the micro-calorimeter vessels containing the cells using an Oroboros titration micro-pump made by Grinzens, Austria to give final vessel concentrations of 1- 200ug/ml. The incubator was set at 37°C and 5% CO₂.

For the micro-calorimetry the cells grown to 75% confluence on glass 2cm diameter coverslips in 5cm diameter Petri-dishes. The medium in the Petri-dishes was replaced 24 hours before the micro- calorimetry. The cover slips were directly placed into the calorimeter in 5ml of fresh warm medium. Compounds were added in a minimum volume of warm medium once the temperature in the vessel was stabilised. A turbine ran continually in the vessel to ensure rapid mixing once compound was added. The change in temperature was measured over 2 hours and additional compound added at various times. Once the micro-calorimeter measurements were made the cover slips were scraped into the 5ml of medium within the vessels using plastic cell scrapers. The number of cells in the 5ml was calculated to determine the number of cells that had been in the micro-calorimeter. The numbers of cells were always between 0.8 and 1.5 million.

Flow Cytometry All flow cytometric analyses were performed using a Coulter Epics Elite Flow Cytometer (Beckman- Coulter UK) using an argon ion laser with excitation at 488nm. The flow cytometer was set up as described in the manufacturer's manual and a logarithmic gain was used in all cases.

Phosphate buffered saline (GIBCO sterile PBS, pH 7.2 -CaCI2, - MgCI2 ref. 20012-019) was used as sheath fluid. Signals representing the forward scatter, side scatter and fluorescence at 525 nm were collected in list mode for each particle analysed. Off-line data analysis was performed using FCS express (De Novo Software, Ontario, Canada). Approximately 2 million cells were grown in 10ml of medium in 25ml treated non-pyrogenic plastic culture vessels (Corning 430639) to which compounds D-casuarine, D-3,7-diepicasuarine or D-swainsonine were added at 50ug/ml or 1ug/ml and cultured for 24 hours prior to washing in warm medium without calf serum and then adding

Serotec rat anti-mouse mannose receptor antibody FITC (MR5D3) 0.5mg/ml made up to suggested working dilution [1/10] and 50OuI added per culture vessel and incubated for 30 minutes prior to washing in cold PBS and removal from the vessels, centrifugation and storage in 1ml of PBS in ice before immediate measuring by flow cytometry. Results

Casuarine at 5ug/ml gave a rapid heat response from J774.E cells starting within seconds and reaching an estimated 4-5 fold over basal heat output within 30 minutes. Subsequent elevations of 5ug/ml gave an immediate elevation of heat production several times. Swainsonine and 3,7- diepicasuarine gave a similar response. The activation of the cells was consistent over several repeat studies of each compound. A threshold of 2.5ug/ml appeared to be required before activation occurred. Once activation had started it appeared to become self-perpetuating and reached near maximum heat production. However, further smaller steps in heat production could be achieved by further additions. Further addition of LPS (E. coli Sigma L-3137) at 5ug/ml did not further increase the heat production after addition of the sugar analogues. LPS was not run by itself. Heat production per cell was calculated as being 135.5 pW per cell (untriggered macrophage hybridoma cells gave 32 pW per cell after 24 hours, Richard Kemp, pers. comm.).

Flow Cytometry With the J774.E cells control there was one main fluorescence population with medium fluorescence (MF) and a smaller LF population. All treatments (3,7-diepicasuarine 1ug/ml, swainsonine 50ug/ml, casuarine at 50 and 1ug/ml) caused an increase in fluorescence. 3,7-diepicasaurine and casuarine at 1ug/ml cause a more marked increase with a second HF population emerging.

It appears, therefore, that imino sugars that are both mannose and rhamnose analogues can activate murine macrophages J774.E and lead to an increase in receptors binding the mannose receptor anti-body. We have also shown that murine bone marrow-derived macrophages from BaIb- C mice show increased expression of the receptor CD40 in response to casuarine and 3,7- diepicasuarine: over a 100% percent increase in CD40 expression on murine bone marrow-derived macrophages in the presence of casuarine and 3,7-diepicasuaime at 10μg/ml has been detected (data not shown). CD40 is a crucial membrane protein found on the surface of B lymphocytes, dendritic cells, follicular dendritic cells and macrophages. Cross-linking of CD40 to its ligand CD40-L induces B cell proliferation, cytokine production from the cell possessing CD40 and also amplification of T cell responses.

Discussion

The micro-calorimetry shows that sugar analogues (imino sugars) such as D-swainsonine, D- casuarine and D-3,7-diepicasuarine can immediately activate immune cells. The murine macrophage cell line J774.E responded to the.imino sugars by increasing metabolic activity. The incubation of the activated macrophages for 24 hours resulted in the increase in expression of mannose receptors on the cell surface (as measured by anti-body binding). Increased metabolic activity of macrophages and other immune cells is associated with increased immune function. It is likely that the increased tumouricidal activity of macrophages reported by Das et a/., (1995) in the presence of swainsonine is due to the increased metabolic activity. The activation is clearly not due to the ability of swainsonine to inhibit mannosidases and change endogenous glycans as assumed by previous publications (reviewed by Olden et al., 1991). 3,7-diepicasuarine does not inhibit any glycosidase (see table below) and yet gives a similar response to D-swainsonine. The interaction of D-swainsonine with mannose receptors is most likely given the potent mannosidase inhibition shown by the compound. 3,7-diepicasuarine and casuarine itself more closely resemble L- swainsonine and L-rhamnose. Rhamnose receptors occur on neutrophils and are likely to be involved in detection of microbial cell wall carbohydrates (which include rhamnose) (Grillon et al., 1990; Nathan 2006). The same receptors are likely to be found on macrophages as they also respond to bacterial infections. Rhamnose receptors have been shown to be increased by GM-CSF (but not by phorbol myristate acetate (a protein kinase C activator) and by incubation of neutrophils with T cells and B cells (Grillon etal., 1990). Neutrophils and macrophages are important decision shapers during the early phases of immune responses and prime responses by other cells. The activation of macrophages by sugar analogues is going to likely to increase immune surveillance and direct other cells. The response is almost certainly due to sugar receptors (C-type lectins) on the cell surface.

Glycosidase Inhibition by Swainsonine, Casuarine and 3,7-Diepicasuarine

Assays run using 0.8mM inhibitors and 5mM p-nitrophenyl-substrates at pH maxima for the glycosidases. Glycosidases and substrates purchased from Sigma. References

Bennouna, S., Bliss, S.K., Curiel, TJ. and Denkers, E.Y. (2003) Journal of Immunology: 6052-6058.

Bleriot, Yves., Gretzke, Dirk., Krϋlle, Thomas M., Butters, Terry D., Dwek, Raymond A., Nash,

Robert J., Asano, Naoki., and Fleet, George W.J. (2005). Carbohydrate Research 340 (2005): 2713- 2718

Davis, B., Bell, A.A., Nash, R.J., Watson, AA, Griffiths, R.C., Jones, M.G., Smith, C₁ Fleet, G.W.J.

(1996) Tetrahedron Letters 37(47): 8565-8568.

Grillon, C, Monsigny, M. and Kieda, C. (1990). Glycobiology 1 (1): 33-38.

McGowan, SA (2006) Modulation of the Immune Response by lmino Sugars. PhD Thesis, University of Strathclyde.

Nathan, C. (2006) Nat. Rev. Immunol. 6: 173-182.

Olden, K., Breton, P., Grzegorwewski, K., Yasuda, Y., Gause, B. L., Oredipe, O.A., Newton, S.A. and

White, S.L. (1991) Pharmac. Ther. 50: 285-290.

Simon, H. U. (2001) Immunol. Rev. 179: 156-162. Chung, N-H, Shepherd, V.L and Stahl, P.D. (1984) J Bio) Chem., 259 (23): 14637-14641.

Das, P.C., Robert, J.D., White, S.L, Olden, K. (1995) Oncol. Res. 7(9): 425-33.

Davis, B., Bell, A.A., Nash, R.J., Watson, AA, Griffiths, R.C., Jones, M.G, Smith, C, Fleet, G.W.J.

(1996) Tetrahedron Letters 37(47): 8565-8568.

Grillon, C₁ Monsigny, M. and Kieda, C. (1990). Glycobiology 1 (1): 33-38. Grzegorzewski, K., Newton, SA, Akiyama, SK, Sharrow, S, Olden, K, White, SL. Cancer Commun.

1989 1(6): 373-9.

Loike, J. D., Silverstein, S.C. and Sturtevant, J. M. (1981) Proc. Natl. Acad. Sci. USA 78 (10): 5958-

5962.

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Nash, R.J., Thomas, P.I., Waigh, R.D., Fleet, G.W.J. , Wormald, M. R., Lilley, P.M. de Q and Watkin,

D.J. (1994). Tetrahedron Letters 35: 7849-7852.

Nathan, C. (2006) Nat. Rev. Immunol. 6: 173-182.

Olden, K., Breton, P., Grzegorwewski, K., Yasuda, Y., Gause, B. L, Oredipe, O.A., Newton, S.A. and White, S.L. (1991) Pharmac. Ther. 50: 285-290.

Watson, A. A., Fleet, G.W.J., Asano, N., Molyneux, R.J. and Nash, R.J. (2001). Phytochemistry 56:

265-295.

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. Use of an imino sugar (or a pharmaceutically acceptable salt or derivative thereof) for inhibiting or postponing neutrophil apoptosis.

2. Use of an imino sugar (or a pharmaceutically acceptable salt or derivative thereof) for the treatment or prophylaxis of a disease or disorder in which the inhibition or postponement of neutrophil apoptosis is indicated.

3. Use of an imino sugar (or a pharmaceutically acceptable salt or derivative thereof) for the treatment or prophylaxis of a disease or disorder characterized by excessive neutrophil apoptosis.

4. Use of an imino sugar (or a pharmaceutically acceptable salt or derivative thereof) for use in PRR (for example C-type lectin)-mediated immunotherapy or immunoprophylaxis.

5. The use of claim 4 wherein the PRR (for example C-type lectin)-mediated immunotherapy or immunoprophylaxis is:

(a) immune response variegation (for example, in the treatment of pathological variegated states, e.g. arising from immune dysfunction or pathogen-mediated immune system subversion);

(b) stimulation of immune surveillance (for example by the innate immune system);

(c) non-specific immunoprophylaxis.

6. Use of an imino sugar PRR (for example C-type lectin) ligand (or a pharmaceutically acceptable salt or derivative thereof) for immune response variegation (for example, in the treatment of pathological variegated states, e.g. arising from immune dysfunction or pathogen-mediated immune system subversion).

7. Use of any one of the preceding claims wherein the imino sugar is a: (a) piperidine;

(b) pyrroline;

(c) pyrrolidine;

(d) pyrrolizidine;

(e) indolizidine; (f) nortropane;

(g) mixture of any two or more of (a) to (f).

8. Use of claim 7 wherein the imino sugar has the formula:

9. Use of any one of the preceding claims wherein the imino sugar is a ligand for a PRR (for example C-type lectin) selected from:

(a) MMR (CD206, macrophage mannose receptor); and/or

(b) DEC-205; and/or (c) Dectin 1 ; and/or

(d) Dectin 2; and/or

(e) Langerin; and/or (I) DC-SIGN; and/or (m) BDCA-2; and/or (n) DCIR; and/or

(0) DLEC; and/or (p) CLEG; and/or

(q) a rhamnose-binding C-type lectin.

10. Use of any one of the preceding claims wherein the PRR (for example C-type lectin) is a DC PRR (for example a DC C-type lectin).

11. Use of any one of the preceding claims for:

(a) the treatment or prevention of viral infection; (b) the treatment or prevention of bacterial infection;

(c) the treatment or prevention of protozoal infection;

(d) the treatment or prevention of fungal infection;

(e) the treatment or prevention of prion infection;

(f) the treatment or prevention of metazoan infection; (g) the treatment or prevention of proliferative disorders (for example cancer);

(h) the treatment or prevention of autoimmune disorders;

(1) the treatment or prevention of pathogen-mediated immune system subversion; (j) the treatment or prevention of allergy; (k) the treatment or prevention of pathological variegated immune system states; (I) non-specific immunoprophylaxis; (m) increasing immune surveillance;

(n) the treatment of neutropenia (e.g. pathogen-mediated neutropenia); (o) haematopoietic recovery following cytoablative therapy (e.g. radio- or chemotherapy).

12. Use of any one of the preceding claims wherein the imino sugar is a sugar (for example a rhamnose (e.g. α-L-rhamnose) or mannose) analogue.

13. Use of claim 12 wherein the imino sugar is an inhibitor of one or more rhamnosidase(s).

14. Use of claim 12 or claim 13 wherein the imino sugar is an inhibitor of one or more mannosidase(s).

15. Use of an imino sugar as defined in any one of the preceding claims for the manufacture of a medicament for use in any of the medical uses as defined in any one of the preceding claims.

16. A method for the treatment or prevention of a disease or disorder as defined in any one of the preceding claims comprising administering an effective amount of an imino sugar as defined in any one of the preceding claims to a patient in need thereof.