WO2007010266A1

WO2007010266A1 - Pyrrolidine compositions

Info

Publication number: WO2007010266A1
Application number: PCT/GB2006/002717
Authority: WO
Inventors: Robert James Nash; Miles William Carroll; Alison Ann Watson; George William John Fleet; Graeme Horne
Original assignee: Mnl Pharma Limited
Priority date: 2005-07-20
Filing date: 2006-07-20
Publication date: 2007-01-25
Also published as: GB0514865D0

Abstract

Disclosed are pyrrolidine compounds and their use in therapy and prophylaxis. In particular, the invention relates to the use of various pyrrolidine compounds (including N-hydroxyethyl-DMDP and certain analogues thereof) as immunomodulatory (immunostimulatory or immunosuppressive) drugs and/or as antivirals (for example, as gylcovirs or alkovirs, as herein defined).

Description

PYRROLIDINE COMPOSITIONS

Field of the Invention

The present invention relates to pyrrolidine compounds and to their use in therapy and prophylaxis. In particular, the invention relates to the use of various pyrrolidine compounds (including N-hydroxyethyl-DMDP and certain analogues thereof) as immunomodulatory (immunostimulatory or immunosuppressive) drugs and/or as antivirals (for example, as glycovirs or alkovirs, as herein defined).

Background to the Invention

Immunity

When the immune system is challenged by a foreign antigen it responds by launching a protective response. This response is characterized by the coordinated interaction of both the innate and acquired immune systems. These systems, once thought to be separate and independent, are now recognized as two interdependent parts that when integrated fulfil two mutually exclusive requirements: speed (contributed by the innate system) and specificity (contributed by the adaptive system).

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

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

Interdependence of innate and adaptive responses

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

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

Polarization of the adaptive immune response

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

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

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

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

Two cytokines in particular appear to have early roles in determining the path of the immune response. lnterleukin-12 (IL-12), secreted by macrophages, drives the type 1 response by stimulating the differentiation of Th1 cells, the helper cells that oversee the type 1 response. Another macrophage cytokine, interleukin-10 (IL- 10) inhibits this response, instead driving a type 2 response. The type 1 and type 2 responses can be distinguished inter alia on the basis of certain phenotypic changes attendant on priming and subsequent polarization of naϊve helper T cells. These phenotypic changes are characterized, at least in part, by the nature of the cytokines secreted by the polarized helper T cells.

Th1 cells produce so-called TM cytokines, which include one or more of TNF, IL-1 , IL-2, IFN-gamma, IL-12 and/or IL-18. The Th1 cytokines are involved in macrophage activation and Th1 cells orchestrate Type 1 responses. In contrast, Th2 cells produce so-called Th2 cytokines, which include one or more of IL-4, IL-5, IL- 10 and IL-13. The Th2 cytokines promote the production of various antibodies and can suppress the type 1 response. The involvement of Th1 and Th2 cells and cytokines in type 1:type 2 immune response polarization has given rise to the terms TM response and Th2 response being used to define the type 1 and type 2 immune responses, respectively. Thus, these terms are used interchangeably herein.

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

Glycoproteins and viral development Glycoproteins are classified into two major classes according to the linkage between sugar and amino acid of the protein. The most common and extensively studied is N-glycosidic linkage between an asparagine of the protein and an N-acetyl-D-glucosamine residue of the oligosaccharide. N-linked oligosaccharides, following attachment to a polypeptide backbone, are processed by a series of specific enzymes in the endoplasmic reticulum (ER) and this processing pathway has been well-characterized.

In the ER, α-glucosidase I is responsible for the removal of the terminal α-1,2 glucose residue from the precursor oligosaccharide and α-glucosidase Il removes the two remaining α-1,3 linked glucose residues, prior to removal of mannose residues by mannosidases and further processing reactions involving various transferases. These oligosaccharide "trimming" reactions enable glycoproteins to fold correctly and to interact with chaperone proteins such as calnexin (CNX) and calreticulin (CRT) for transport through the Golgi apparatus.

This glycoprotein processing is vital for the proper folding of many virus-encoded glycoproteins and inhibitors of key enzymes in this biosynthetic pathway, particularly those blocking α-glucosidases and α-mannosidase, have been shown to prevent replication of several enveloped viruses. Such inhibitors may act by interfering with the folding of the viral envelope glycoprotein, so preventing the initial virus-host cell interaction or subsequent fusion. They may also prevent viral duplication and/or secretion by preventing the construction of the proper glycoprotein required for the completion of the viral membrane.

Alkaloids

The term alkaloid is used sensu stricto to define any basic, organic, nitrogenous compound which occurs naturally in an organism. The term alkaloid may also be used 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.

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.

Most alkaloids are classified structurally on the basis of the configuration of the N-heterocycle. Examples of some important alkaloids and their structures are set out in Kutchan (1995) The Plant Cell 7:1059-1070. Watson et al. (2001) Phytochemistry 56: 265-295 have classified a comprehensive range of polyhydroxylated alkaloids inter alia as piperidine, pyrroline, pyrrolidine, pyrrolizidine, indolizidine and nortropanes alkaloids (see Figs. 1-7 of Watson et al. (2001), the disclosure of which is incorporated herein by reference).

Watson et al. (2001), ibidem also show that a functional classification of at least some alkaloids is possible on the basis of their glycosidase inhibitory profile: many polyhydroxylated alkaloids are potent and highly selective glycosidase inhibitors. These alkaloids can mimic the number, position and configuration of hydroxyl groups present in pyranosyl or furanosyl moieties and so bind to the active site of a cognate glycosidase, thereby inhibiting it. This area is reviewed in Legler (1990) Adv. Carbohydr. Chem. Biochem. 48: 319-384 and in Asano et al. (1995) J. Med. Chem. 38: 2349-2356.

It has long been recognized that many alkaloids are pharmacologically active, and humans have been using alkaloids (typically in the form of plant extracts) as poisons, narcotics, stimulants and medicines for thousands of years. The therapeutic applications of polyhydroxylated alkaloids have been comprehensively reviewed in Watson et al. (2001), ibidem: applications include cancer therapy, immune stimulation, the treatment of diabetes, the treatment of infections (especially viral infections), therapy of glycosphingolipid lysosomal storage diseases and the treatment of autoimmune disorders (such as arthritis and sclerosis).

Both natural and synthetic mono- and bi-cyclic nitrogen analogues of carbohydrates are known to have potential as therapeutic agents. Alexine (1) and australine (2) were the first pyrrolizidine alkaloids to be isolated with a carbon substituent at C-3, rather than the more common C-1 substituents characteristic of the necine family of pyrrolizidine alkaloids.

Aiexine (1)

Australine (2)

The alexines occur in all species of the genus Alexa and also in the related species Castanospermum australe. Stereoisomers of aiexine, including 1,7a-diepialexine (3), have also been isolated (Nash et al. (1990) Phytochemistry (29) 111) and synthesised (Choi et al. (1991 ) Tetrahedron Letters (32) 5517 and Denmark and Cottell (2001) J. Org. Chem. (66) 4276-4284).

1 ,7a-diepialexine (3)

Because of the reported weak in vitro antiviral properties of one 7,7a-diepialexine (subsequently defined as 1,7a-diepialexine), there has been some interest in the isolation of the natural products and the synthesis of analogues.

As an indolizidine alkaloid (and so structurally distinct from the alexines), swainsonine (4) is a potent and specific inhibitor of α-mannosidase and is reported to have potential as an antimetastic, tumour anti-proliferative and immunoregulatory agent (see e.g. US5650413, WOOO/37465, WO93/09117).

Swainsonine (4)

Another indolizidine alkaloid, castanospermine (5), is a potent α-glucosidase inhibitor. This compound, along with certain 6-O-acyl derivatives (such as that known as Bucast (6)), has been reported to exhibit anti-viral and antimetastic activities.

Castanospermine (5)

Bucast (6)

The effect of variation in the size of the six-membered ring of swainsonine on its glycosidase inhibitory activity has been studied: derivatives (so-called "ring contracted swainsonines") have been synthesised. However, these synthetic derivatives (1S, 2R, 7R, 7aR)-1 ,2,7-trihydroxy(7) and the 7S-epimer (8)) were shown to have much weaker inhibitory activity relative to swainsonine itself (see US5075457).

1S, 2R, 7R₁ 7aR)-1,2,7-trihydroxy(7)

7S-epimer (8)

Another compound, 1α,2α,6α,7α,7αβ-1 ,2,6,7-tetrahydroxy(9) is an analogue of 1 ,8-diepiswainsonine and described as a "useful" inhibitor of glycosidase enzymes in EP0417059.

1a, 2a, 6a, 7a, 7aβ- 1, 2, 6, 7-tetrahydroxy(9)

Casuarine, (1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1 ,2,6,7-tetrahydroxy(10) is a highly oxygenated bicyclic alkaloid that can be regarded as a more highly oxygenated analogue of the 1 ,7a-diepialexine (shown in 3) or as a C(3) hydroxymethyl-substituted analogue of the 1α,2α,6α,7α,7αβ-1,2,6,7-tetrahydroxy(shown in 9).

Casuarine (10)

Casuarine can be isolated from several botanical sources, including the bark of Casuarina equisetifolia (Casuarinaceae), the leaves and bark of Eugenia jambolana (Myrtaceae) and Syzygium guineense (Myrtaceae) (see e.g. Nash et al. (1994) Tetrahedron Letters (35) 7849-7852). Epimers of casuarine, and probably casuarine itself, can be synthesised by sodium hydrogen telluride-induced cyclisation of azidodimesylates (Bell et al. (1997) Tetrahedron Letters (38) 5869-5872).

Casuarina equisetifolia wood, bark and leaves have been claimed to be useful against diarrhoea, dysentery and colic (Chopra et al. (1956) Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research (India), New Delhi, p. 55) and a sample of bark has recently been prescribed in Western Samoa for the treatment of breast cancer. An African plant containing casuarine (identified as Syzygium guineense) has been reported to be beneficial in the treatment of AIDS patients (see Wormald et al. (1996) Carbohydrate Letters (2) 169-174). The casuarine-6-α-glucoside (casuarine-6-α-D-glucopyranose, 11) has also been isolated from the bark and leaves of Eugenia jambolana (Wormald et al. (1996) Carbohydrate Letters (2) 169-174).

Casuarine-6-α-D-glucopyranose (11)

Eugenia jambolana is a well-known tree in India for the therapeutic value of its seeds, leaves and fruit against diabetes and bacterial infections. Its fruit have been shown to reduce blood sugar levels in humans and aqueous extracts of the bark are claimed to affect glycogenosis and glycogen storage in animals (Wormald et al. (1996) Carbohydrate Letters (2) 169-174).

Various immunotherapeutic applications for casuarine and certain analogues and derivatives thereof have recently been described in WO2004/064715.

Some pyrrolidine alkaloids appear to be fairly widespread secondary metabolites: for example, 2R.5R- dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) (12) and 1 ,4-dideoxy-1 ,4-imino-D-arabinitol (D-AB1) (13) have been isolated from species of both temperate and tropical plants from quite unrelated families, and DMDP is also produced by a species of the filamentous bacterium Streptomyces.

H

DMDP (12)

D-AB1 (13) DMDP has been shown to have nematocidal activity: WO 92/09202 describes the use of the compound in controlling diseases caused by parasitic nematodes in both plants and mammals.

Alkaloids as αlvcosidase inhibitors

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 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 so-called polyhydroxylated alkaloids.

Most alkaloids are classified structurally on the basis of the configuration of the N-heterocycle. Examples of some important alkaloids and their structures are set out in Kutchan (1995) The Plant Cell 7:1059-1070. Watson ef al. (2001) Phytochemistry 56: 265-295 have classified a comprehensive range of polyhydroxylated alkaloids inter alia as piperidine, pyrroline, pyrrolidine, pyrrolizidine, indolizidine and nortropanes alkaloids (see Figs. 1-7 of Watson et al. (2001), the disclosure of which is incorporated herein by reference).

Watson ef al. (2001), ibidem also show that a functional classification of at least some alkaloids is possible on the basis of their glycosidase inhibitory profile: many polyhydroxylated alkaloids are potent and highly selective glycosidase inhibitors. These alkaloids can mimic the number, position and configuration of hydroxyl groups present in pyranosyl or furanosyl moieties and so bind to the active site of a cognate glycosidase, thereby inhibiting it. This area is reviewed in Legler (1990) Adv. Carbohydr. Chem. Biochem. 48: 319-384 and in Asano et al. (1995) J. Med. Chem. 38: 2349-2356.

Both natural and synthetic mono- and bi-cyclic nitrogen analogues of carbohydrates are known to have potential as chemotherapeutic agents. Alexine and australine were the first pyrrolizidine alkaloids to be isolated with a carbon substituent at C-3, rather than the more common C-1 substituents characteristic of the necine family of pyrrolizidines. The alexines occur in all species of the genus Alexa and also in the related species Castanospermum australe. Stereoisomers of alexine, including 1,7a-diepialexine, have also been isolated (Nash βt al. (1990) Phytochemistry (29) 111) and synthesised (Choi et al. (1991) Tetrahedron Letters (32) 5517 and Denmark and Cottell (2001) J. Org. Chem. (66) 4276-4284). Because of the reported weak in vitro antiviral properties of one 7,7a-diepialexine (subsequently defined as 1 ,7a-diepialexine), there has been some interest in the isolation of the natural products and the synthesis of analogues.

As an indolizidine alkaloid (and so structurally distinct from the pyrrolidine alexines), swainsonine is a potent and specific inhibitor of α-mannosidase and is reported to have potential as an antimetastic, tumour antiproliferative and immunoregulatory agent (see e.g. US5650413, WO00/37465, WO93/09117). The effect of variation in the size of the six-membered ring of swainsonine on its glycosidase inhibitory activity has been studied: pyrrolizidine derivatives (so-called "ring contracted swainsonines") have been synthesised. However, these synthetic derivatives (1S, 2R, 7R, 7aR)-1,2,7-trihydroxypyrrolizidine and the 7S-epimer) were shown to have much weaker inhibitory activity relative to swainsonine itself (see US5075457).

Another indolizidine alkaloid, castanospermine, is a potent α-glucosidase inhibitor. This compound, along with certain 6-O-acyl derivatives (such as that known asCelgosivir or Bucasf), has been reported to exhibit anti-viral and antimetastatic activities.

Casuarine, (1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1,2,6,7-tetrahydroxypyrrolizidine (10) (also known as casuarin) is a highly oxygenated bicyclic pyrrolizidine alkaloid that can be regarded as a more highly oxygenated analogue of the 1 ,7a-diepialexine (shown in 3) or as a C(3) hydroxymethyl-substituted analogue of the 1α,2α,6α,7α,7a(S)-1 ,2,6,7-tetrahydroxypyrrolizidine. Casuarine can be isolated from several botanical sources, including the bark of Casuarina equisetifolia (Casuarinaceae), the leaves and bark of Eugenia jambolana (Myrtaceae) and Syzygium guineense (Myrtaceae) (see e.g. Nash et al. (1994) Tetrahedron Letters (35) 7849-52). Casuarina equisetifolia wood, bark and leaves have been claimed to be useful against diarrhoea, dysentery and colic (Chopra ef a/. (1956) Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research (India), New Delhi, p. 55) and a sample of bark has recently been prescribed in Western Samoa for the treatment of breast cancer. An African plant containing casuarine (identified as Syzygium guineense) has been reported to be beneficial in the treatment of AIDS patients (see Wormald ef a/. (1996) Carbohydrate Letters (2) 169-74). The casuarine-6-α-glucoside (casuarine-6-α-D-glucopyranose) has also been isolated from the bark and leaves of Eugenia jambolana (Wormald et al. (1996) Carbohydrate Letters (2) 169-74). Eugenia jambolana is a well known tree in India for the therapeutic value of its seeds, leaves and fruit against diabetes and bacterial infections. Its fruit have been shown to reduce blood sugar levels in humans and aqueous extracts of the bark are claimed to affect glycogenosis and glycogen storage in animals (Wormald et al. (1996) Carbohydrate Letters (2) 169-74).

Alkaloids as immunomodulators

Some alkaloids have immunomodulatory activity that is independent of any glycosidase inhibitory activity. Examples of such alkaloids are described, for example, in WO2004/064715, WO2005/070415 and WO2005/070418. It is thought that this immunomodulatory activity may arise from the stimulation of secretion of various cytokines (e.g. IL-12 and/or IL-2) by immune cells (e.g. dendritic cells and/or macrophages). As described in WO2004/064715, WO2005/070415 and WO2005/070418 (the content of which relating to the structure of the various alkaloids described and their biological activity is hereby incorporated herein by reference), the immunomodulatory activity of such alkaloids can itself confer antiviral activity.

The recognition that alkaloids may exert some or all of their biological effects, including antiviral activity, independently of glycosylation inhibition has led to the recent classification of imino sugars mediating an antiviral effect via α-glucosidase inhibition (for example, DNJ and NB-DNJ) as glucovirs, whereas those (such as NN-DGJ and Λ/-7-oxanonyl-6-deoxy-DGJ) mediating an antiviral effect independently of α-glucosidase inhibition (for example by interfering with viral p7 protein as described infra) have been dubbed alkovirs (see Block and Jordan (2001 ) Antivir. Chem. Chemother. 12(6): 317-325).

Dendritic Cells and their lmmunotherapeutic Uses

(a) Introduction

Dendritic cells (DCs) are a heterogeneous cell population with distinctive morphology and a widespread tissue distribution (see Steinman (1991) Ann. Rev. Immunol. 9: 271-296). They play an important role in antigen presentation, capturing and processing antigens into peptides and then presenting them (together with components of the MHC) to T cells. T cell activation may then be mediated by the expression of important cell surface molecules, such as high levels of MHC class I and Il molecules, adhesion molecules, and costimulatory molecules.

Dendritic cells therefore act as highly specialized antigen-presenting cells (APCs): serving as "nature's adjuvants", they potentiate adaptive T-cell dependent immunity as well as triggering the natural killer (NK and NKT) cells of the innate immune system. Dendritic cells therefore play a fundamental and important regulatory role in the magnitude, quality, and memory of the immune response. As a result, there is now a growing interest in the use of dendritic cells in various immunomodulatory interventions, which are described in more detail below.

Dendritic cells can be classified into different subsets inter alia on the basis of their state of maturation (mature or immature) and their cellular developmental origin (ontogeny). Each of these subsets appear to play distinct roles in vivo, as described below.

(b) Dendritic Cell Maturation

Immature (or resting) DCs are located in non-lymphoid tissue, such as the skin and mucosae, are highly phagocytic and readily internalize soluble and particulate antigens. It is only when such antigen-loaded immature DCs are also subject to inflammatory stimuli (referred to as maturation stimuli) that they undergo a maturation process that transforms them from phagocytic and migratory cells into non-phagocytic, highly efficient stimulators of naϊve T cells.

Immature DCs are characterized by high intracellular MHC Il in the form of MIICs, the expression of CD1a, active endocytosis for certain particulates and proteins, presence of FcgR and active phagocytosis, deficient T cell sensitization in vitro, low/absent adhesive and costimulatory molecules (CD40/54/58/80/86), low/absent CD25, CD83, p55, DEC-205, 2A1 antigen, responsiveness to GM-CSF, but not M-CSF and G-CSF and a sensitivity to IL-10, which inhibits maturation. Upon maturation, mature DCs, loaded with antigen and capable of priming T cells, migrate from the non- lymphoid tissues to the lymph nodes or spleen, where they process the antigen load and present it to the resident naϊve CD4⁺ T cells and CD8⁺ cytotoxic T cells. This latter interaction generates CTLs, the cellular arm of the adaptive immune response, and these cells eliminate virally infected cells and tumour cells. The naϊve CD4⁺ T cells differentiate into memory helper T cells, which support the differentiation and expansion of CD8⁺ CTLs and B cells. Thus, helper T cells exert anti-tumour activity indirectly through the activation of important effector cells such as macrophages and CTLs. Having activated the T cells in this way, the mature DCs undergo apoptosis within 9-10 days.

Mature DC cells are characterized morphologically by motility and the presence of numerous processes (veils or dendrites). They are competent for antigen capture and presentation (exhibiting high MHC class I and Il expression) and express a wide range of molecules involved in T cell binding and costimulation, (e.g. CD40, CD54/ICAM-1, CD58/LFA-3, CD80/B7-1 and CD86/B7-2) as well as various cytokines (including IL-12). They are phenotypically stable: there is no reversion/conversion to macrophages or lymphocytes. Thus, mature DCs play an important role in T cell activation and cell-mediated immunity. In contrast, immature DCs are involved in regulating and maintaining immunological tolerance (inducing antigen-specific T cell anergy).

(c) Dendritic Cell Ontogenic Subsets

Dendritic cells are not represented by a single cell type, but rather comprise a heterogeneous collection of different classes of cells, each with a distinct ontogeny. At least three different developmental pathways have been described, each emerging from unique progenitors and driven by particular cytokine combinations to DC subsets with distinct and specialized functions.

At present it is thought that the earliest DC progenitors/precursors common to all DCs originate in the bone marrow. These primitive progenitors are CD34⁺, and they are released from the bone marrow to circulate through both the blood and lymphoid organs.

Once released from the bone marrow, the primitive CD34⁺ DC progenitors are subject to various stimulatory signals. These signals can direct the progenitors along one of at least three different pathways, each differing with respect to intermediate stages, cytokine requirements, surface marker expression and biological function.

• Lymphoid DCs are a distinct subset of DCs that are closely linked to the lymphocyte lineage. This lineage is characterized by the lack of the surface antigens CD11b, CD13, CD14 and CD33. Lymphoid DCs share ancestry with T and natural killer (NK) cells, the progenitors for all being located in the thymus and in the T cell areas of secondary lymphoid tissues. The differentiation of lymphoid DCs is driven by interleukiπs 2, 3 and 15 (IL-3, IL-2 and IL-15), but not by granulocyte macrophage colony- stimulating factor (GM-CSF). Functionally, lymphoid promote negative selection in the thymus (possibly by inducing fas-mediated apoptosis) and are costimulatory for CD4⁺ and CD8⁺T cells. More recently, lymphoid-like DCs derived from human progenitors have also been shown to preferentially activate the Th2 response. Because of their capacity to induce apoptosis and their role in eliminating potentially self-reactive T cells, it has been suggested that lymphoid DCs primarily mediate regulatory rather than stimulatory immune effector functions. • Myeloid DCs are distinguished by a development stage in which there is expression of certain features associated with phagocytes. There appear to be at least two structurally and functionally distinct subsets. The first is defined antigenically as CD14^", CD34⁺, CD68^" and CDIa⁺ and sometimes referred to as DCs of the Langerhans cell type. This subset appears to prime T cells to preferentially activate Th1 responses and IL-12 appears implicated in this process. The subset may also activate naϊve B cells to secrete IgM and may therefore be predominantly associated with an inflammatory Th1 response. A second myeloid DC subset, sometimes referred to as interstitial DCs, is defined antigenically as CD14⁺, CD68^* and CD1a^"and related to monocytes (as a result they are also referred to as monocyte-derived DCs or Mo-DCs).

(d) Dendritic Cell Vaccines

In one dendritic cell-based treatment paradigm (reviewed in Schuler et al. (2003) Current Opinion in Immunol 15: 138-147), DC cells are taken from a patient (for example by apheresis) and then pulsed (primed or spiked) with a particular antigen or antigens (for example, tumour antigen(s)). They are then re-administered as an autologous cellular vaccine to potentiate an appropriate immune response.

In this treatment paradigm, the responding T cells include helper cells, especially Th1 CD4⁺ cells (which produce IFN-γ) and killer cells (especially CD8⁺ cytolytic T lymphocytes). The DCs may also mediate responses by other classes of lymphocytes (B, NK, and NKT cells). They may also elicit T cell memory, a critical goal of vaccination.

At present, little is known about the identity of the DC subset(s) required for optimum effectiveness of DC vaccines, beyond the recognition that maturation is required and immature DCs are to be avoided (Dhodapkar and Steinman (2002) Blood 100: 174-177). Hsu et al. (1996) Nat Med 2: 52-58 used rare DCs isolated ex vivo from blood. These DCs were highly heterogeneous with respect to their ontogenic subsets but matured spontaneously during the isolation procedure. However, the yields were very low.

The yield problem has been addressed by the development of techniques for expanding the DCs ex vivo, for example with Flt3 ligand (Fong et al. (2001) PNAS 98: 8809-8814), but this is of limited effectiveness.

However, most studies have used Mo-DCs. These cells are obtained by exposing monocytes to GM-CSF and IL-4 (or IL-13) to produce immature Mo-DCs, which are then matured by incubation in a maturation medium. Such media comprise one or more maturation stimulation factor(s), and typically comprise Toll-like receptor (TLR) ligands (e.g. microbial products such as lipopolysaccharide and/or monophosphoryl lipid), inflammatory cytokines (such as TNF-α), CD40L, monocyte conditioned medium (MCM) or MCM mimic (which contains IL- 1β, TNF- α, IL-6 and PGE₂).

Although little is known at present about the influence of maturation medium on DC vaccine performance, MCM or MCM mimic currently represent a standard: Mo-DCs matured using these media are homogenous, have a high viability, migrate well to chemotactic stimuli and induce CTLs both in vitro and in vivo.

Techniques have been developed for generating large numbers of Mo-DCs (300 to 500 million mature DCs per apheresis) from adherent monocytes within semi-closed, multilayered communicating culture vessels offering a surface area large enough to cultivate one leukapheresis product. These so-called cell factories can be used to produce cryopreserved aliquots of antigen preloaded DCs which are highly viable on thawing, and optimised maturation and freezing procedures have been described (Berger et al (2002) J. Immunol. Methods 268: 131- 140; Tuyaerts et al. (2002) J. Immunol. Methods 264: 135-151).

Dendritic cells for vaccination have also been prepared from CD34⁺-derived DCs comprising a mixture of interstitial and DCs of the Langerhans cell type. Some workers believe that the latter DC subset are more potent than Mo-DCs when used as DC vaccines.

With regard to antigen selection, various approaches have been used. Both defined and undefined antigens can be employed. The antigens can be xenoantigens or autoantigens. One or more defined neoantigen(s) may be selected: in the case of cancer treatment, the enoantigen(s) may comprise a tumour-associated antigen. However, most popular are 9-11 amino acid peptides containing defined antigens (either natural sequences or analogues designed for enhanced MHC binding): such antigens can be manufactured to good manufacturing practice (GMP) standard and are easily standardized. Other approaches have employed antigens as immune complexes, which are delivered to Fc-receptor-bearing DCs and which results in the formation of both MHC class I and MHC class Il peptide sequences. This offers the potential for inducing both CTLs and Th cells (Berlyn et al. (2001) Clin Immunol 101: 276-283).

Methods have also been developed for exploring the whole antigenic repertoire of any given tumour (or other target cell, such as a virally-infected cell). For example, DC-tumour cell hybrids have been successfully used to treat renal cell carcinoma (Kugler et al. (2000) 6: 332-336), but the hybrids are difficult to standardize and shortlived. Necrotic or apoptotic tumour cells have been used, as have various cellular lysates.

It appears that the selection of patient-specific antigens may be important in the treatment of at least some cancers, and antigens derived from fresh tumour cells rather than tumour cell lines or defined antigens may prove important (Dhodapkar et al. (2002) PNAS 99: 13009-13013).

As regards delivery of the selected antigen(s) to the DCs, various techniques are available. Since the number and quality of MHC-peptide complexes directly influences the immunogenicity of the DC, the antigen loading technique may prove critical to DC vaccine performance (van der Burg et al. (1996) J Immunol 156: 3308-

3314). It seems that prolonged presentation of MHC-peptide complexes by the DCs enhances immunogenicity and so loading techniques which promote prolonged presentation may be important. This has been achieved by loading the DCs internally through the use of peptides linked to cell-penetrating moieties (Wang and Wang (2002) Nat Biotechnol 20: 149-154).

Antigens can also be loaded by transfecting the DCs with encoding nucleic acid (e.g. by electroporation) such that the antigens are expressed by the DC, processed and presented at the cell surface. This approach avoids the need for expensive GMP proteins and antibodies. RNA is preferred for this purpose, since it produces only transient expression (albeit sufficient for antigen processing) and avoids the potential problems associated with the integration of DNA and attendant long-term expression/mutagenesis. Such transfection techniques also permit exploration of the whole antigenic repertoire of a target cell by use of total or PCR-amplified tumour RNA. There is some evidence that helper proteins (for example, keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT)) can provide unspecific help for CTL induction (Lanzavecchia (1998) Nature 393: 413-414) and it may prove advantageous to pulse DC with such helper proteins prior to vaccination.

With regard to posology, the dose, frequency and route of DC vaccine administration have not yet been optimised in clinical trials. Clearly, the absolute number of cells administered will depend on the route of administration and effectiveness of migration after infusion. In this respect there are indications that intradermal or subcutaneous administration may be preferred for the development of Th1 responses, although direct intranodal delivery has been employed to circumvent the need for migration from the skin to the nodes (Nestle et al. (1998) Nat Med 4: 328-332).

Quite distinct from the antigen-pulsed DC vaccine paradigm described above is an approach in which dendritic cells secreting various chemokines are injected directly into tumours where they have been shown to prime T cells extranodally (Kirk et al. (2001) Cancer Res 61: 8794-8802). Thus, in another treatment paradigm, DCs are targeted to a tumour and activated to elicit immune responses in situ without the need for ex vivo antigen loading.

In situ DC vaccination constitutes yet another distinct (but related) approach (Hawiger et al. (2001) J Exp Med 194: 769-779. In this therapeutic paradigm, antigen is targeted to DCs in vivo which are expanded and induced to mature in situ. This approach depends on efficient targeting of antigen to endogenous DCs (for example, using exosomes - see Thery et al. (2002) Nat Rev Immunol 2: 569-579) and the development of maturation stimulants that can effectively trigger maturation (preferably of defined DC subset(s)) in vivo.

(e) Use of Dendritic Cells in Adoptive CTL Immunotherapy Cytotoxic T lymphocytes (CTLs) can be administered to a patient in order to confer or supplement an immune response to a particular disease or infection (typically cancer). For example, tumour specific T cells can be extracted from a patient (e.g. by leukapheresis), selectively expanded (for example by tetramer-guided cloning - see Dunbar et al. (1999) J Immunol 162: 6959-6962) and then re-administered as an autologous cellular vaccine.

The clinical effectiveness, applicability and tractability of this type of passive immunotherapy can be greatly increased by using dendritic cells to prime the T cells in vitro prior to administration.

(f) Dendritic Cell-based Approaches to the Treatment of Autoimmune Disorders Dendritic cells are also involved in regulating and maintaining immunological tolerance: in the absence of maturation, the cells induce antigen-specific silencing or tolerance. Thus, in another dendritic cell-based treatment paradigm, immature DCs are administered as part of an immunomodulatory intervention designed to combat autoimmune disorders. In such applications, the suppressive potential of the DCs has been enhanced by in vitro transfection with genes encoding cytokines.

(q) The Role of IL-2 in Dendritic Cell Function

Granucci et al. (2002) Trends in Immunol. 23: 169-171 have reported transient upregulation of mRNA transcripts for IL-2 in dendritic cells following microbial stimulus. In WO03012078 Granucci describes the important role played by DC-derived IL-2 in mediating not only T cell activation but also that of NK cells and goes on to suggest that DC-derived IL-2 is a key factor regulating and linking innate and adaptive immunity.

Moreover, systemic administration of IL-2 has recently been shown to enhance the therapeutic efficacy of a DC vaccine (Shimizu et al. (1999) PNAS 96: 2268-2273), while the presence of IL-2 was shown to be essential for specific peptide-mediated immunity mediated by dendritic cells in at least some DC vaccination regimes (Eggert et al. (2002) Eur J Immunol 32: 122-127). In their recent review, Schuler et al. (ibidem) conclude that "... it might be worthwhile to explore the combination of DC vaccination with IL-2 administration, as the T-cell responses induced by DC vaccination appear enhanced and therapeutically more effective.".

It will be clear from the foregoing discussion that dendritic cells are now proven as valuable tools in immunotherapy (particularly in the treatment of cancer), but that DC vaccination is still at a relatively early stage. Methods for preparing DCs are improving continuously and an increasing number of Phase I₁ Il and III clinical trials are driving intense research and development in this area. However, there is still a need to improve efficacy at the level of DC biology.

The present inventors have now surprisingly discovered that certain polyhydroxylated pyrrolidine compounds have unexpected immunomodulatory activity, and that this activity may be independent of any glycosidase inhibititory activity.

Summary of the Invention

According to the invention there is provided a pyrrolidine compound, for use in therapy or prophylaxis, having the formula:

wherein. R₁-R₅ is hydrogen or any group provided that at least three of R₁-R5 is a group comprising X, wherein X is selected from: -OH, -NH₂, -CN, -NO₂ or a halogen (e.g. Br, F, I or Cl), or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

In another aspect, there is provided a substituted or unsubstituted, N-hydroxyethyl triethanolamine pyrrolidine compound, for use in therapy or prophylaxis.

The pyrrolidine compound is preferably isolated.

The compound may be immunomodulatory. The therapy or prophylaxis is preferably any of the therapies or prophylactic methods described herein.

In another aspect, there is provided a pharmaceutical composition comprising a substituted or unsubstituted, N- hydroxyethyl triethanolamine pyrrolidine compound, optionally further comprising a pharmaceutically acceptable excipient.

The pharmaceutical compositions of the invention may be parenteral or enteral compositions. They may also be vaccine or vaccine adjuvant compositions. The pharmaceutical compositions may be sterile and/or non- pyrogenic. Parenteral compositions are preferably sterile and non-pyrogenic.

In another aspect, there is provided a plurality of dose units of a pharmaceutical composition, the composition comprising a substituted or unsubstituted, N-hydroxyethyl triethanolamine pyrrolidine compound, wherein the inter-dose co-efficient of variation in the concentration of pyrrolidine compound in said dose units is less than 50%. Particularly preferred are dose units wherein the inter-dose co-efficient of variation in the concentration of pyrrolidine compound in said dose units is less than 40%, 30%, 20%, 10% or 5%. The plurality of dose units may constitute a batch of dose units.

In another aspect, there is provided a process for producing a plurality of dose units of a pharmaceutical composition (for example, for producing a batch of said dose units), the process comprising the step of introducing a substituted or unsubstituted, N-hydroxyethyl triethanolamine pyrrolidine compound as defined in any one of the preceding claims into each dose unit such that the inter-dose co-efficient of variation in the concentration of pyrrolidine compound in said dose units is less than 50%. Particularly preferred are processes wherein the inter-dose co-efficient of variation in the concentration of pyrrolidine compound is less than 40%, 30%, 20%, 10% or 5%.

The invention also contemplates the use, for the manufacture of a medicament, of the pyrrolidines of the invention for use in therapy or prophylaxis.

The invention also contemplates methods of therapy and prophylaxis comprising the step of administering the pyrrolidine compound to a patient.

The pyrrolidine compound may have the general formula:

or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof. Pyrrolidine compounds conforming to this general formula may be substituted (e.g. with a straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, cycloalkyl, alkenyl, alkynyl, aryl, acyl, ether or amine group) at C5 and/or C6 and/or C7 and/or C8.

In preferred embodiments, the pyrrolidine compound of the invention has the formula:

or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, cycloalkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

Pyrrolidine compounds conforming to this formula may also be substituted (e.g. with a straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, cycloalkyl, alkenyl, alkynyl, aryl, acyl, ether or amine group) at C5 and/or C6 and/or C7 and/or C8.

Particularly preferred as a pyrrolidine compound of the invention is selected from:

(a) N-hydroxyethylDMDP having the formula:

(b)

In another aspect the invention provides a pyrrolidine compound having the formula:

or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

In such embodiments, the compound preferably has the formula:

In anotehr aspect, the invention provides a compound having the formula:

In such embodiments, the compound preferably has the formula:

H or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or uπsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

wherein G is a group comprising OH (for example being (CHk)_nOH), wherein n is 1-4; or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

In such embodiments, the compound preferably has the formula:

In a particularly preferred aspect, the invention provides an antiviral composition comprising a compound of the formula:

for example having the formula:

In the latter embodiments the invention contemplates methods for the therapy or prophylaxis of viral infections (for example HCV infections) comprising administration of an effective amount of a compound of the formula:

for example of the formula:

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 alkovir\s a term of art (see Block and Jordan (2001) Antivir. Chem. Chemother. 12(6): 317-325) and is used herein to define a family of iminosugars which exert antiviral activity independently of ER α-glucosidase inhibition. Alkovirs therefore include iminosugars which act to inhibit antiviral activity by mechanisms which are wholly independent of ER α-glucosidase inhibition (such alkovirs not being ER α-glucosidase inhibitors), as well as iminosugars which exert antiviral activity by a combination of ER α-glucosidase inhibition and one or more other modes of action (for example, interference with viral p7 protein or by immunomodulatory activity). However, it should be noted that, as used herein, the term alkovir is also used sensu lato to include (and where context permits) not only iminosugars which exert antiviral activity independently of ER α-glucosidase inhibition but also other alkaloids having this activity as defined herein. Thus, the term alkovir is used herein to define a class of alkaloids (including, but not limited to, iminosugars) which exert antiviral activity independently of ER α- glucosidase inhibition.

The term glucovir is a term of art (see Block and Jordan (2001) Antivir. Chem. Chemother. 12(6): 317-325) and is used herein to define a family of iminosugars which exert antiviral activity, at least in part, by ER α- glucosidase inhibition. Glucovirs therefore include iminosugars which act to inhibit antiviral activity by ER α- glucosidase inhibition, as well as iminosugars which exert antiviral activity by a combination of ER α- glucosidase inhibition and one or more other modes of action (for example, interference with viral p7 protein or by immunomodulatory activity). Thus, the alkovir and glucovir iminosugar families as herein defined partially overlap. However, it should be noted that, as used herein, the term glucovir is also used sensu lato (and where context permits) to include any alkaloid which exerts antiviral activity, at least in part, by glucosidase (particularly glucosidase I) inhibition.

The analagous term glycovir is used herein as a more generic term than glucovir (as defined above) to define a class of alkaloids (including, but not limited to, iminosugars) which exert antiviral activity, at least in part, by glycosidase inhibition. Thus, glucovirs form a subclass of the broader glycovir class of alkaloid antivirals. Thus, glycovirs and glucovirs suitable for use according to the invention may be glycosylation modulators as herein defined.

The term alkaloid is used herein sensu stricto to define any basic, organic, nitrogenous compound which occurs naturally in an organism. In this sense, the term embraces naturally occurring imino sugars (see infra). 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-occurring basic, organic, nitrogenous compounds but also derivatives and analogues thereof which are not naturally occurring (and which may not be basic). In this context, the term imino sugar defines a saccharide (e.g. a mono- or disaccharide) analogue in which the ring oxygen is replaced by a nitrogen.

The term tumour-associated antigen is used herein to define an antigen present in transformed (malignant or tumourous) cells which is absent (or present in lower amounts or in a different cellular compartment) in normal cells of the type from which the tumour originated. Oncogenic viruses can also induce expression of tumour antigens, which are often host proteins induced by the virus.

The term maturation medium is used herein to define a composition (either defined or undefined) comprising one or more compounds which induce the maturation of dendritic cells from immature dendritic cells. Typically, the maturation medium comprises one or more Toll-like receptor (TLR) ligands and/or one or more inflammatory cytokines (such as TNF-α). Other growth factors may also be present.

The term neoantigen is used herein to define any newly expressed antigenic determinant. Neoantigens may arise upon conformational change in a protein, as newly expressed determinants (especially on the surfaces of transformed or infected cells), as the result of complex formation of one or more molecules or as the result of cleavage of a molecule with a resultant display of new antigenic determinants. Thus, as used herein, the term neoantigen covers antigens expressed upon infection (e.g. viral infection, protozoal infection or bacterial infection), in prion-mediated diseases (e.g. BSE and CJD), an on cell transformation (cancer), in which latter case the neoantigen may be termed a tumour-associated antigen.

As used herein, the term co-administration, as used in the context of the administration of the various components of the alkaloid compositions, vaccines etc. of the invention, is intended to cover the sequential, concurrent or separate administration of the referenced components. Concurrent administration therefore covers the case where the referenced components are physically mixed prior to administration. Sequential administration covers circumstances in which the referenced components are administered separately with some degree of temporal separation (typically from several minutes to several hours, although in some embodiments the administration of the co-administered components may be separated by a period of one or more days).

The term batch defines a plurality of dose units intended to have uniform character and quality, within specified limits, produced in the same manufacturing run. The term covers plural dose units produced by both batch manufacturing processes and continuous manufacturing processes. The co-efficient of vanation (or C V ) as applied to the dose units of the pharmaceutical compositions of the invention is term of art defining a key statistic of the quality of batches of a formulated pharmaceutical composition Specifically, the C V is the standard deviation divided by the mean multiplied by 100

The term alkyl defines the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched- chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e g , C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20, 10, 5 or fewer than 5 Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure

The terms alkenyl and alkynyl define unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively

The term aryl defines 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms Examples include benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, tπazole, pyrazole, pyridine, pyrazine, pyπdazine and pyπmidine The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3 or -CN The term also covers polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining (or fused) rings wherein at least one of the rings is aromatic In such cases the other cyclic rιng(s) can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls

The term herbal medicine is used herein to define a pharmaceutical composition in which at least one active principle is not chemically synthesized and is a phytochemical constituent of a plant In most cases, this non- synthetic active principle is not isolated (as defined herein), but present together with other phytochemicals with which it is associated in the source plant In some cases, however, the plant-derived bioactive prιncιple(s) may be in a concentrated fraction or isolated (sometimes involving high degrees of purification) In many cases, however, the herbal medicine comprises a more or less crude extract, infusion or fraction of a plant or even an unprocessed whole plant (or part thereof), though in such cases the plant (or plant part) is usually at least dried and/or milled

The term bioactive principle is used herein to define a phytochemical which is necessary or sufficient for the pharmaceutical efficacy of the herbal medicament in which it is comprised In the case of the present invention, the bioactive principle comprises the immunostimulatory alkaloid of the invention (e g casuarine, casuarine glucoside or mixtures thereof)

The term standard specification is used herein to define a characteristic, or a phytochemical profile, which is correlated with an acceptable quality of the herbal medicine In this context, the term quality is used to define the overall fitness of the herbal medicament for its intended use, and includes the presence of one or more of the bioactive principles (at an appropriate concentration) described above or else the presence of one or more bioactive markers or a phytochemical profile which correlates with the presence of one or more of the bioactive principles (at an appropriate concentration).

The term phytochemical profile is used herein to define a set of characteristics relating to different phytochemical constituents.

The term adjunctive (as applied to the use of the drugs of the invention in therapy) defines uses in which the pyrrolidine alkaloid 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 pyrrolidine compound of the invention and the other treatment(s). Thus, in some embodiments, adjunctive use of the pyrrolidine compound 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 pyrrolidine compound 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 pyrrolidine compound of the invention may be reflected in the composition of the pharmaceutical kits of the invention, wherein the pyrrolidine compound 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 pyrrolidine compound of the invention may be reflected in the content of the information and/or instructions co-packaged with the pyrrolidine compound relating to formulation and/or posology.

The term non-pyrogenic as applied to the pharmaceutical compositions of the invention defines compositions which do not elicit undesirable inflammatory responses when administered to a patient.

The term isolated as applied to the compounds of the invention is used herein to indicate that the compound exists in a physical milieu distinct from that in which it occurs in nature or in a state of at least partial purification from reagents used in its synthesis. 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 isolated material 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, 99% w/w or higher. In some circumstances, the isolated compound forms 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 compound may be purified to essential homogeneity, for example as determined spectrophotometrically, by NMR or by chromatography (for example GC-MS).

The term pharmaceutically acceptable derivative as applied to the pyrrolidine compounds of the invention define compounds which are obtained (or obtainable) by chemical derivatization of the parent pyrrolidine compounds of the invention. The pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with the tissues of humans 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 pyrrolidine compounds of the invention. The derivatives may be immunostimulatory perse, 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 immunostimulatory activity of the parent compound. In some cases, the immunostimulatory activity is increased by derivatization. Derivatization may also augment other biological activities of the compound, for example bioavailability and/or glycosidase inhibitory activity and/or glycosidase inhibitory profile. For example, derivatization may increase glycosidase inhibitory potency and/or specificity. The derivatives of the invention may include bioisosteres (as hereinbelow defined).

The term pharmaceutically acceptable salt as applied to the pyrrolidine compounds of the invention defines any non-toxic organic or inorganic acid addition salt of the free base compounds which are suitable for use in contact with the tissues of humans and lower animals 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 pyrrolidine drugs 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 pyrrolidine compounds 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.

These salts and the free base compounds can exist in either a hydrated or a substantially anhydrous form. Crystalline forms of the compounds of the invention are also contemplated and in general the acid addition salts of the pyrrolidine compounds 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 pyrrolidine compounds of the invention. Those skilled in the art will appreciate that, owing to the asymmetrically substituted carbon atoms present in the compounds of the invention, the pyrrolidine compounds of the invention may exist and be synthesised and/or isolated in optically active and racemic forms. Thus, references to the pyrrolidine compounds of the present invention encompass the pyrrolidine compounds 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 compounds of the invention, and unless indicated otherwise (e.g. by use of dash-wedge structural formulae) the compounds shown herein are intended to encompass ail possible optical isomers of the compounds so depicted. In cases where the stereochemical form of the compound is important for pharmaceutical utility, the invention contemplates use of an isolated eutomer. The term bioisostere (or simply isostere) is a term of art used to define alkaloid analogues in which one or more atoms (or groups of atoms) have been substituted with replacement atoms (or groups of atoms) having similar steric and/or electronic features to those atoms which they replace. The substitution of a hydrogen atom or a hydroxyl group with a fluorine atom is a commonly employed bioisosteric replacement. Sila-substitution (C/Si- exchange) is a relatively recent technique for producing isosteres. This approach involves the replacement of one or more specific carbon atoms in a compound with silicon (for a review, see Tacke and Zilch (1986) Endeavour, New Series 10: 191-197). The sila-substituted isosteres (silicon isosteres) may exhibit improved pharmacological properties, and may for example be better tolerated, have a longer half-life or exhibit increased potency (see for example Englebienne (2005) Medicinal Chemistry, Vol. 1(3): 215-226). In its broadest aspect, the present invention contemplates all bioisosteres (and specifically, all silicon bioisosteres) of the pyrrolidine compounds of the invention.

The term immunomodulatory as applied to the compounds of the invention is intended to define the ability to stimulate and/or suppress one or more components or activities of the immune system (e.g. the mammalian immune system) in vivo or in vitro. Immunomodulatory activity may be determined by in vitro cytokine release assays (for example using one or more immune cells, e.g. macrophage, dendritic or spleen cells). Preferred immunomodulatory compounds of the invention stimulate the release of one or more cytokines (e.g. IL-12) in vitro (for example, in spleen cells, macrophages and/or dendritic cells). Other preferred compounds suppress the activity of one or more cytokines (e.g. IL-12, TNF-alpha and/or IFN-g): such compounds may find particular application in the treatment or prophylaxis of autoimmune and allergic disorders (as described in more detail infra).

The term p7-viroporin virus defines a virus which express one or more p7 proteins (for example, the HCV p7 protein or homologues thereof), thought to mediate cation permeability across host membranes and so facilitate virion release or maturation. Examples of p7-viroporin viruses include members of the genera Pβstivirus and Hβpacivirus, which includes the causative agents of numerous human diseases and a variety of animal diseases which cause significant losses to the livestock industry. In particular, p7-viroporin viruses include pestiviruses such as bovine viral diarrhoea virus (BVDV), classical swine fever virus and border disease virus and hepaciviruses such as HCV (which causes hepatitis C in man).

The term a/Ay/ defines the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched- chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain, C3-C30 for branched chain), and more preferably up to 20, 15, 12, 10, 8 or 6. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

The terms alkenyl and alkynyl define unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term aryl as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. Those aryl groups having heteroatoms in the ring structure may also be referred to as heteroaryls, aryl heterocycles or heteroaromatics.

The term aralkyl defines an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

As used herein, the term "pharmaceutical kit" defines an array of one or more unit doses of a pharmaceutical composition together with dosing means (e.g. measuring device) and/or delivery means (e.g. inhaler or syringe), optionally all contained within common outer packaging. In pharmaceutical kits comprising a combination of two or more compounds, the individual compounds may unitary or non-unitary formulations.

Biological activities of the compounds of the invention

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

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

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

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

• Modification of cell-surface protein glycosylation in infected host cells

• Modification of bacterial cell walls

Thus, the compounds of the invention may:

(a) modify tumour cell glycosylation (e.g. tumour antigen glycosylation); and/or

(b) modify viral protein glycosylation (e.g. virion antigen glycosylation); and/or

(c) modify cell-surface protein glycosylation in infected host cells; and/or (d) modify bacterial cell walls,

when administered in vivo.

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

The compounds of the invention may inhibit ER α-glucosidases. Such compounds may be identified by standard enzymological assay. Preferred are compounds which specifically inhibit ER α-glucosidases (for example, which specifically inhibit ER α-glucosidase I and/or ER α-glucosidase II, relative to other mammalian glycosidase enzymes). Most preferably, the compounds of the invention inhibit ER α-glucosidase I and/or ER α-glucosidase Il with a degree of specificity such that gastrointestinal toxicity via disaccharidase inhibition on administration at antiviral concentrations in humans is absent (or present at clinically acceptable or subtoxic levels).

Thus, the compounds of the invention may be (but are not necessarily) glycosidase inhibitors. Particularly preferred are compounds which exhibit specificity of glycosidase inhibition, for example Glucosidase I rather than mannosidase. Such compounds are therefore be quite different in their glycosidase inhibitory profile to swainsonine and its analogues, since the latter are potent and specific inhibitors of mannosidase.

Viral p7 protein inhibition and ion channel interference

Alternatively, or in addition, the compounds may inhibit the activity of a viral p7 protein (for example, acting as viral ion channel blockers). Such compounds may be identified by the methods described for example in Pavlovic et al. (2003) PNAS 100(10): 6104-6108 (the relevant methodological disclosure of which is incorporated herein by reference).

In such embodiments, the compounds of the invention may not inhibit ER α-glucosidases at physiologically significant levels in vivo (and may not exhibit significant ER α-glucosidase I or Il inhibitory activity in vitro). Indeed, in such embodiments the compounds of the invention may exhibit poor glucosidase inhibitory activity (relative to castanospermine and DNJ as reference glucosidase inhibitors) and may therefore exhibit levels of glucosidase inhibition which are so low as to permit viral glycoprotein processing on administration at antiviral concentrations in humans (the antiviral activity in such embodiments being mediated independently of glucosidase inhibition).

Without wishing to be bound by any theory, it is thought that antiviral activity in such embodiments of the invention may arise from: (a) direct interaction of the compounds of the invention with viral p7molecules, either blocking the p7-derived ion channels or preventing them from forming and/or opening; and/or (b) effecting a change to the membrane bilayer (for example by accumulating therein), so preventing p7 molecules from assembling into channel-forming pores.

In this embodiment, the invention finds particular application in the treatment or prevention of any infection mediated by p7-viroporin viruses, which include pestiviruses and hepaciviruses (so including the treatment or prevention of infections involving members of the genera Pestivirus and Hepacivirus, including the HCV and BVDV viruses, as discussed infra).

Cytokine modulation

Without wishing to be bound by any theory, it is thought that the pyrrolidine compounds of the invention mediate immunomodulatory activity via the modulation of cytokine secretion patterns in vivo. In particular, it is thought that the pyrrolidine compounds of the invention may act at least in part by stimulating the activity of one or more cytokines and/or the suppressing the activity of one or more cytokines in vivo. In preferred embodiments, the pyyrolidine compounds of the invention may act at least in part by stimulating the activity of one or more Th1 cytokines and/or suppressing the activity of one or more Th2 cytokines in vivo. For example, the compounds may stimulate IL12 and/or IL-2 activity or secretion in vivo or in vitro (for example in lymphocytes and/or dendritic cells). Particularly preferred are compounds that stimulate the production of 1L-2 in dendtritic cells in vitro.

IL-2 is a Th1 cytokine involved in mediating type-1 responses. It appears to be involved not only in T cell activation but also in the activation of Inter alia NK cells, so functioning to regulate and link innate and adaptive immunity. Thus, stimulation of IL-2 in dendritic cells may directly potentiate a Th1 immune response. Stimulation of IL-2 may also indirectly potentiate a Th1 response (and so increase the Th1:Th2 response ratio) by stimulating the activity of endogenous dendritic cells, which cells then trigger responses by other classes of lymphocytes (CTL, B, NK, and NKT cells) and also elicit T cell memory (a critical goal of vaccination).

IL-12 is the primary mediator of type-1 immunity (the Th1 response). It induces natural killer (NK) cells to produce IFN-γ as part of the innate immune response and promotes the expansion of CD4⁺ TM cells and cytotoxic CD8⁺ cells which produce IFN-γ. It therefore increases T-cell invasion of tumours as well as the susceptibility of tumour cells to T-cell invasion. Stimulation of IL-12 (for example in dendritic cells and/or macrophages) may therefore also potentiate a immune response. Stimulation of the expression of IL-12 can overcome the suppression of innate and cellular immunities of HIV-1 -infected individuals and AIDS patients.

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

Other activities

Alternatively, or in addition, the compounds may exert antiviral activity independently of α-glucosidase inhibition or p7 interference. For example, the compounds of the invention may exert an antiviral effect mediated by an immunomodulatory activity (as proposed in Mehta et al. (2004) Antimicrobial Agents and Chemotherapy 48(6): 2085-2090), for example by activating components of the innate immune system by a TLR-distinct or NF-KB- independent mechanism, by inducing interferon expression or by acting as interferon surrogates in vivo.

The compounds of the invention may exert an antiviral effect mediated by inhibition of other enzymes, for example viral enzymes involved or required for viral pathogenicity (for example neuraminidase).

Medical applications of the compounds of the invention

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

These medical applications may be applied to any warm-blooded animal, including humans. The applications include veterinary applications, wherein the pyrrolidine compounds of the invention are administered to non- human animals, including primates, dogs, cats, horses, cattle and sheep. The pyrrolidine compounds of the invention may act as immunomodulators. Thus, they find general application in the treatment or prophylaxis of conditions in which stimulation, augmentation or induction of the immune system is indicated or in which suppression or elimination of part or all of the immune response is indicated.

The pyrrolidine compounds of the invention may also act have antiviral activity. Thus, they find general application in the treatment or prophylaxis of viral infections. In such applications, the pyrrolidine compounds of the invention may act as glycovirs, glucovirs and/or alkovirs.

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

1. Increasing the Th1 :Th2 response ratio

General considerations

The immune response comprises two distinct types: the Th1 response (type-1, cellular or cell mediated immunity) and Th2 response (type-2, humoral or antibody mediated immunity).

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

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

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

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

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

Accordingly, in the mouse, a cell that makes IFN-gamma and not IL-4 is classified as Th1, whereas a CD4⁺ cell that expresses IL-4 and not IFN-gamma is classified as Th2. Although this distinction is less clear in humans (T cells that produce only Th1 or Th2 cytokines do not appear to exist in humans), the phenotype of the T cell response (Th1 or Th2) can still be distinguished in humans on the basis of the ratio of Th1 to Th2 cytokines expressed (usually, the ratio of IFN-gamma to IL-4 and/or IL-5). There is an increasing realization that the type of immune response is just as important in therapy and prophylaxis as its intensity or its duration. For example, an excess Th1 response can result in autoimmune disease, inappropriate inflammatory responses and transplant rejection. An excess Th2 response can lead to allergies and asthma. Moreover, a perturbation in the Th1 :Th2 ratio is symptomatic of many immunological diseases and disorders, and the development of methods for altering the Th1:Th2 ratio is now a priority.

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

Thus, the compounds of the invention find application in methods of therapy and/or prophylaxis which comprise increasing the Th1:Th2 response ratio (for example, by preferentially promoting a Th1 response and/or preferentially suppressing a Th2 response).

The medical applications contemplated herein therefore include any diseases, conditions or disorders in which an increase in the Th1:Th2 response ratio is indicated or desired. For example, the medical applications contemplated include diseases, conditions or disorders in which stimulation of a Th 1 response and/or suppression of a Th2 response is indicated or desired.

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

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

The compounds of the invention may also suppress or inactivate (either directly or indirectly) the release and/or activity (in vitro and/or in vivo) of one or more Th2 cytokines (for example one or more cytokines selected from IL-4, IL-5 and IL-13). Particularly preferred are compounds which suppress or inactivate the release and/or activity (in vitro and/or in vivo) of IL-5. Thus, particularly preferred are compounds which exhibit a Th1 cytokine stimulatory activity together with a complementary Th2 cytokine inhibitory activity.

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

Th1-related diseases

Th1 -related diseases are diseases, disorders, syndromes, conditions or infections in which Th1 cells are involved in preventing, curing or alleviating the effects of the disease, disorder, syndrome, condition or infection. Thus, the compounds of the invention find application in the treatment or prophylaxis of Th1 -related diseases.

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

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

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

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

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

Th2-related diseases and allergy Th2-related diseases are diseases, disorders, syndromes, conditions or infections in which Th2 cells are implicated in (e.g. support, cause or mediate) the effects of the disease, disorder, syndrome, condition or infection. Thus, the compounds of the invention find application in the treatment or prophylaxis of Th2-related diseases. One important class of Th2-related diseases treatable with the compounds of the invention is allergic disease.

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

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

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

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

Allergic disorders

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

Particularly preferred for use in the treatment or prophylaxis of allergic disorders are compounds which are specific glucosidase inhibitors. Such compounds may specifically inhibit ER α-glucosidases (for example, which specifically inhibit ER α-glucosidase I and/or ER α-glucosidase II, relative to other mammalian glycosidase enzymes). Most preferably, the compounds of the invention inhibit ER α-glucosidase I and/or ER α-glucosidase Il with a degree of specificity such that gastrointestinal toxicity via disaccharidase inhibition on administration at antiviral concentrations in humans is absent (or present at clinically acceptable or subtoxic levels). Particularly important is the treatment or prophylaxis of asthma.

2. Haemorestoration

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

Haemorestoration may be indicated following 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 pyrrolidine compounds of the invention as haemorestoratives may be adjunctive to other treatments which tend to depress splenic and bone marrow cell populations. Particularly preferred adjunctive therapies according to the invention include the administration of an immunorestorative dose of the pyrrolidine compound of the invention adjunctive to: (a) chemotherapy; and/or (b) radiotherapy; and/or (c) bone marrow transplantation; and/or (d) haemoablative immunotherapy. 3. Alleviation of immunosuppression

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

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

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

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

4. Cytokine stimulation

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

Accordingly, the pyrrolidine compounds of the invention find general application in the treatment or prophylaxis of conditions in which the in vivo induction, potentiation or activation of one or more cytokines (e.g. IL-12) is indicated. Such applications may be employed to stimulate particular elements of the cellular immunity system, including macrophages (e.g. tissue-specific macrophages), NK and LAK cells. 5. Cytokine suppression

The pyrrolidine compounds of the invention may be used to suppress, repress, block or inactivate various cytokines in vivo, including IFN-g and/or TNF-alpha.

Accordingly, the pyrrolidine compounds of the invention find general application in the treatment or prophylaxis of conditions in which the in vivo suppression, repression, blockade or inactivation of one or more cytokines (e.g. IFN-g) is indicated. Such applications may be employed in the treatment of allergic and autoimmune disorders. 6. Treatment of proliferative disorders

Thus, the invention finds application in the treatment or prophylaxis of any proliferative disorder, including various cancers and cancer metastasis. For example, the invention finds application in the treatment of breast cancer, colon cancer, lung cancer and prostate cancer. It also finds application in the treatment or prophylaxis of cancers of the blood and lymphatic systems (including Hodgkin's Disease, leukemias, lymphomas, multiple myeloma, and Waldenstrom's disease), skin cancers (including malignant melanoma), cancers of the digestive tract (including head and neck cancers, salivary gland, oesophageal cancer, stomach cancer, cancer of the pancreas, liver cancer, colon and rectal cancer, anal cancer), lung cancers, cancers of the genital and urinary systems (including kidney cancer, bladder cancer, testis cancer, prostate cancer), cancers in women (including breast cancer, ovarian cancer, gynecological cancers (e.g. cervico-uterine cancers and choriocarcinoma) as well as in brain, adenomas, sarcomas (e.g. Kaposi's Sarcoma, particularly when associated with AIDS), bone carcinoid, nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. It also finds application in the treatment or prophylaxis of cancers of unknown primary site.

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

7. Use as adjuvant

The pyrrolidine compounds of the invention find utility as vaccine adjuvants, in which embodiments they may promote, induce or enhance an immune response to antigens, particularly antigens having low intrinsic immunogenicity (for example in subunit vaccines).

They may also be used as adjuvants to skew the immune response towards a Th-1 response (for example in applications where a cell-based immune response component is desired).

Without wishing to be bound by any theory, the pyrrolidine compounds of the invention may augment vaccine immunogenicity by stimulating cytokine release, thereby promoting T-cell help for B cell and CTL responses. They may also change glycosylation of cancer or viral antigens and increase vaccine effectiveness. When used as adjuvant, the compounds of the invention may be administered concurrently, separately or sequentially with administration of the vaccine. Thus, in some embodiments, the pyrrolidine compound of the invention may be present in admixture with other vaccine component(s), or else co-packaged (e.g. as part of an array of unit doses) with the other vaccine components with which it is to be used as adjuvant. In yet other embodiments, the use of the pyrrolidine compounds of the invention as adjuvant is simply reflected in the content of the information and/or instructions co-packaged with the vaccine components and relating to the vaccination procedure, vaccine formulation and/or posology.

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

Particularly preferred is the treatment or prophylaxis of infections in which the pathogen occupies an intracellular compartment, including HIV/AIDS, influenza, tuberculosis and malaria.

8. Dendritic cell-based applications

As explained earlier, dendritic cells are a rare and heterogeneous cell population with distinctive morphology and a widespread tissue distribution (see Steinman (1991) Ann. Rev. Immunol. 9: 271-296). They play an important role in antigen presentation, capturing and processing antigens into peptides and then presenting them (together with components of the MHC) to T cells.

Dendritic cells therefore play an important regulatory role in the magnitude, quality, and memory of the immune response and there is growing interest in the use of dendritic cells in various immunomodulatory interventions.

Dendritic cell vaccines

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

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

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

In particular, the compounds of the invention can induce the production of one or more Th1 cytokines (for example IL-12 and/or IL-2) in dendritic cells.

The compounds of the invention may therefore be used as components in a dendritic cell based vaccine. As such, they may act to enhance the function of the dendritic cell component, for example by stimulating the production of one or more cytokine(s) (preferably one or more TM cytokines).

Thus, the invention contemplates a dendritic cell based vaccine comprising a pyrrolidine compound of the invention in admixture therewith. Preferably, the dendritic cell based vaccine of the invention comprises dendritic cells which are primed with antigen(s).

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

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

Thus, the invention finds application in the treatment or prophylaxis of breast cancer, colon cancer, lung cancer and prostate cancer. It also finds application in the treatment or prophylaxis of cancers of the blood and lymphatic systems (including Hodgkin's Disease, leukemias, lymphomas, multiple myeloma, and Waldenstrom's disease), skin cancers (including malignant melanoma), cancers of the digestive tract (including head and neck cancers, oesophageal cancer, stomach cancer, cancer of the pancreas, liver cancer, colon and rectal cancer, anal cancer), cancers of the genital and urinary systems (including kidney cancer, bladder cancer, testis cancer, prostate cancer), cancers in women (including breast cancer, ovarian cancer, gynecological cancers and choriocarcinoma) as well as in brain, bone carcinoid, nasopharyngeal, retroperitoneal, thyroid and soft tissue tumours. It also finds application in the treatment or prophylaxis of cancers of unknown primary site. The dendritic cell based vaccines of the invention also find application in the treatment or prophylaxis of various infections, including bacterial, viral, fungal, protozoan and metazoan infections. For example, the vaccines may be used in the treatment or prophylaxis of infection with respiratory syncytial virus (RSV), Epstein-Barr, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, hantann virus (hemorrhagic fever), human papilloma virus (HPV), tuberculosis, leprosy and measles.

Dendritic cell-based approaches to autoimmune disorders

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

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

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

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

9. Wound healing

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

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

10. Antiviral applications

Target viral infections

The invention finds broad application in the treatment or prevention of all viral infections, including for example infections, diseases and disorders in which any of the following viruses (or virus classes) are implicated:

Retroviridae (e.g. the human immunodeficiency viruses, including HIV-1); Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses, including SARS coronavirus); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus);

Orthomyxoviridaβ (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridaβ (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adβnoviridae (most adenoviruses); Herpβsvlridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the HCV virus (causing non-A, non-B hepatitis); Norwalk and related viruses, and astroviruses). Of the foregeoing, particularly preferred are HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus, poliovirus, influenza virus (including influenza A and influenza B virus), meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, haemorrhagic fever viruses and Herpes viruses, particularly, varicella, cytomegalovirus and Epstein-Barr virus.

The invention finds particular application in the treatment or prevention of infections mediated by enveloped viruses. Examples of enveloped virus families and some human species within the families include Poxviridae, e.g. vaccinia and smallpox, Iridoviridae, Herpesviridae, e.g. Herpes simplex, Varicella virus, cytomegalovirus and Eppstein-Barr virus, Togaviridae, e.g. Yellow fewer virus, thick-borne encephalitis virus, Rubella virus and tropical encephalitis virus, Coronaviridae, e.g. Human coronovirus, Paramyxoviridae, e.g. Parainfluenza, mumps virus, measles virus and respiratory syncytial virus, Rabdoviridae, e.g. vesicular stomatitis virus and rabies virus, Filoviridae, e.g. Marburg virus and Ebola virus, Orthomyxoviridae, e.g. Influenza A, B and C viruses, Bunyaviridae, e.g. Bwamba virus, California encephalitis virus, sandfly fever virus and Rift Valley fever virus, Arenaviridae, e.g. LCM virus, Lassa virus and Juni virus, Hepnadnaviridae, e.g. hepatitis B-virus, and Retroviridae, e.g. HTLV and HIV-1 and HIV-2; Flaviviridae; Rhabdoviridae. These viruses and others are responsible for such diseases as encephalitis, intestinal infections, immunosuppressive disease, respiratory disease, hepatitis and pox infections. The Paramyxoviridae are enveloped viruses that include, among others, mumps virus, measles virus, Sendai virus, Newcastle disease virus (NDV), human respiratory syncytial virus (RSV), parainfluenza virus 5 (SV5) and human parainfluenza viruses 1-4 (hPIV)1. Many members of this viral family are significant human and animal pathogens, and newly emergent deadly paramyxoviruses (Nipah and Hendra viruses) have been identified.

The flavivirus group (family Flaviviridae) comprises the genera Flavivirus, Pestivirus and Hepacivirus and includes the causative agents of numerous human diseases and a variety of animal dieases which cause significant losses to the livestock industry.

The family Flaviviridae (members of which are referred to herein as flaviviruses) include the genera Flavivirus (e.g. yellow fever virus, dengue viruses, Japanese encephalitis virus, Murray Valley encephalitis virus, West Nile fever virus, Rocio virus, St. Louis encephalitis virus, Louping ill virus, Powassan virus, Omsk hemorrhagic fever virus, Kyasanur forest disease virus and tick-borne encephalitis virus), Pestivirus (e.g. bovine viral diarrhoea virus, rubella virus, classical swine fever virus, hog cholera virus and border disease virus), Hepacivirus (hepatitis C virus) and currently unclassified members of the Flaviviridae (e.g. GB virus types A, B and C).

The full list of members of the Flaviviridae are defined in detail by the International Committee on Taxonomy of Viruses (the currently accepted taxanomic definition is described in: Virus Taxonomy: The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses (M.H.V. van Regenmortel, CM. Fauquet, D.H.L. Bishop, E.B. Carstens, M. K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, DJ. McGeoch, CR. Pringle, R.B. Wickner (2000). Virus Taxonomy, Vllth report of the ICTV. Academic Press, SanDiego), the content of which relating to the constitution of the family Flaviviridae is hereby incorporated by reference.

One particularly important flavivirus is the hepatitis C virus (HCV). HCV is an enveloped plus-strand RNA virus belonging to the Flaviviridae family, but classified as a distinct genus Hepacivirus. It was first identified in 1989 and it has since become clear that this virus is responsible for most cases of post-transfusion non-A, non-B hepatitis. Indeed, HCV is now recognised as one of the commonest infections causing chronic liver disease and The World Health Organisation estimates that 170 million people are chronically infected. HCV infection results in a chronic infection in 85% of infected patients and approximately 20-30% of these will progress to cirrhosis and end stage liver disease, frequently complicated by hepatocellular carcinoma.

The study of HCV has been hampered by the inability to propagate the virus efficiently in cell culture. However, in the absence of a suitable cell culture system able to support replication of human HCV, BVDV is an accepted cell culture model. HCV and BVDV share a significant degree of local protein homology, a common replication strategy and probably the same subcellular location for viral envelopment.

The invention therefore finds particular application in the treatment or prevention of HCV infection (e.g. in the treatment or prevention of hepatitis C).

The combination therapy of the invention may therefore be applied to other viral infections involving glycosylated envelope proteins, such as Hepatitis A, B and C, Herpes Simplex virus 1 and 2, Epstein Barr Virus, Herpes zoster virus, other Herpesviridiae, Influenza virus and Newcastle disease virus infections. Particularly preferred is the treatment or prophylaxis of HIV (particularly HIV-1) infection, influenza A and B, SARS coronavirus and HCV. The invention (and in particular the combined use of a glycosylation modulator and a membrane fusion inhibitor) will be of particular use in the treatment of HIV-infected patients, in particular such patients who have previously been treated with other known anti-HIV therapies (and where for example the viral infection has not been effectively controlled by the existing treatment regime, for example because of viral resistance).

Thus, the invention finds broad application in the treatment or prevention of all viral infections, including for example infections, diseases and disorders in which any of the following viruses (or virus classes) are implicated:

Retroviridae (e.g. the human immunodeficiency viruses, including HIV-1); Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridaβ (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reovlridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hβpadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovavlrldae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridaβ (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the HCV virus (causing non-A, non-B hepatitis); Norwalk and related viruses, and astroviruses). Of the foregeoing, particularly preferred are HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus, poliovirus, influenza virus (including influenza A and influenza B virus), meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, haemorrhagic fever viruses and Herpes viruses, particularly, varicella, cytomegalovirus and Epstein-Barr virus.

In particular, the invention finds application in the treatment or prevention of infections mediated by:

(a) a virus which acquires its envelope from a membrane associated with the intracellular membrane of an infected cell; and/or (b) a virus which replicates via cooperation with the endoplasmic reticulum (or the membrane surrounding the lumen of the endoplasmic reticulum) in the host cell; and/or

(c) a virus which replicates via cooperation with the Golgi apparatus (or the membrane of the lumen of the Golgi apparatus) in the host cell; and/or

(d) a virus which encodes one or more glycoproteins which depend on calnexin and/or calreticulin interaction for proper folding; and/or

(e) a p7-viroporin virus (as hereinbefore defined); and/or

(f) a virus which requires neuraminidase for pathogenicity. Viruses of classes (a)-(d)

The invention finds broad application in the treatment or prevention of any infection mediated by viruses of these classes. In particular, the invention finds application in the treatment or prevention of infections involving flaviviruses.

The family Flaviviridae (members of which are referred to herein as flaviviruses) include the genera Flavivirus (e.g. yellow fever virus, dengue viruses, Japanese encephalitis virus, Murray Valley encephalitis virus, West Nile fever virus, Rocio virus, St. Louis encephalitis virus, Louping ill virus, Powassan virus, Omsk hemorrhagic fever virus, Kyasanur forest disease virus and tick-borne encephalitis virus), Pestivirus (e.g. bovine viral diarrhoea virus, rubella virus, classical swine fever virus, hog cholera virus and border disease virus), Hepacivirus (hepatitis C virus) and currently unclassified members of the Flaviviridae (e.g. GB virus types A, B and C). The full list of members of the Flaviviridae are defined in detail by the International Committee on Taxonomy of Viruses (the currently accepted taxanomic definition is described in: Virus Taxonomy: The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses (M.H.V. van Regenmortel, CM. Fauquet, D.H.L. Bishop, E.B. Carstens, M. K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, CR. Pringle, R.B. Wickner (2000). Virus Taxonomy, Vllth report of the ICTV. Academic Press, SanDiego), the content of which relating to the constitution of the family Flaviviridae is hereby incorporated by reference.

P7-viroporin viruses of class (e)

The invention finds broad application in the treatment or prevention of any infection mediated by p7-viroporin viruses, which include pestiviruses and hepaciviruses. Thus, the invention finds particular application in the treatment or prevention of infections involving members of the genera Pestivirus and Hepacivirus (including the HCV and BVDV viruses, as discussed above).

Neuraminidase viruses of class ffl Influenza virus neuraminidase (NA) is a subtype-specific, transmembrane glycoprotein of the class Il type and, like haemagglutinin (HA), undergoes antigenic variation. Neuraminidase is also functionally important for the removal of sialic acid residues from various glycoproteins on the host-cell surface that potentially bind viral glycoproteins and hence restrict virion egress. NA activity is necessary to prevent clumping and allow the release of virus progeny from the host cell.

Thus, NA is a potential target in the treatment of influenza virus infection and NA inhibitors have recently found application in the treatment of influenza virus infection.

Auxiliary antiviral agents for use in combination with the pyrrolidine compounds of the invention In addition to the viral entry inhibitor and adjunctive agent selected from: (a) a glycosylation modulator; (b) an alkovir; and (c) a glycovir (e.g. a glucovir), the invention also contemplates the use of one or more of the following auxiliary antiviral agents as further components of the invention. This is particularly advantageous in the case where the invention is applied to the treatment of HIV infection (AIDS). In the following list, both the trade name, the various generic name(s) and drug code(s) are listed, together with the manufacturing pharmaceutical company.

Protease Inhibitors (PIs)

One or more of the following protease inhibitors may be used:

(a) Agenerase® amprenavir APV 141W94 or VX-478 (GlaxoSmithKline) (b) Aptivus® tipranavir TPV PNU-140690 (Boehringer Ingelheim)

(c) Crixivan® indinavir IDV MK-639 (Merck & Co)

(d) Fortovase® saquinavir (Soft Gel Cap) SQV (SGC) (Hoffmann-La Roche)

(e) Invirase® saquinavir SQV Ro-31-8959 (Hoffmann-La Roche)

(f) Kaletra® lopinavir + ritonavir LPV ABT-378/r (Abbott Laboratories) (g) Lexiva® fosamprenavir FPV GW-433908 or VX-175 (GlaxoSmithKline)

(h) Norvir® ritonavir RTV ABT-538 (Abbott Laboratories)

(i) Reyataz® atazanavir ATZ BMS-232632 (Bristol-Myers Squibb)

G) Viracept® nelfinavir NFV AG-1343 (Pfizer)

(k) Brecanavir™ GW640385 or VX-385 (GlaxoSmithKline) (I) Darunavir™ TMC-114 (Tibotec)

Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIsI One or more of the following NRTIs may be used:

(a) Combivir® zidovudine + lamivudine AZT + 3TC (GlaxoSmithKline) (b) Emtriva® emtricitabine FTC (Gilead Sciences)

(c) Epivir® lamivudine 3TC (GlaxoSmithKline)

(d) Epzicom™ abacavir + lamivudine ABC + 3TC (GlaxoSmithKline)

(e) Hivid® zalcitabine ddC (Hoffmann-La Roche)

(f) Retrovir® zidovudine AZT or ZDV (GlaxoSmithKline) (g) Trizivir® abacavir + zidovudine + lamivudine ABC + AZT + 3TC (GlaxoSmithKline)

(h) Truvada® tenofovir DF + emtricitabine TDF + FTC (Gilead Sciences)

(i) Videx® didanosine: buffered versions ddl BMY-40900 (Bristol-Myers Squibb)

0) Videx® EC didanosine: delayed-release capsules ddl (Bristol-Myers Squibb) (k) Viread® tenofovir disoproxil fumarate (DF) TDF or Bis(POC) PMPA (Gilead Sciences)

(I) Zerit® stavudine d4T BMY-27857 (Bristol-Myers Squibb)

(m) Ziagen® abacavir ABC 1592U89 (GlaxoSmithKline)

(n) Reverset™ dexelvucitabine DFC (Pharmasset and Incyte)

(o) Alovudine™ MIV-310 (Boehringer Ingelheim) (p) Amdoxovir™ DAPD (Gilead Sciences)

(q) Elvucitabine™ Beta-L-Fd4C ACH-126,443 (Achillion Pharmaceuticals)

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) One or more of the following NNRTIs may be used: (a) Rescriptor® delavirdine DLV U-90152S/T (Pfizer)

(b) Sustiva® efavirenz EFV DMP-266 (Bristol-Myers Squibb)

(c) Viramune® nevirapine nivirapine NVP BI-RG-587 (Boehringer Ingelheim)

(d) (+)-calanolide A (Sarawak Medichem)

(e) etravirine TMC-125 (Tibotec) (f) TMC-278 (Tibotec)

(g) BMS-561390 or DPC-083 (Bristol-Myers Squibb)

Immune-Based Therapies

One or more of the following may also be used: (a) Proleukin® aldesleukin, or lnterleukin-2 IL-2 (Chiron Corporation)

(b) Remune® HIV-1 Immunogen, or SaIk vaccine AG1661 (The Immune Response Corporation)

(c) One or more interferons.

Other Classes of Anti-HIV Drugs (a) Integrase Inhibitors: e.g. MK-0518 (Merck & Company)

(b) Maturation Inhibitors: e.g. PA-457 (Panacos Pharmaceuticals)

(c) Cellular Inhibitors: e.g. Droxia® hydroxyurea HU (Bristol-Myers Squibb)

In embodiments where HIV infection (AIDS) is treated or prevented by the compounds of the invention, two or more auxiliary antiviral agents independently selected from two or more distinct classes (viz. PIs, NRTIs and NNRTIs) are preferably used. Thus, if desired, a glycosylation modulator and a membrane fusion inhibitor may be used in further combination with other anti-HIV therapeutics such as, but not limited to, zidovudine, lamivudine, nelfinavir, indinavir and efavirenz.

In embodiments where HIV infection (AIDS) is treated or prevented by the compounds of the invention, the use of the combinations of the invention may advantageously form part of a HAART or E-HAART treatment regimen (combination of several (typically three or four) antiretroviral drugs is known as Highly Active Anti-Retroviral Therapy (HAART). Where one or more of these drugs acts extracellularly, then the regimen is known as E- HAART). Other co-therapeutic agents for use with the pyrrolidine compounds of the invention The compounds of the invention may be co-administered with a variety of other co-therapeutic agents which treat or prevent side effects arising from the anti-viral treatment and/or presenting as sequelae of the viral infection. For example (and particularly in the treatment of HIV infection (AIDS), co-therapeutic agents which treat or prevent any of the following side effects may be used as part of the same treatment regimen as the compounds of the invention: (a) lipodystrophy and wasting; (b) facial lipoatrophy; (c) hyperlipidemia; (d) fatigue; (e) anemia; (f) peripheral neuropathy; (g) nausea; (h) diarrhoea; (i) hepatotoxicity; (j) osteopenia and (k) osteoporosis.

The compounds of the invention may be co-administered with a variety of antimicrobial agents as co- therapeutic agents which treat or prevent opportunistic infections arising from the anti-viral treatment and/or presenting as sequelae of the viral infection. For example (and particularly in the treatment of HIV infection (AIDS), antimicrobial agents which treat or prevent bacterial, fungal, metazoan or protozoan infections may be used as part of the same treatment regimen as the compounds of the invention.

11. Autoimmune disorders

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

Particularly preferred for use in the treatment or prophylaxis of autoimmune disorders are compounds which are specific glucosidase inhibitors. Such compounds may specifically inhibit ER α-glucosidases (for example, which specifically inhibit ER α-glucosidase I and/or ER α-glucosidase II, relative to other mammalian glycosidase enzymes). Most preferably, the compounds of the invention inhibit ER α-glucosidase I and/or ER α-glucosidase Il with a degree of specificity such that gastrointestinal toxicity via disaccharidase inhibition on administration at antiviral concentrations in humans is absent (or present at clinically acceptable or subtoxic levels).

The compounds are preferably formulated for oral delivery in applications where delivery to the Gl tract is indicated (for example, in the treatment or prophylaxis of Crohn's disease and inflammatory bowel disease).

The pyrrolidine compounds of the present invention can be administered by enteral (typically 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 pyrrolidine compound 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 pyrrolidine compound selected.

Moreover, the pyrrolidine compounds 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 pyrrolidine compound 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 pyrrolidine compound, and can be taken one or more times per day. The pyrrolidine compound 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 pharmaceutical compositions of the invention comprise the pyrrolidine compound of the invention, optionally together with a pharmaceutically acceptable excipient.

The pyrrolidine compound of the invention may take any form. It may be synthetic, purified or isolated from natural sources. When isolated from a natural source, the pyrrolidine compound of the invention may be purified. However, the compositions of the invention may take the form of herbal medicines, as hereinbefore defined. 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 herbal medicine 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 pyrrolidine compound of the invention is formulated together with a pharmaceutically acceptable excipient, any suitable excipient may be used, including for example inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc.

The pharmaceutical compositions may take any suitable form, and include for example tablets, elixirs, capsules, solutions, suspensions, powders, granules and aerosols. The pharmaceutical composition may take the form of a kit of parts, which kit may comprise the 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 pyrrolidine compound 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 pyrrolidine compound 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 pyrrolidine compound 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 compounds 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 compounds of the invention may also be presented as liposome formulations.

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

When used adjunctively, the pyrrolidine compounds of the invention may be formulated for use with one or more other drug(s). In particular, the pyrrolidine compounds of the invention may be used in combination with antitumor agents, antimicrobial agents, antiinflammatories, antiproliferative agents and/or other immunostimulatory agents. For example, the pyrrolidine compounds of the invention may be used with antiviral 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 pyrrolidine compound 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 pyrrolidine compound 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 pyrrolidine compound with antitumor agents, antimicrobial agents and/or antiinflammatories.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described.

Examples 1-3: Anti-BVDV activity

The hepatitis C virus (HCV) was first identified in 1989 and it has since become clear that this virus is responsible for most cases of post-transfusion non-A, non-B hepatitis. Indeed, HCV is now recognised as one of the commonest infections causing chronic liver disease and The World Health Organisation estimates that 170 million people are chronically infected. HCV infection results in a chronic infection in 85% of infected patients and approximately 20-30% of these will progress to cirrhosis and end stage liver disease, frequently complicated by hepatocellular carcinoma.

The study of HCV has been hampered by the inability to propagate the virus efficiently in cell culture. However, in the absence of a suitable cell culture system able to support replication of human HCV, bovine diarrhoea virus (BVDV) is an accepted cell culture model. HCV and BVDV share a significant degree of local protein homology, a common replication strategy and probably the same subcellular location for viral envelopment.

The ability of various compounds of the invention to exert a direct anti-BVDV effect in vitro was therefore tested and activity demonstrated in a BVDV plaque inhibition assay (as detailed below).

For these assays a confluent monolayer of MDBK cells is produced in a flat bottomed well of a tissue culture plate. The monolayer is infected with BVDV. Sufficient virus is added to eventually form approximately 20-30 plaques. After allowing approximately 1 hr for the virus to infect, the cells are washed and liquid agar is added and allowed to set as a thin layer over the cell surface (the 'overlay'). The infected cells are then left for a period of days to allow the virus to replicate and cells to shed virus, detach or lyse . Cells in the immediate vicinity of the initial virus infection are therefore infected - localized by the agar layer. Hence a clear plaque devoid of cells is eventually formed which after staining uninfected cells around it with neutral red is visible and can be scored.

The test compound is added at appropriate dilutions with the virus. An antiviral effect of the compound is scored by the reduction of plaque number or size. The concentration of compound required to produce a 50% (IC50) reduction of plaque number or size is noted. Controls of no compound added are included. A control of a known antiviral compound (castanospermine) are carried out to calibrate the antiviral activity.

Castanospermine, a known viral inhibitor (see infra), was used as a positive control. The following data was obtained:

Example 4: Toxicity assay

The compounds tested above were assayed for toxicity using a standard 'XTT colorimetric assay. In this assay the test compound, in the absence of virus was added to the cell monolayer. The cells and compound (and controls of cells without compound) were incubated for a period equivalent to the time required for viral plaques to be formed in the standard antiviral assay. XTT reagents are then added. XTT is metabolized by the mitochondria of viable cells producing an increase in absorbance at 450 nm. The effect of toxic compounds is to reduce this metabolism and generate less absorbance at 450 nm.

All compounds assayed at 200μg/ml showed approximately 15% reduction in absorbance with respect to no compound controls. This is in the range of a designation of 'not toxic' in this assay.

Example 5: Inhibition of qlvcosidase activity

All enzymes were purchased from Sigma, as were the appropriate p-nitrophenyl substrates. Assays were carried out in microtitre plates. Enzymes were assayed in 0.1 M citric acid/0.2M di-sodium hydrogen phosphate (Mcllvaine) buffers at the optimum pH for the enzyme. All assays were carried out at 2O⁰C. For screening assays the incubation assay consisted of 10 μl of enzyme solution, 10 μl of inhibitor solution (made up in water) and 50 μl of the appropriate 5 mM p-nitrophenyl substrate (3.57mM final cone.) made up in Mcllvaine buffer at the optimum pH for the enzyme.

The reactions were stopped with 0.4M glycine (pH 10.4) during the exponential phase of the reaction, which was determined at the beginning of the assay using blanks with water, which were incubated for a range of time periods to measure the reaction rate using 5 mM substrate solution. Endpoint absorbances were read at 405nm with a Biorad microtitre plate reader (Benchmark). Water was substituted for the inhibitors in the blanks.

The enzymes tested are shown in the table below.

The compounds tested are shown in the table below.

Notes to the table

¹The position of the methoxy group in this compound is tentative. The results (% inhibition) for these pyrroline and indolizidine compounds (all at 1mg/ml) are shown in the table below:

Further studies showed that the K_/ for casuarine (8) with yeast α-D-glucosidase was 217μM (castanospermine not being inhibitory at a concentration of 800μM). The K; for castanospermine (20) with almond β-D-glucosidase was 9μM (casuarine not being inhibitory at 800μM). Moreover, casuarine also inhibited rabbit gut mucosa α-D- glucosidase with an ICso value of 210μM, as compared with an IC50 value of 8μM for castanospermine. Both casuarine and castanospermine inhibited rabbit small intestine sucrase at a concentration of 700μM. Castanospermine also inhibited rabbit small intestine lactase and trehalase by over 50% at this concentration.

Example 6: Reduction of LPS-stimulated IL-12 (DCs) and IFN-q from anti-CD3 activated Splenocvtes (Th-1 depression)

The effect of the anti-CD3 antibody in combination with the polyhydroxylated alkaloids on the splenic cell populations has been investigated. The rational behind this approach is that CD3 cross-linking non-specifically activates the T cells. On the other hand, DCs are known to take up antigens via different groups of receptor families, including C-type lectin receptors, which are involved in recognition of a wide range of carbohydrate structures on antigens. Therefore, the aim of this study was to investigate whether the compounds of the invention in combination with anti-CD3 antibody can have an immunomodulatory effect on splenic cell populations, and the approach selected was to test the cytokine production by spleen cells upon exposure to the alkaloids. Materials and methods

Alkaloid Compounds The various alkaloids (Molecular Nature Ltd., Aberystwyth) were diluted in complete medium (RPMI, 1% L- Glutamine, 1% Penicillin / Streptomycin and 10% FCS) without GM-CSF, made up to 10mg/ml in most cases. Compounds were diluted in complete medium to the required concentration and then filter sterilised (Schleicher & Schuell, Germany). The chemical structures of the alkaloids used in the study are shown in the results below.

Animals

BALB/c male and female mice bred and maintained at the University of Strathclyde under conventional conditions were used at 8 weeks old.

Isolation of Spleen Cells The spleens were removed aseptically and placed in a Petri dish containing 5ml of complete medium. Cell suspensions were prepared by grinding a spleen against a wire mesh. The cells were then centrifuged for 5 minutes at lOOOrpm. Next, Boyle's solution (Tris 1.03g in 50 ml distilled water; NH4CI 4.15 g in 500 ml distilled water; mixed 1 :9 and filter sterilised) was added to remove the erythrocytes and centrifuged for 5 minutes. A further two washes in complete medium were carried out to ensure the Boyle's solution was removed. The pellet was then re-suspended and a cell concentration was adjusted to 5 x 10^s /ml.

Stimulation of Spleen Cells

Spleen cells were stimulated using anti-CD3 (α-CD3 / Clone C363. 29B Southern Biotechnology Associates, USA) at 0.5μg/ml. All experiments were carried out in 96-well tissue culture plates. 100μ1 of cell suspension were added per well, and each well had a final volume of 200μl. The control cells were incubated with 100μl complete medium. The test cells were incubated with 50μl α-CD3, or with 50μl of an alkaloid compound giving a final concentration of 50μg/ml, or with 50μl α-CD3 and 50μl of an alkaloid. Plates were then incubated for 72 hours at 37⁰C, 5% CO2 for subsequent analysis of the supernatant for the presence of the cytokines.

Measurement of cytokine production by ELISA

IL-12 concentration in the supematants was measured by an enzyme linked immunosorbent assay (ELISA). All reagents used in this assay were from PharMingen. 96 well flat-bottomed ELISA plates were coated with 2μg/ml of purified rat anti-mouse IL-12 (p40/p70) MAb (Cat no. 554478) diluted in PBS. The plates were incubated at 4⁰C overnight. Following incubation, the plates were washed 3 times in washing buffer (0.05% Tween 20 in PBS buffer at pH 9.0) and dried manually by hitting against paper towels. 200μl of blocking buffer (10% foetal calf serum in PBS pH 7.0) was added to each well and the plates were incubated at 37⁰C for 45 minutes. After three washes recombinant mouse IL-12 standard was added at 30μl in duplicate wells, with 20, 10, 5, 2.5, 1.25, 0.625, 0.31, 0.156, 0.078, 0.039, 0.02 and 0.01ng/ml. Standards were diluted in blocking buffer. The supernatant samples were added in at 50μl /well. The plates were incubated for 2 hours at 37⁰C, washed 4 times and dried, then the secondary antibody was added. Biotin labeled anti-mouse IL-12 (p40/p70) MAb (Cat No. 18482D) at 1μg/ml (diluted in blocking buffer) was added to each well at a volume of 100μl /well. The plates were incubated at 37⁰C for 1 hour, washed 5 times and dried and the conjugate was added. Streptavidin-AKP (Cat No. 13043E) at 100μl/well was added at a dilution of 1/2000 in blocking buffer, and the 7

plates again were incubated at 37⁰C for 45 minutes. The plates were finally washed 6 times and dried, and the substrate was added. pNPP (Sigma) in glycine buffer (7.51g glycine, 0.203g MgCI₂, 0.136g ZnCI₂ in 1 litre water at pH 10.4) at 1mg/ml was added at 100μl/well. Then the plates were covered in tinfoil, incubated at 37⁰C and checked every 30 minutes for a colour change; absorbance was then read at 405nm using a SPECTRAmax 190 machine.

Production of IL-2 and IFN-γ cytokines was also tested using the method described above.

Statistical Analysis Statistical significance was determined using an independent T Test and a Mann-Whitney U Test was used for antibody analysis. Results were considered significant at P<0.05. Data are represented by a mean value and error bars represent the standard error.

Results

HomoDMDP (referenced as MNLP10 in Figure 1 below) reduced anti-CD3 induced production of IFN-g by splenocvtes

This activity was shared by the related glucosidase-inhibiting pyrrolidine alkaloids DAB (MNLP20) and L-DMDP (MNLP21) (see Figure 2). DAB and MNLP10 are potent glucosidase inhibitors (Watson et a/., 2001) Such reductions in stimulated production of IFN-g by splenocytes had been reported for other glucosidase inhibitors, including bromoconduritol, /V-Methyl-DNJ and DNJ (MNLP22) (our data shown in Figure 3) (Kosuge et al., 2000) although the mechanism was not proven. Such activity has never before been ascribed to pyrrolidine alkaloids.

Figure 1: Reduction of anti-CD3 stimulated IFN-g (1a) but not LPS-stimulated IL-12 by MNLP10 (50ug/ml) (1b). (a)

IFN-g Production from Spleen Cells With MNLP 10

Anti- CD3

(b) IL-12 Production from Dendritic Cells with MNLP 10

LPS

Figure 2. IFN-g production from spleen cells following 72 hours incubation with anti-CD3 (0.5ug/ml), anti-CD3 with either MNLP 20 (50ug/ml) or MNLP 21 (50ug/ml). Results are expressed as mean cytokine concentration +/- standard error. N = 3; *P<0.05.

Anti-CD3 only WINLP 20 + Anti-CD3 WINLP 21 + Anti-CD3

Compound (50μg/ml)

The compounds reducing Th-1 cytokines, particularly evident when measuring anti-CD3 stimulated IFN-g from splenocytes, are glucosidase inhibitors. Thus, cytokine suppressive activity may be linked to glucosidase inhibitory activity. This effect has been reported by others (e.g. Kosuge, T., Tamura, T., Nariuchi, H. And Toyoshima, S. (2000). Biol. Pharm. Bull. 23 (1): 1-5) but the mode of action has not been elucidated and the selectivity of the reported compounds as glucosidase inhibitors (e.g. DNJ and castanospermine) is poor. They therefore cause Gl upset when given orally at high doses (Watson, A. A., Fleet, G.W.J. , Asano, N., Molyneux, R.J. and Nash, R.J. (2001). Polyhydroxylated Alkaloids - Natural Occurrence and Therapeutic Applications. Phytochemistry 56: 265-295). The compounds of the invention may be more selective and, therefore, less detrimental if used to reduce over activation of Th-1 responses, such as in Crohn's disease. Moreover, these monocyclic compounds are chemically more tractable than the bicyclic compounds of the prior art and hence production costs would be relatively low.

Equivalents

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

Claims

CLAIMS:

1. A pyrrolidine compound, for use in therapy or prophylaxis (for example in the treatment or prophylaxis of viral infection, allergic disorders or autoimmune disease), which pyrolidine compound:

(a) has the formula:

or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof; or

(b) has the formula:

wherein R1-R₅ is hydrogen or any group provided that at least three of R1-R5 is a group comprising X, wherein X is selected from: -OH, -NH2, -CN, -NO2 or a halogen (e.g. Br, F, I or Cl), or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

2. The compound of claim 1(b) wherein:

(a) R₅ is (CH₂)_nX and

Ri and R₄ are independently selected from X or (CH₂)_nX; and/or R₂ and R₃ are independently selected from X or (CHk)_nX; wherein n is 1-4; (b) R₂ = R₃ = X and at least one of Ri, R4 and R5 is a group comprising X (for example being X or (CH₂)_nX, wherein n is 1- 4.

3. The compound of claim 2 wherein: R₅ is (CHa)_nX; and

and/or R₂=R₃=X or (CHz)_nX.

4. The compound of claim 2 wherein: R₅ is (CH₂)_nX; and Ri=R₄=(CH₂)_nX; and/or R₂=R₃=X.

5. The compound of any one of claims 2 to 4 wherein n is 1 or 2.

6. The compound of any one of the preceding claims wherein X is -OH.

7. The compound of any one of the preceding claims wherein

and n is 2.

8. The compound of any one of the preceding claims wherein R₂=R₃=X.

9. The compound of any one of the preceding claims wherein Rs is (CH₂)₂X.

10. The compound of claim 6 as dependent on claim 2(b) and claim 1 (b) and wherein Rs is hydrogen, the compound having the formula:

11. The compound of claim 10 having the formula:

for example, having the formula:

12. The compound of claim 6 as dependent on claims 5 and 2(b) wherein n is 2 and FU and Rs are hydrogen, the compound having the formula:

13. The compound of claim 12 having the formula:

for example, having the formula:

H or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

14. The compound of claim 6 as dependent on claim 2(b) and wherein Rs is hydrogen, the compound having the formula:

wherein G is a group comprising OH (for example being (CH₂)nOH), wherein n is 1-4; or a pharmaceutically acceptable salt or derivative (e.g. straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, alkenyl, alkynyl, aryl, acyl, ether or amine derivative) thereof.

15. The compound of claim 14 having the formula:

for example, having the formula:

16. The compound of any one of the preceding claims (for example any one of claims 10 to 15) which is an alkovir and/or glycovir (e.g. glucovir).

17. The compound of any one of the preceding claims (for example any one of claims 10 to 16) for use in therapy or prophylaxis.

18. An antiviral composition comprising the compound of any one of the preceding claims (for example any one of claims 10 to 17), optionally further comprising an adjunctive antiviral agent.

19. Use of the compound of any one of the preceding claims (for example any one of claims 10 to 17) or the composition of claim 18 for the manufacture of a medicament for use in the treatment or prophylaxis of a viral infection.

20. The use of claim 19 wherein the viral infection involves one or more of the following viruses (or virus classes): Retroviridae (e.g. the human immunodeficiency viruses, including HIV-1); Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Cahiviridae (e.g. strains that cause gastroenteritis); Togaviήdae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridaβ (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses);

Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesvlridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the HCV virus (causing non-A, non-B hepatitis); Norwalk and related viruses, and astroviruses). Of the foregeoing, particularly preferred are HlV, Hepatitis A, Hepatitis B, Hepatitis C, rabies virus, poliovirus, influenza virus (including influenza A and influenza B virus), meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, haemorrhagic fever viruses and Herpes viruses, particularly, varicella, cytomegalovirus and Epstein-Barr virus.

21. A pharmaceutical kit of parts comprising a compound as defined in any one of the preceding claims (for example any one of claims 10 to 16) and an adjunctive antiviral agent, wherein for example the compound and adjunctive are in unit dosage form.

22. A pyrrolidine compound, for use in therapy or prophylaxis (for example in antiviral therapy or prophylaxis), which is a substituted or unsubstituted, N-hydroxyethyl triethanolamine.

23. A pharmaceutical composition comprising a compound as defined in any one of the preceding claims, optionally further comprising a pharmaceutically acceptable excipient.

24. A plurality of dose units of a pharmaceutical composition, the composition comprising a compound as defined in any one of the preceding claims, wherein the inter-dose co-efficient of variation in the concentration of pyrrolidine compound in said dose units is less than 50%.

25. The invention of any one of the preceding claims wherein the pyrrolidine compound has the general formula:

26. The invention of claim 25 wherein the pyrrolidine compound is substituted (e.g. with a straight or branched, substituted or unsubstituted, saturated or unsaturated alkyl, cycloalkyl, alkenyl, alkynyl, aryl, acyl, ether or amine group) at C5 and/or C6 and/or C7 and/or C8.

27. The invention of claim 26 wherein the pyrrolidine compound has the formula:

OH

28. The invention of any one of the preceding claims wherein the pyrrolidine compound is selected from:

(a) N-hydroxyethylDMDP having the formula:

(b) a compound having the formula:

29. The invention of any one of the preceding claims wherein the pyrrolidine compound is immunomodulatory and optionally induces, potentiates or activates one or more cytokines (e.g. Th1 cytokines) in vivo and/or suppresses one or more cytokines (e.g. Th1 and/or Th2 cytokine(s)) in vivo).

30. The invention of claim 29 wherein:

(a) the one or more cytokines comprises one or more interleukins; and/or

(b) the one or more Th1 cytokine(s) comprises IFN-gamma, IL-12 and/or IL-2; and/or

(c) the one or more Th2 cytokine(s) comprise IL-5.

31. A process for producing a plurality of dose units of a pharmaceutical composition (for example, for producing a batch of said dose units), the process comprising the step of introducing a pyrrolidine compound as defined in any one of the preceding claims into each unit dose such that the inter-dose co-efficient of variation in the concentration of pyrrolidine compound in said dose units is less than 50%.

32. Use of a compound as defined in any one of the preceding claims for the manufacture of a medicament for use in immunotherapy or immunoprophylaxis (for example in any of the immunotherapeutic or immunoprophylactic or antiviral methods described herein).

33. Use of a compound as defined in any one of the preceding claims for the manufacture of a medicament for the treatment or prophylaxis of an autoimmune disease.

34. Use of a compound as defined in any one of the preceding claims for the manufacture of a medicament for the treatment or prophylaxis of an allergic disorder (e.g. asthma).

35. Use of claim 33 or claim 34 wherein the compound suppresses the level and/or activity of IFN-gamma and/or TNF-alpha In vivo.

36. Use of any one of claims 33 to 35 wherein the compound is a specific glucosidase inhibitors (for example which specifically inhibit ER α-glucosidases).

37. Use of any one of claims 33 to 36 wherein the compound has a formula selected from: (a)

(b)

for example, having a formula selected from: (a)

(b)