Connect public, paid and private patent data with Google Patents Public Datasets

Antiviral drug combinations

Info

Publication number
WO2006077427A2
WO2006077427A2 PCT/GB2006/000209 GB2006000209W WO2006077427A2 WO 2006077427 A2 WO2006077427 A2 WO 2006077427A2 GB 2006000209 W GB2006000209 W GB 2006000209W WO 2006077427 A2 WO2006077427 A2 WO 2006077427A2
Authority
WO
Grant status
Application
Patent type
Prior art keywords
invention
alkaloids
virus
viral
cells
Prior art date
Application number
PCT/GB2006/000209
Other languages
French (fr)
Other versions
WO2006077427A3 (en )
Inventor
Robert James Nash
Jason H. Slingsby
Miles William Carroll
Original Assignee
Mnl Pharma Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Abstract

There is disclosed a combined preparation comprising a glycosylation modulator and a membrane fusion inhibitor for combined, simultaneous or sequential use in the treatment of infections caused by viruses bearing glycosylated envelope proteins.

Description

ANTIVIRAL DRUG COMBINATIONS

Field of the Invention

The present invention relates to combinations of a viral entry inhibitor and a glycosylation modulator, alkovir or glycovir and to various medical uses of said combinations. In particular, the present invention relates to the use of glycosylation modulators, such as n-butyldeoxynojirimycin, in combination with membrane fusion inhibitors, such as enfuvirtide, for treatment of infections caused by viruses bearing glycosylated envelope proteins.

Background to the Invention

A number of viral pathogens display heavily glycosylated envelope proteins on their surface. These glycosylated envelope proteins are central to the initial binding event between the virus particle and the target cell. In addition, the glycosylated envelope proteins are often centrally involved in the post-binding membrane fusion event required for a productive infection. This application describes inter alia the use of pharmaceutical compounds which have been pharmacologically characterised as modulators of viral envelope glycosylation, in a novel therapeutic combination with inhibitors of viral-host cell membrane fusion events. This novel combination therapy is predicted to have enhanced therapeutic effect, in particular in the field of anti-HIV therapy. The general approach is predicted to have wider clinical application in other viral infection processes where membrane fusion events can be modulated by alterations to carbohydrate structures on glycoproteins.

Viral entry into host cells

The plasma membrane of eukaryotic cells acts as a barrier against invading viruses. Thus, in order to infect a eukaryotic cell, an invading virus must first bind to the target host cell and then transport its genome and accessory proteins across its plasma membrane. In the case of enveloped viruses, entry into the host cell typically involves three steps: (i) attachment (typically to one or more host cell virus receptors); (ii) co- receptor binding and (iii) membrane fusion. Specificity for one or more virus receptors may give rise to cell tropism. For example, viruses typically restrict the host cells they infect by targeting receptors which are restricted to particular compartments, for example the gut (coronaviruses) or immune cells (HIV-1 ).

Membrane fusion may occur by two different general mechanisms: (1 ) fusion of viral envelope and host cell plasma membrane; and (2) fusion of endosomal membrane with viral envelope following virus internalization by receptor-mediated host cell endocytosis. In both cases, membrane fusion is mediated by specific viral surface glycoproteins. Thus, many viral pathogens display heavily glycosylated envelope proteins on their surface.

Glycosylated envelope. proteins are central to the initial binding event between the virus particle and the target cell. In addition, the glycosylated envelope proteins are often centrally involved in the post-binding membrane fusion event required for a productive infection Viral fusion proteins undergo structural reorganization, changing from a nonfusogenic to fusogenic conformation

Viral fusion glycoproteins are type I integral membrane proteins comprising a large ectodomain, a single transmembrane sequence and a small C-terminal endodomain They contain N-linked carbohydrates and form oligomers at high density in the viral membrane The particular segment involved in membrane fusion is known as the fusion peptide

At least two classes of viral fusion peptide can be recognized on the basis of structural criteria Class I fusion proteins are trimeric and have a predominantly α-helical secondary structure The fusion peptide is located at the N-terminus Class I fusion proteins are found in many important pathogens, for example retroviruses (including HIV, SIV, MoLV, HTLV-1), orthomyxoviruses (including influenza viruses), paramyxoviruses (including Sendai, SV5 and HRSV) and filoviruses (including Ebola) Class Il fusion proteins are dimeric and have a predominantly β-sheet secondary structure The fusion peptide is located internally Class Il fusion proteins are also found in important pathogens, including for example alphaviruses (including SFV) and flaviviruses (including dengue and TBE)

Despite their structural differences, both class I and class Il fusion proteins are believed to function by an essentially identical mechanism the proteins exist in a metastable, prefusion conformation in the isolated virus particle and an irreversible transition to the post-fusion conformation provides the energy required for membrane fusion A third class of fusion proteins (exemplified by the rhabdovirus fusion glycoprotein) has recently been recognized and is thought to function in a completely different manner from the class I and class Il fusion peptides described above

Viral entry inhibitors as antiviral drugs

Viral entry is an attractive target for therapeutic or prophylactic intervention, since drug activity is independent of intracellular access The extracellular site of action renders such agents unsusceptible to cellular efflux transporters that lower the intracellular concentration of other classes of antiviral drugs and is thought to confer a low toxicity profile Moreover, the distinct site of action relative to intracellularly-acting agents in other classes minimizes cross-resistance when entry inhibiting drugs are used with such other classes

Each of the three discrete steps of viral entry described above (attachment, co-receptor binding and fusion) represents a unique drug target and a large number of drugs have now been developed in each class For example, and in the case of HIV-1 , several attachment inhibitors have been produced that block the binding of gp120 to the CD4 receptor and a number are in pre-clinical or clinical development (including BMS-806, BMS-043, PRO 2000, TNX-35525 and PRO 542) Agents that act as chemokine co-receptor binding inhibitors are in various stages of development These inhibitors include those that block the CCR5 receptor (e g TAK-779, SCH-C and SCH-D, PRO-140, UK-427, GW873140 and AMD887) and others which block the CXCR4 receptor (e g AMD310039 and AMD070) The third and final step in the viral entry process, fusion, is particularly attractive as a target, since it combines a virus-specific target with an extracellular mode of action. Toxicity is therefore likely to be inherently lower because the target is exclusively viral and elements of the host cell receptor systems (in the case of HIV-1, elements of the host immune system) are not targeted.

Fusion inhibitors can be designed as peptide mimetics of an essential region within viral fusion proteins that block the structural rearrangements by forming a complex with the pre-fusion conformation so preventing adoption of the post-fusion conformation and blocking membrane fusion. The first fusion inhibitor to be approved for clinical use is Enfuvirtide™ (FUZEON™, formerly known as T-20 or DP178), which blocks the entry of HIV-1. Enfuvirtide is a peptide homologous to a segment of the HR2 region of gp41 and binds to the HR1 region, so blocking the formation of the six-helix bundle necessary for fusion. Enfuvirtide exhibits potent and selective inhibition of HIV-1 both in vitro and in vivo.

Other fusion inhibitors include T-1249 (a slightly longer peptide than Enfuvirtide™) which is active against HIV-1 and HIV-2 as well as simian immunodeficiency virus. Several other fusion inhibitor therapeutics have been developed, most of them being peptide mimetics. These include T-649 (which blocks hairpin structure formation in a similar way to Enfuvirtide™; C34 peptide; D-peptide, a cyclic molecule designed to bind pocket region within the six-helix structure, DP-107 and DP-178. Other fusion inhibitors include 5-helix and RPR 103611 , a non-peptide triterpene compound that targets the loop region linking the two halves of the gp41 leucine zipper so disrupting the association of gp120-gp41 in CXCR4-tropic HIV viruses.

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 as qlvcosidase 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 ef a/. (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 a/. (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 ef a/. (2001 ), the disclosure of which is incorporated herein by reference).

Watson ef a/. (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 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 ef al. (1990) Phytochemistry (29) 111 ) and synthesised (Choi ef 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 pyrrolizidine alexines), swainsonine 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). 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 Bucast), has been reported to exhibit anti-viral and antimetastatic activities.

Casuarine, (1 R,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 ef 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 al. (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 ef 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).

Summary of the Invention

According to the present invention there is provided a combination of a viral entry inhibitor and an adjunctive agent selected from: (a) a glycosylation modulator; (b) an alkovir; (c) a glycovir (e.g. a glucovir); and (d) an alkaloid as herein defined.

Any of the adjunctive agents disclosed herein may be used according to the invention, and in particular any of the agents described in the sections entitled "Glycosylation modulators for use in the combinations of the invention" and "Alkaloids for use in the combinations of the invention" set out below. Thus, the adjunctive agent is preferably selected from the following structural classes:

(a) piperidine alkaloids;

(b) pyrroline alkaloids;

(c) pyrrolidine alkaloids;

(d) pyrrolizidine alkaloids;

(e) indolizidine alkaloids; and (f) nortropane alkaloids.

In preferred embodiments, the adjunctive agent is a polyhydroxylated alkaloid. Particularly preferred are adjunctive agents which are imino sugars.

The viral entry inhibitor may be selected from: (a) an attachment inhibitor; (b) a co-receptor binding inhibitor; and (c) a membrane fusion inhibitor. The combinations preferably further comprise one or more auxiliary antiviral agent(s). Such auxiliary antiviral agents may be selected from one or more of: (a) protease inhibitors; (b) nucleoside/nucleotide reverse transcriptase inhibitors; (c) non-nucleoside reverse transcriptase inhibitors; (d) integrase inhibitors; (e) maturation inhibitors; and (f) cytokines or cytokine stimulatory factors.

In another aspect, the invention provides various therapeutic and prophylactic methods and uses based upon the combinations of the invention. Such methods and uses are set out in the claims appended hereto.

Other aspects of the invention and specific embodiments thereof are set out in the claims appended hereto.

Detailed Description of the Invention

Definitions and general preferences

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:

As used herein, the term glycosylation modulator encompasses any pharmaceutical agent which alters relinked or O-linked oligosaccharide structures on viral envelope glycoproteins. Preferably the glycosylation modulator is a glucosidase I or glycosidase I inhibitor. Most preferably, the glycosylation modulator is an imino sugar. In other embodiments, the glycosylation modulator is an alkaloid (for example a polyhydroxylated alkaloid). Particularly preferred glycosylation inhibitors are glycovirs. Most preferred glycosylation inhibitors are glucovirs.

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 glucovirls 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.

Most known alkaloids are phytochemicals, present as secondary metabolites in plant tissues (where they may play a role in defence), but some occur as secondary metabolites in the tissues of animals, microorganisms and fungi. There is growing evidence that the standard techniques for screening microbial cultures are inappropriate for detecting many classes of alkaloids (particularly highly polar alkaloids, see below) and that microbes (including bacteria and fungi, particularly the filamentous representatives) will prove to be an important source of alkaloids as screening techniques become more sophisticated.

Structurally, alkaloids exhibit great diversity. Many alkaloids are small molecules, with molecular weights below 250 Daltons. The skeletons may be derived from amino acids, though some are derived from other groups (such as steroids). Others can be considered as sugar analogues. It is becoming apparent (see Watson et al. (2001) Phytochemistry 56: 265-295) that the water soluble fractions of medicinal plants and microbial cultures contain many interesting novel polar alkaloids, including many carbohydrate analogues. Such analogues include a rapidly growing number of polyhydroxylated alkaloids.

The term immunomodulatory alkaloid is used herein to define any alkaloid which can 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 alkaloids of the invention stimulate the release of one or more cytokines (e.g. IL-12) in w'fra (for example, in spleen cells, macrophages and/or dendritic cells). They may act as PRR ligands (as herein defined).

The term cytokine stimulatory alkaloid is used herein to define a subclass of immunomodulatory alkaloids which are capable of stimulating the activity of one or more cytokine(s) in a PRR-bearing cell. Such alkaloids are said to exhibit a cytokine stimulation profile in that PRR-bearing cell. Typically, the immunomodulatory alkaloids of the invention are capable of stimulating the activity of one or more cytokines in macrophages and/or dendritic cells. This stimulatory activity may be observable In vitro and/or in vivo. The stimulation may occur directly or indirectly via any mechanism and at any level (e.g. at the level of transcription, translation, post-translational modification, secretion, activation, shedding, stabilization or sequestration). Preferred cytokine stimulatory alkaloids are PRR ligands (as herein defined). Typically, the stimulation comprises an increase in the production of the cytokine(s) by the PRR-bearing cell. Typically, the one or more cytokine(s) stimulated by the immunomodulatory alkaloids for use according to the invention comprise one or more Th1 cytokines (as herein defined and described). Particularly preferred are immunomodulatory alkaloids that stimulate IL-2 and/or IL-12 in dendritic cells and/or macrophages {in vivo and/or in vitro).

The term PRR ligand as used herein in relation to the preferred immunomodulatory alkaloids that act as PRR ligands) defines compounds which can act as binding partners for a PRR. Such immunomodulatory alkaloids therefore include those which bind (or directly physically interact) with a PRR in vivo irrespective of the physiological consequences of that binding. Thus, the PRR ligands of the invention may bind a PRR as part of a cellular signalling cascade in which the PRR forms a part. Alternatively, they may bind PRR in the context of some other aspect of cellular physiology. In the latter case, the ligands may for example bind PRR at the cell surface without triggering a signalling cascade, in which case the binding may effect other aspects of cell function. Thus, the ligands of the invention may bind PRRs and thereby effect an increase in the concentration of functional PRR at the cell surface (for example mediated via an increase in PRR stability, absolute receptor numbers and/or PRR activity). Alternatively, the ligands may bind PRR (or PRR precursors) intracellular^, in which case they may act as molecular chaperones to increase the expression of active PRR.

In preferred embodiments, the PRR ligands of the invention are PRR agonists. The term agonist is used herein in relation to the PRR ligands of the invention to define a subclass of ligands which productively bind PRR to trigger the cellular signalling cascade of which the PRR forms a part.

As used herein, the term PRR-bearing cell defines any cell which expresses one or more pathogen-(or pattern-) recognition receptors (PRRs). The term PRR is a term of art used to define a class of receptors which are expressed on various cells (e.g. epithelial cells and effector cells of the innate immune system, including the professional antigen-presenting cells, macrophages and dendritic cells) and which recognize a few, highly conserved structures present in diverse groups of microorganisms known as pathogen- associated molecular patterns (PAMPs). Thus, PRR-bearing cells as described herein may comprise epithelial cells, macrophages, dendritic cells or other effector cells of the innate immune system. In preferred embodiments, the PRR-bearing cell for use in relation to the invention are dendritic cells or macrophages. Thus, those functional attributes of the immunomodulatory alkaloids of the invention that are defined by reference to inter alia a PRR-bearing cell are to be understood to relate to any of a wide variety of different PRR-bearing cells of diverse cytological properties and biological functions, including inter alia epithelial cells, dendritic cells, macrophages, various APCs, natural killer (NK) cells and other cells of the innate immune system (including e.g. neutrophils, granulocytes and monocytes). Preferably, however, the PRR-bearing cells described herein (and used for example to define a parameter of the reference conditions under which the functional properties of the immunomodulatory alkaloid are manifest) are macrophages or dendritic cells.

The term cytokine stimulation profile is used herein to define a functional attribute of the immunomodulatory alkaloids of the invention which is characterized by reference to the identity of one or more cytokines stimulated (and optionally the identity of one or more cytokines unstimulated) in a PRR-bearing cell when contacted with the relevant immunomodulatory alkaloid. Preferably, the cytokine stimulation profile is characterized by reference to the presence or absence of stimulation of two or more cytokines, more preferably four or more. Even more preferably, the cytokine stimulation profile is characterized by reference to the presence or absence of stimulation of one or more Th1 cytokines and/or one or more Th2 cytokines. Alternatively, or in addition, the stimulation profiles which functionally define the immunomodulatory alkaloids may be characterized by the degree of stimulation of one or more reference cytokine(s) (or classes thereof). The degree of stimulation may be expressed as an induction ratio with respect to: (a) the levels of the reference cytokine(s) (or markers thereof, such as encoding nucleic acids) in the PRR-bearing cell in the absence of the relevant test immunomodulatory alkaloid; and/or (b) the level of one or more other cytokine(s) (or classes thereof) also present in the PRR-bearing cell (whether stimulated or not by the immunomodulatory alkaloid). The cytokine stimulation profile of the immunomodulatory alkaloids for use according to the invention is preferably characterized by the stimulation of one or more Th1 cytokines (and optionally the absence of stimulation of one or more Th2 cytokines).

The term Th1 cytokine (or Type-1 cytokine) is a term of art used to define those cytokines produced by Th1 T-helper cells. Th1 cytokines include, for example, IL2, IFN-γ, IFN-α/β, IL12, IL-18, IL-27 and TNF-β. The term Th2 cytokine (or Type-2 cytokine) is a term of art used to define those cytokines produced by Th2 T- helper cells. Th2 cytokines include, for example, IL-4, IL-5, IL-9, IL-13, IL-25 and TSLP. The term Treg cytokine is a term of art used to define those cytokines produced by regulatory T-cells. Treg cytokines include, for example, IL-10, TGF-β and TSP1.

The term isolated as applied to the alkaloids of the invention is used herein to indicate that the alakloid exists in a physical milieu distinct from that in which it occurs in nature or in a purified form. For example, the isolated material may be substantially isolated (for example purified) with respect to the complex cellular milieu in which it naturally occurs. When the alkaloid is purified, the absolute level of purity is not critical and those skilled in the art can readily determine appropriate levels of purity according to the use to which the material is to be put. Preferred, however, are purity levels of 90% w/w or higher. In some circumstances, the isolated alkaloid may form part of a composition (for example a more or less crude extract containing many other substances) or buffer system, which may for example contain other components. In other circumstances, the isolated alkaloid 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 alkaloids of the invention define alkaloids which are obtained (or obtainable) by chemical derivatization of the parent alkaloids of the invention. The pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with mammalian tissues without undue toxicity, irritation or allergic response (i.e. commensurate with a reasonable benefit/risk ratio). Preferred derivatives are those obtained (or obtainable) by alkylation, esterification or acylation of the parent alkaloids. The derivatives may be active per se, or may be inactive until processed in vivo. In the latter case, the derivatives of the invention act as pro-drugs. Particularly preferred pro-drugs are ester derivatives which are esterified at one or more of the free hydroxyls and which are activated by hydrolysis in vivo. The pharmaceutically acceptable derivatives of the invention retain some or all of the activity of the parent alkaloid. In some cases, the activity is increased by derivatization. Derivatization may also augment other biological activities of the alkaloid, for example bioavailability.

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. SiIa- 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 immunomodulatory alkaloids of the invention.

The term pharmaceutically acceptable salt as applied to the alkaloids of the invention defines any non-toxic organic or inorganic acid addition salt of the free base alkaloid which are suitable for use in contact with mammalian tissues without undue toxicity, irritation, allergic response and which are commensurate with a reasonable benefit/risk ratio. Suitable pharmaceutically acceptable salts are well known in the art. Examples are the salts with inorganic acids (for example hydrochloric, hydrobromic, sulphuric and phosphoric acids), organic carboxylic acids (for example acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic, 2- acetoxybenzoic and mandelic acid) and organic sulfonic acids (for example methanesulfonic acid and p- toluenesulfonic acid). The imino sugars of the invention may also be converted into salts by reaction with an alkali metal halide, for example sodium chloride, sodium iodide or lithium iodide. Preferably, the alkaloids 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 alkaloids can exist in either a hydrated or a substantially anhydrous form. Crystalline forms of the alkaloids of the invention are also contemplated and in general the acid addition salts of alkaloids 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 alkaloids of the invention. Those skilled in the art will appreciate that, owing to the asymmetrically substituted carbon atoms present in the alkaloids of the invention, they may exist and be synthesised and/or isolated in optically active and racemic forms. Thus, references to the alkaloids of the present invention encompass alkaloids 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 alkaloids of the invention, and unless indicated otherwise (e.g. by use of dash-wedge structural formulae) the alkaloids shown herein are intended to encompass all possible optical isomers of the alkaloids so depicted. In cases where the stereochemical form of the alkaloid is important for pharmaceutical utility, the invention contemplates use of an isolated eutomer.

As used herein, the term combination is applied in relation to two or more different compounds to define material in which the two or more compounds are associated. The terms "combined" and "combining" in this context are to be interpreted accordingly.

The association of the two or more compounds in a combination may be physical or non-physical. Examples of physically associated combined compounds include:

• compositions (e.g. unitary formulations) comprising the two or more compounds in admixture (for example within the same unit dose);

• compositions comprising material in which the two or more compounds are chemically/physicochemically linked (for example by crosslinking, molecular agglomeration or binding to a common vehicle moiety);

• compositions comprising material in which the two or more compounds are chemically/physicochemically co-packaged (for example, disposed on or within lipid vesicles, particles (e.g. micro- or nanoparticles) or emulsion droplets); • pharmaceutical kits, pharmaceutical packs or patient packs in which the two or more compounds are co-packaged or co-presented (e.g. as part of an array of unit doses);

Examples of non-physically associated combined compounds include: • material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds together with instructions for the extemporaneous association of the at least one compound to form a physical association of the two or more compounds;

• material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds together with instructions for combination therapy with the two or more compounds;

• material comprising at least one of the two or more compounds together with instructions for administration to a patient population in which the other(s) of the two or more compounds have been (or are being) administered;

• material comprising at least one of the two or more compounds in an amount or in a form which is specifically adapted for use in combination with the other(s) of the two or more compounds.

As used herein, the term "combination therapy" is intended to define therapies which comprise the use of a combination of two or more compounds (as defined above). Thus, references to "combination therapy", "combinations" and the use of compounds "in combination" in this application may refer to compounds that are administered as part of the same overall treatment regimen. As such, the posology of each of the two or more compounds may differ: each may be administered at the same time or at different times. It will therefore be appreciated that the compounds of the combination may be administered sequentially (e.g. before or after) or simultaneously, either in the same pharmaceutical formulation (i.e. together), or in different pharmaceutical formulations (i.e. separately). Simultaneously in the same formulation is as a unitary formulation whereas simultaneously in different pharmaceutical formulations is non-unitary. The posologies of each of the two or more compounds in a combination therapy may also differ with respect to the route of administration.

The combinations of the invention may produce a therapeutically efficacious effect relative to the therapeutic effect of the individual compounds when administered separately. The term 'efficacious' includes advantageous effects such as additivity, synergism, reduced side effects, reduced toxicity, increased time to disease progression, increased time of survival, or the sensitization or resensitization of one agent to another. Advantageously, an efficacious effect may allow for lower doses of each or either component to be administered to a patient, thereby decreasing the toxicity of chemotherapy, whilst producing and/or maintaining the same therapeutic effect.

A "synergistic" effect in the present context refers to a therapeutic effect produced by the combination which is larger than the sum of the therapeutic effects of the components of the combination when presented individually.

An "additive" effect in the present context refers to a therapeutic effect produced by the combination which is larger than the therapeutic effect of any of the components of the combination when presented individually.

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. The unit dose(s) may be contained within a blister pack. The pharmaceutical kit may optionally further comprise instructions for use.

As used herein, the term "pharmaceutical pack" defines an array of one or more unit doses of a pharmaceutical composition, optionally contained within common outer packaging. In pharmaceutical packs comprising a combination of two or more compounds, the individual compounds may unitary or non-unitary formulations. The unit dose(s) may be contained within a blister pack. The pharmaceutical pack may optionally further comprise instructions for use.

As used herein, the term "patient pack" defines a package, prescribed to a patient, which contains pharmaceutical compositions for the whole course of treatment. Patient packs usually contain one or more blister pack(s). Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.

Viral entry inhibitors for use in the combinations of the invention

Attachment inhibitors - non-specific

Polyanionic molecules

Certain sulphated polysaccharides (dextran-sulphate, pentosan-sulphate or heparin) can inhibit viral replication in vitro . The anti-HIV activity of compounds with structures which are so heterogeneous seems to be due to the fact that they have a high density of negative charges (polyanions). Dextran-sulphate binds to the V3 loop of gp120 in CXCR4 strains and prevents binding to the CD4 receptor.

Other substances which share this mechanism of action are PRO 2000 and cyanovirin-N. Although they are unlikely to be used in clinical practice, different polyanions are currently being evaluated as topical agents.

Attachment inhibitors - specific

Soluble recombinant CD4 (srCD4) srCD4 was one of the first antiretroviral drugs to be studied. It acts by blocking gp120 and has proven to very active in vitro . Nevertheless, its activity is much lower against viral strains from patients, possibly because there are viral variants of lower affinity. Therefore, srCD4 has not been developed as an anti-HIV agent.

Anti-CD4 monoclonal antibodies

These are monoclonal antibodies anti determined epitopes of the CD4 receptor or of gp120 which, on binding, block interaction and repress the replication of several viral sub-types. Even though blockade of the CD4 receptors could lead to some degree immunosuppression, at least the so-called TNX-355 antibody has been well tolerated and CD4 lymphocytopenia has not been reported. A first clinical trial (phase l/ll) at a single dose of TNX-355 showed a reduction in viral load and an increase in CD4 cells at 21 days. Nevertheless, longer clinical trials are necessary to confirm the usefulness of this antibody. PRO-542 is a hybrid tetramer which contains domains of the CD4 receptor bound to IgG2 and which acts as a decoy of the CD4 receptor to which viral gp120 binds, thus preventing it from binding to the real CD4 receptor. In vitro , it has shown activity with both laboratory and clinical strains, and this activity has been confirmed in vivo in a small number of advanced-stage patients.

BMS-806

This substance forms part of other molecules which can bind competitively and reversibly to gp120 by blocking the interaction with the CD4 receptor. It has been evaluated in vitro and is active against different strains of HIV, although the early emergence of mutation-related resistance at the point of action of gp120 leads it to lose efficacy.

Other attachment inhibitors include BMS-043, PRO 2000, TNX-35525 and PRO 542.

Co-receptor binding inhibitors

CCR5 receptor antagonists

Tak-220 Pre-clinical studies of this molecule have shown high specificity for CCR5 with no affinity for other ligands. Similarly, oral administration enables suitable levels of the drug in blood to be reached.

SCH-C/SCH-D These are substances have a high intrinsic activity against R5 strains and are synergic with other antiretroviral drugs. SCH-C has undergone a phase l/ll trial in 12 patients for 10 days and has shown a reduction in viral load of between 0.5 and 1.0 log. Nevertheless, its development has been stopped because of the risk of cardiac arrhythmias after reports of a lengthening of the QTc interval. It has been replaced by SCH-D, which has recently been reported to be more potent (mean reduction in viral load of 1.3 Iog10), is better tolerated and has no effect on heart ratei 8 . Nevertheless, it is important to note that one of the 48 patients studied developed a mixed viral strain, R5/X4 and another developed an X4 strain on finishing therapy.

UK-427,857 This drug is active against a wide number of viral strains and is specific for the CCR5 receptor (not active against CXCR4 strains). It is currently in phase ll/lll studies.

Other substances Other co-receptor binding inhibitors include Pro-140 (a specific and potent monoclonal antibody which inhibits entry of the virus and does not block CCR4 receptor activity). GW873140 has shown good tolerance (digestive symptoms) and a prolonged half-life after oral adminstration19 . AMD887 is active and potent in vitro , both as the only drug and in combination with AMD070 (CXCR4 co-receptor inhibitor). Other examples include Tak-779. CXCR4 receptor antagonists

Bicyclams These molecules are so named because they are composed of two macrocyclical rings (cyclams) bound by an aliphatic or aromatic chain. They are very potent and are active against HIV-1 and HIV-2 in cell culture.

KRH-1636 and KRH-2731 are potent antagonists of the CXCR4 receptor, can be administered orally and are active in pre-clinical studies. Other CXCR4 receptor antagonists include AMD310039 and AMD070.

Fusion inhibitors

Membrane fusion inhibitors are particularly preferred as viral entry inhibitors for use according to the invention. Preferably, the membrane fusion inhibitor inhibits a membrane fusion event between the virion and a host cell.

Enfuvirtide (Fuzeon™) is a 36-aminoacid peptide which camouflages a sequence of the HR-2 region and is active against strains X4 and R5. It binds to the HR-1 domain of the gp41 virus by preventing the formation of the 6-helix structure necessary to start the conformational changes which finish in membrane fusion. The drug is active during the phase where the virus approaches the target cell in which gp41 , and specifically the HR-1 domain, are accessible. This "therapeutic window" can vary according to the affinity of the virus for the receptor, in such a way that, if affinity is very high, the drug can act more quickly and its efficacy is lower in these viral strains. The efficacy of enfuvirtide is not modified independently of the co-receptor used by the virus.

Enfuvirtide has been developed by Trimeris Inc. as a novel class of HIV-1 therapeutic. Currently marketed by Roche worldwide for approximately $10,000 per yearly treatment course, this sub-cutaneously administered compound is a 36 amino acid synthetic peptide with the N-terminus acetylated and the C- terminus as a carboxamide. It has a molecular weight of 4,492. Enfuvirtide is formulated as a single use vial for reconstitution with sterile water. A dose of 90 mg is delivered daily by sub-cutaneous injection in a 1 ml volume. Enfuvirtide interferes with the entry of HIV-1 into cells by inhibiting the fusion of cellular and viral membranes. Enfuvirtide binds to the first heptad repeat (HR1) in the gp41 subunit of the viral envelope glycoprotein and prevents the conformational changes required for the fusion of viral and cellular membranes. Enfuvirtide has exhibited IC5O values in laboratory and primary isolates of HIV-1 ranging from 4 to 280 nM. Enfuvirtide has no activity against HIV-2. Enfuvirtide has exhibited synergistic effects in cell culture assays when combined with individual members of various anti-retroviral classes, including zidovudine, lamivudine, nelfinavir, indinavir and efavirenz. A significant number of patent applications describe the use of enfuvirtide in HIV-infected populations.

Like all current HIV therapies, Enfuvirtide suffers from resistance problems, caused by high HIV mutation rates. Genotypic analysis has identified amino acid substitutions at the Enfuvirtide binding HR1 domain positions 36 to 38 in the HIV-1 gp41 envelope protein in resistant HIV-1 isolates. Site directed mutants in positions 36 to 38 have shown a 5-fold to 680-fold decrease in susceptibility to Enfuvirtide. Similar findings have been identified in clinical trials, with decreases in sensitivity ranging from 4-fold to 420-fold, and showed genotypic changes in gp41 amino acids 36 to 45. However, HIV-1 clinical isolates previously resistant to nucleoside analogue reverse transcriptase inhibitors (NRTIs), non- nucleoside analogue reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (Pl) were susceptible to Enfuvirtide in cell culture.

It is possible that the resistance patterns experienced during clinical treatment with Enfuvirtide might also be modulated by co-administration of the alkaloids described herein (e.g. Bu-DNJ). This is due to the potential for novel carbohydrate structures on gp41/gp120 to render the typical HIV-1 resistance mutants with altered amino acids in amino acids 36 to 45 of gp41 still sensitive to co-administration treatment. This might be due to Enfuvirtide still being able to interact with the first heptad repeat (HR1) under alkaloid (e.g. Bu-DNJ) treatment or to the moderately inhibited membrane fusion event being further inhibited by the synergistic action of the alkaloid (e.g. Bu-DNJ).

Enfuvirtide is marketed in many countries. Initial studies using the drug in intravenous monotherapy showed a potent antiretroviral effect with few adverse effects. This activity was proven both for sub-type B (dominant in Europe and the USA) and against other viral sub-types. The optimal subcutaneous dose was initially determined in a continuous perfusion pump and later in two daily doses in 16 adults who achieved a negative viral load ( < 500 cop/ml) when the dose was above 100 mg. Studies in vitro have shown the synergy of enfuvirtide with other binding blockers such as AMD-3100, SCH-C and PRO-54225 .

T-1249 This is a second-generation fusion inhibitor. It is a 39-aminoacid peptide designed from different HR-2 regions of HIV-1 , HIV-2 and SIV. As with enfuvirtide, T-1249 has a high capacity for reducing viral replication in HlV strains which are multi-resistant to current drugs, even those which are resistant to T-20, and in sub-types A to G. In naϊve patients and those on monotherapy, T-1949 has proven to be very potent at a single daily dose (fall in viral load of 2.0 log). Nevertheless, the clinical development program of this product has been temporarily stopped because of technical difficulties.

Several other fusion inhibitor therapeutics have been developed, most of them being peptide mimetics. These include T-649 (which blocks hairpin structure formation in a similar way to Enfuvirtide™; C34 peptide; D-peptide, a cyclic molecule designed to bind pocket region within the six-helix structure, DP-107 and DP- 178. Other fusion inhibitors include 5-helix and RPR103611 , a non-peptide triterpene compound that targets the loop region linking the two halves of the gp41 leucine zipper so disrupting the association of gp120-gp41 in CXCR4-tropic HlV viruses.

Other preferred fusion inhibitors include TR-291144 and TR-290999 (Roche/Trimeris) which are peptides derived from HR2 sequences of HIV. These need less frequent dosing than enfuvirtide.

Suitable fusion inhibitors for use in the combinations of the invention may therefore be selected from any of those described above. Alternatively, they may be obtained by any of a wide number of screening and synthesis processes described in the prior art. Examples of such processes include those described in, for example, US2005208678 and WO0006599 (the disclosure of which relating to particular fusion inhibitors and their identification and syntehsis is hereby incorporated by reference).

Glycosylation modulators for use in the combinations of the invention

Any agent which can alter N-linked or O-linked oligosaccharide structures on viral glycoproteins (particularly viral envelope glycoproteins) can be used in the combinations of the invention. Preferred glycosylation modulators are pharmaceutical agents which alter (e.g. eliminate, truncate or debranch) N-linked or O-linked oligosaccharide structures on viral envelope glycoproteins. Most preferably, the glycosylation modulator is a glycosylation inhibitor. The glycosylation inhibitors of the invention may eliminate, truncate or debranch oligosaccharide structures on viral envelope proteins.

The glycosylation modulators may modulate the activity of one or more glycosidase(s). Preferred are glycosylation inhibitors which inhibit the activity of one or more glycosidase(s). Particularly preferred are glycosylation modulators or inhibitors which modulate or inhibit the activity of glycosidase I (particularly glucosidase I).

The glycosylation modulator for use in the combination of the invention is preferably an alkaloid (for example a polyhydroxylated alkaloid) as herein defined. Preferred alkaloid glycosylation inhibitors are imino sugars. Suitable alkaloids and imino sugars which act as glycosylation modulators may be selected from the various alkaloids and imino sugars described herein, and in particular in the section below.

Particularly preferred glycosylation inhibitors for use in the combinations of the invention are glycovirs, and more particularly glucovirs (as described and defined herein)

Glycosylation modulators may be identified by standard enzymological assay. Preferred are agents 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 glycosylation modulators 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 preferred glycosylation modulators for use according to the invention are commonly derived from plants or microorganisms. Novel sugars have been synthesised and naturally occurring ones have also been modified. The common structural feature is that they are analogues of monosaccharides with a ring oxygen replaced by a ring nitrogen. They include polyhydroxylated derivatives of piperidine (Nojiromycin, deoxynojiromycin, N-butyldeoxynojiromycin, deoxymannojiromycin), pyrrolidines (DMDP, LAB), indolizidines (swainsonine, castanospermine, 6-O-Butanoylcastanospermine) and pyrolizidines (Australine).

It is conjectured that the mechanism of action of the glycosylation modulators in HIV-1 infection is that of glucosidase I inhibition. A number of imino sugars are inhibitors of glucosidase I in vitro and also act as anti- HIV-1 agents, whereas mannosidase inhibitors, such as deoxymannojiromycin and swainsonine, display no anti-viral activity. There is structural evidence that glucosidase I inhibition occurs at the in vitro anti-viral concentration of 0.5 mM Bu-DNJ (Karlsson et al., 1993, Journal of Biological Chemistry 268: 570-576). Recombinant gp120/gp41 was expressed in CHO cells with Bu-DNJ present at 0.5 mM. The gp120 produced contained altered N-linked oligosaccharide structures with the terminal sequence

Glcα1 ,2Glcα1 ,3Glcα1 ,3 being present. The three structures found on gp120 were GlcsMang, GlcaManβ and Glc3Man7. Other experiments have more directly suggested inhibition of the post-virion binding membrane fusion event as the anti-HIV mechanism of the imino sugars (Gruters et al., Nature 330: 74-77; Walker et al., PNAS USA 84: 8120-8124). Virus particles produced in the presence of both castanospermine and deoxynojiromycin (two imino sugars related to Bu-DNJ) could still bind to the HIV-1 receptor CD4, but the infectivity of virions was reduced. This implicates the inhibition of a post-CD4 binding event , such as the necessary membrane fusion event.

Thus, the present invention utilises glycosylation modulators such as, but not limited to, Bu-DNJ and 6-BuCS (BuCast), in combination with a membrane fusion inhibitor which interferes with, modulates or otherwise has a biological effect on post virion-binding membrane fusion events. Such compounds which modulate fusion events include, but are not limited to, Enfuvirtide. It is the expectation that the biological effect of coadministration of, for instance, Bu-DNJ and Enfuvirtide will be synergistic; that is to say the effect of both compounds in a treated individual will be greater than the effect of either compound used individually in the patient.

Alkaloids for use in the combinations of the invention

Any alkaloid may be used according to the invention providing that it is therapeutically efficacious when administered in combination with a viral entry inhibitor (as herein defined).

Preferred alkaloids for use in the combinations of the invention: (a) are glycosylation modulators as defined herein and described in the previous section; (b) have antiviral activity (e.g. being an alkovir, glycovir or glucovir as herein defined); and/or (c) have immunomodulatory activity (e.g. being an immunomodulatory or cytokine activating alkaloid as herein defined).

Antiviral alkaloids for use according to the invention may be readily identified by routine screening assays (e.g. cell-based plaque reduction assays, assays based on the reduction of cytopathic effects or a reduction of viral antigen expression). Those skilled in the art will readily be able to identify appropriate conditions for such assays, including inter alia the nature, source and number of the target cells and viruses, the relative concentrations of cells and virus (the MOl), the duration and conditions of incubation and the methods used to detect viral activity.

Immunomodulatory alkaloids for use according to the invention may be readily identified by screening assays designed to detect the induction of one or more cytokine(s) (for example, IL-12 production in dendritic cells) in vitro. Such assays conveniently involve immune assays or microarray analysis (the latter being especially useful in embodiments where immunomodulatory alkaloids which stimulate a large number of different cytokines or which differentially stimulate a specific subclass of cytokines (e.g. Th1 cytokines) are to be selected). Those skilled in the art will readily be able to identify appropriate conditions for such assays, including inter alia the nature, source and number of the PRR-bearing cell (e.g. macrophages or dendritic cells), the relative concentrations of alkaloid and cells, the duration of stimulation with alkaloid and the methods used to detect the induction of the cytokine(s).

Alkaloid glycosylation modulators, glucovirs and glycovirs may be identified by standard enzymological assay. Preferred are alkaloids 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).

Examples of various antiviral and immunomodulatory alkaloids and alkaloid classes suitable for use according to the invention will now be described.

General considerations

Many alkaloids suitable for use according to the invention are phytochemicals, present as secondary metabolites in plant tissues (where they may play a role in defence), but some may 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 so microbes (including bacteria and fungi, particularly the filamentous representatives) may also be a useful source of immunomodulatory alkaloids for use according to the invention (see below).

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 a/. (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 (including sugar analogues or imino sugars). 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.

Alexine (1) and australine (2) were the first 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.

Alexine (1)

Australine (2)

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 (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 antiproliferative and immunoregulatory agent (see e.g. US5650413, WO00/37465, 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 antimetastatic 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 (1S1 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, 7R, 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.

1α, 2α, 6α, 7ct, 7αβ- 1,2,6, 7-tetrahydroxy(9)

Casuarine, (1 R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1,2,6,7-ietrahydroxy(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).

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.

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.

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

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

Glucose analogues

The alkaloids for use according to the invention may be imino sugars which act as glucose analogues. Such imino sugars share some or all of the binding properties of glucose in vivo (without necessarily sharing all of the attendant functional properties thereof). Examples of such compounds are described in e.g. WO9929321 (the disclosure of which relating to specific piperidine imino sugars and their structure is hereby incorporated by reference).

Thus, in embodiments where the adjunctive agent for use in the combinations of the invention exhibit an inhibitory effect on glucosidases, the adjunctive agent may be a structural analogue of glucose. One example of such an analogue is the imino sugar designated 1 ,5-dideoxy-1 ,5-imino-D-glucitol (alternately designated deoxynojirimycin), hereinafter "DNJ." Numerous DNJ derivatives have been described. DNJ and its alkyl derivatives are potent inhibitors of the N-linked oligosaccharide processing enzymes, a-glucosidase I and a-glucosidase Il (Sauπier et al. (1982) J Biol Chem 257:14155-14161; Elbein (1987) Ann Rev Biochem 56:497534). These glucosidases are associated with the endoplasmic reticulum of mammalian cells. The N- butyl and N-nonyl derivatives of DNJ may also inhibit glucosyltransferases associated with the Golgi. DNJ and various derivatives thereof are described in greater detail infra.

Mannose and/or rhamnose analogues

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

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

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

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

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

Structural considerations

Particularly preferred are alkaloids selected from the following structural classes:

(g) piperidine alkaloids;

(h) pyrroline alkaloids;

(i) pyrrolidine alkaloids;

(j) pyrrolidine alkaloids;

(k) indolizidine alkaloids; (I) nortropane alkaloids;

(m) non-protein amino acids (e.g. canavanine).

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

Preferred immunomodulatory alkaloids are polyhydroxylated alkaloids and imino sugars. Particularly preferred are alkaloids having a small molecular weight, since these may exhibit desirable pharmacokinetics.

Thus, the alkaloid may have a molecular weight of 100 to 400 Daltons, preferably 150 to 300 Daltons and most preferably 200 to 250 Daltons.

In a preferred embodiment, the alkaloid has the formula:

wherein R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof. In such embodiments, the compound may be an acyl derivative. Thus, the alkaloid for use in the combinations of the invention may have the formula shown above and be peracylated, acylated at C-3 hydroxy methyl, acylated at C-6 or acylated at C-3 hydroxy methyl and C-6. Other acyl derivatives include alkanoyl or aroyl derivatives. Thus, the acyl derivative may be selected from an alkanoyl (e.g. acetyl, propanoyl or butanoyl).

Also preferred are alkaloids having the formula:

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

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

or a pharmaceutically acceptable salt or derivative thereof.

In the formulae above, R may be a saccharide moiety (e.g. a glucoside or arabinoside moiety).

Thus, the alkaloid for use in the combinations of the invention may be a casuarine glycoside, or a pharmaceutically acceptable salt or derivative thereof. In such embodiments, the alkaloid is preferably casuarine-6-α-D-glucoside of the formula:

or a pharmaceutically acceptable salt or derivative thereof. Other suitable alkaloids for use according to the invention are selected from:

(a) 3,7-diepi-casuarine;

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

(d) 6,7-diepi-casuarine;

(e) 3-epi-casuarine;

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

(g) 7-epi-casuarine-6-α-D-glucoside; (h) 3,6,7-triepi-casuarine-6-α-D-glucoside;

(i) 6,7-diepi-casuarine-6-α-D-glucoside; and

(j) 3-epi-casuarine-6-a-D-glucoside,

or a pharmaceutically acceptable salt or derivative thereof.

In another preferred embodiment the alkaloid has the formula:

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

In such embodiments, most preferred are alkaloids having the formula:

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

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

or a pharmaceutically acceptable salt or derivative thereof. In another embodiment, the alkaloid has the formula:

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

In such embodiments, the alkaloid preferably has the formula:

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

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

In another embodiment, the alkaloid has the formula:

or a pharmaceutically acceptable salt or derivative thereof.

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

or a pharmaceutically acceptable salt or derivative thereof.

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

or a pharmaceutically acceptable salt or derivative thereof.

Other preferred immunomodulatory alkaloids for use in the combinations of the invention have 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: -OH1 -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 preferred embodiments of the latter case:

(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 (CH2)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 (CHa)nX, wherein n is 1-4.

In yet other preferred embodiments:

Rs is (CH2)nX; and Ri=R4=(CH2)nX; and/or R2=R3=X or (CH2)nX.

In yet other embodiments:

R5 is (CH2)πX; and R1=R4=(CHz)nX; and/or R2=R3=X.

Another preferred pyrrolidine alkaloid is: (a) N-hydroxyethylDMDP having the formula:

and (b) :

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

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

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

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

1S, 2R1 7R, 7aR)-1,2,7-trihydroxy(7)

or of the formula:

7S-epimer (8)

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

Suitable non-protein amino acids for use in the combinations of the invention are described for example in US5110600 (the disclosure of which is hereby incorporated by reference). Preferred as a non-protein amino acid for use according to the invention is L-canavanine:

However, other natural and synthetic amino acid analogs capable of incorporation into proteins synthesized by mammalian cells, but that do not inhibit protein synthesis, may be used. These analogs include less basic analogs of basic amino acids including arginine, lysine and histidine. (Noe, J. Biol. Chem. 265:4940-4946 (1981) and Christner et al., J. Biol. Chem. 250:7623-7630 (1975)).

Deoxynojirimvcin (DNJ) and its derivatives

The use of iminosugars containing the glucose analogue DNJ as antiviral agents against different viruses has been suggested since the late 1980s. While the action of two of them, DNJ and NB-DNJ, has been extensively described in the literature, the discovery of the antiviral action of a longer-alkyl-chain derivative of DNJ, NN-DNJ, was reported only relatively recently (see Zitzmann et al. (1999) PNAS 96: 11878-11882).

DNJ and its N-alkylated derivatives have been shown to inhibit α-glucosidase I and/or α-glucosidase II, so preventing the interaction of CNX and/or CRT with folding glycoproteins. N-alkylation of DNJ has been shown to increase its inhibitory potency: N-nonyl-DNJ (NN-DNJ), a 9-carbon alkyl derivative of DNJ, has been found to be at least 20 times more potent than the non-alkylated DNJ in inhibiting hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) in cell based assays. Other N-substituted DNJ derivatives (including N-methoxy-nonyl-DNJ and N-butyl-cyclohexyl DNJ) have also been shown to have improved potency (the N-methoxy analogue being the most potent, exhibiting micromolar antiviral activity).

However, ER α-glucosidase inhibition does not correlate precisely with antiviral activity: the less active NB- DNJ is a more effective ER α-glucosidase inhibitor than NN-DNJ. Moreover, the short-chain N-butyl-DGJ (NB-DGJ) exhibits no antiviral activity, whereas its long-chain derivative NN-DGJ is a potent antiviral. Thus, an additional mechanism of action appears to be associated with the length of the N-alkyl side chain, and it has recently been suggested that this may be based on the inhibition of an ion channel formed by the HCV p7 protein (Pavlovic ef a/. (2003) PNAS 100(10): 6104-6108; see also WO2004/047719). However, further studies (Mehta ef a/. (2004) Antimicrobial Agents and Chemotherapy 48(6): 2085-2090) have shown that at least one alkovir (N-9-oxadecyl-6-methyl-DGJ) inhibits HCV under conditions where p7 is not present, suggesting that p7 inhibition may not be the sole mechanism of alkoviral activity.

lmino sugars mediating an antiviral effect via α-glucosidase inhibition (for example, DNJ and NB-DNJ) have been dubbed 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). The use of iminosugar α-glucosidase inhibitors in general (and DNJ and other piperidine derivatives in particular) as antiviral drugs is limited by toxicity arising from coinhibition of gastrointestinal α-glycosidases at the concentrations required for therapeutic effects. There is therefore much interest in alkovirs, since toxicity arising from coinhibition of gastrointestinal α-glycosidases may be avoided by members of this class. Indeed, the N-substituted iminosugar Λ/-7-oxanonyl-6-deoxy-DGJ (Λ/-7-oxanonyl-6- methyldeoxygalactonojirimycin; Λ/-7-oxanonyl-6-lvleDGJ) was entered into phase I clinical studies (as UT 231-B) in 2002.

Bu-DNJ (also known as SC-48334, Miglustat, OGT 918 or Zavesca™) is a small molecule, which has most recently been developed by Oxford Glycosciences for the treatment of mild to moderate Type I Gaucher's disease, in patients for whom enzyme replacement therapy is not suitable. Use of Bu-DNJ is described in EP1321143 and WO0107078. Bu-DNJ is described as an orally available inhibitor of glycoceramide synthase, a key enzyme in the biosynthesis of glycosphingolipids (GSLs). Gaucher's disease is marked by an accumulation of GSLs in various organs. It leads to enlarged spleen and liver and a number of clinical symptoms. It is administered in 100mg hard capsules between 2 and 3 times per day. A number of Gl related side effects are common, but respond to loperamide. However, weight gain/loss, peripheral neuropathy, dizziness and cognitive complications have also been identified.

Bu-DNJ has also been previously documented in the scientific literature as an inhibitor of glucosidase I, a key cellular enzyme in the biosynthetic pathway of N-linked oligosaccharides. As a nascent polypeptide is extruded into the lumen of the Endoplasmic Reticulum, the consensus glycosylation sequon Asn-Xaa- Ser/Thr signals to the cellular machinery to transfer a dolichol-linked Glc3MangGlcNAc2 carbohydrate precursor to the asparagine residue of the sequon. Glucosidase I is responsible for the initial trimming reactions that remove the terminal α-1 ,2 linked glucose residue from the carbohydrate precursor. Bu-DNJ has been previously shown to have strong anti-HIV-1 activity in vitro at non-cytotoxic dose ranges (Fleet et al. (1988) FEBS Letters 237: 128-132; Karpas et al. (1988) PNAS USA 85: 9229-9233). The use of Bu-DNJ in inhibiting HIV replication in humans is described in US 4,849,430.

Clinical trials of Bu-DNJ in HIV-1 positive patients were carried out during 1993 and 1994 (Fischl MA, et al.). The safety and efficacy of combination N-butyl-deoxynojirimycin (SC-48334) and zidovudine in patients with HIV-1 infection and 200-500 CD4 cells/mm3. J Acquir Immune Defic Syndr Hum Retroviral 1994; 7: 139-47). A double-blind, randomized Phase Il study was used to evaluate the safety and activity of the combination therapy of Bu-DNJ and zidovudine versus zidovudine alone. Patients with 200 to 500 CD4 cells/ mm3 who had tolerated less than or equal to 12 weeks of prior zidovudine therapy received Bu-DNJ (1000 mg every 8 h) and zidovudine (100 mg every 8 h) or zidovudine and placebo. 60 patients received combination therapy and 58 received zidovudine and placebo. Twenty-three patients (38%) and 15 (26%), in the combination and zidovudine groups, respectively, discontinued therapy (p = 0.15). The mean Bu-DNJ steady-state trough level (4.04 +/- 0.99 micrograms/ml) was below the in vitro inhibitory concentration for human immunodeficiency virus (HIV). The mean increase in CD4 cells at week 4 was 73.8 cells/ mm3and 52.4 cells/ mm3 for the combination and zidovudine groups, respectively (p > 0.36). For patients with prior zidovudine therapy, the mean change in CD4 cells in the combination and zidovudine groups was 63.7 cells/ mm3and 4.9 cells/ mm3 at week 8 and 6.8 cells/ mm3 and -45.1 cells/ mm3 at week 16, respectively. The number of patients with suppression of HIV p24 antigenemia in the combination and zidovudine groups was six (40%) and two (11%) at week 4 (p = 0.10) and five (45%) and two (14%) at week 24 (p = 0.08), respectively. Diarrhoea, flatulence, abdominal pain and weight loss were common for combination recipients.

It would appear that the trials failed as steady state levels of Bu-DNJ achieved in the trial were below the inhibitory concentration for HIV previously shown in vitro. While there was circumstantial evidence of clinical efficacy of Bu-DNJ in this trial, statistical significance was not reached with the comparatively weak clinical effect and small patient numbers.

A further Phase I dose escalation trial on 29 patients was carried out to identify the maximum tolerated dose (MTD) (Tierney et al., J Acquir Immune Defic. Syndr. Hum. Retrovirol. 1995 Dec 15;10(5):549-53). Dosing was begun at 8 mg/kg/day and subsequent doses were 16, 32, 48 and 64 mg/kg/day. The maximum tolerated dose was not achieved because of slow accrual and because the study was stopped after the finding of cataracts in initial long-range rat toxicology studies. These cataracts were later shown to be transient and not found in other animals. The most common side effects were gastrointestinal, with diarrhoea and flatulence occurring in most subjects, which seemed to partially improve on a modified diet that excluded complex carbohydrates. Grade III elevations in liver function tests were seen in two patients. Grade III leukopenia and neutropenia were seen in seven patients, but were only severe enough in two to require discontinuation.

Given the failure of the development of Bu-DNJ as an anti-HIV therapy, it was redeveloped using a lower dose for its current marketed use in Type I Gaucher's disease. The dose used in Gaucher's disease is 100mg, up to three times per day. Therefore, Bu-DNJ appears to have been redeveloped on the basis of its pharmacology as a glycoceramide synthase inhibitor in a new dose range, as opposed to its original application in HIV research, on the basis of its pharmacology as a glucosidase I inhibitor. However, the precise mechanism whereby Bu-DNJ has potent anti-HIV activity in vitro has not been conclusively demonstrated.

Thus, as described above, Bu-DNJ has shown partial activity in HIV-infected individuals at doses of up to 3 g per day in combination with zidovudine, but clinical endpoints for efficacy in clinical trials were not attained with statistical power (p values were above 0.05). To date, the use of Bu-DNJ for HIV infection in a clinical environment has not been successfully achieved. However, the molecule has shown sufficient safety, ADMET characteristics and profile for EU launch for Type I Gaucher's disease, with a lower dose range of 200-300 mg per day.

Bu-DNJ causes a significant alteration of the N-linked carbohydrate structures on gp41 and gp120. gp120 is heavily glycosylated and contains at least 20 consensus glycosylation sequons across viral isolates. Recombinant gp120 contains N-linked oligosaccharides at all these sequons. In addition, gp41 contains 4 consensus glycosylation sequons (Myers et al. (1989) Human Retroviruses and AIDS: A compilation and analysis for nucleic acid and amino acid sequences, Los Alamo National Laboratories) and is central to the post CD4-binding membrane fusion event. The present invention is based on the prediction that moderately altered carbohydrate structures on gp41 or potentially gp120, following administration of a glycosylation modulator, such as Bu-DNJ, will potentiate the activity of other pharmaceuticals involved in gp41 -mediated events, such as membrane fusion. The activity of a membrane fusion inhibitor, such as Enfuvirtide, will be enhanced by co-administration of Bu-DNJ, especially but not only due to the alteration of N-linked oligosaccharide structures on gp41. These altered carbohydrate structures in combination with membrane fusion inhibitor activity will additionally prevent the conformational changes in gp120 and gp41 required for the viral envelope and cellular membrane fusion event.

Mutational analysis on the consensus glycosylation sequons in gp120 (Lee et al., 1992, PNAS USA 89: 2213-2217) and gp41 (Lee et al., 1992, Journal of Virology 66: 1799-1803) has also been important in suggesting the mechanism of anti-HIV activity of Bu-DNJ. Five consensus glycosylation sequons in the 24 glycosylation sequons in gp120 are important in viral infectivity because, if they are individually removed, there is a significant reduction in viral infectivity. Similarly, removing the extracellular glycosylation sequon of gp41 at amino acids 637 to 639 (according to gp160 polyprotein numbering system) had a severe impact on viral infectivity. It is speculated here that alterations to the N-linked oligosaccharide at this extracellular gp41 sequon that may be caused by Bu-DNJ treatment are central to enhancing or complementing the anti-HIV activity of Enfuvirtide. H is additionally proposed that alterations to N-linked oligosaccharide structures in gp41 or gp120, through inhibition of glycosidase I, alter and enhance the way that Enfuvirtide interacts with the first freptad repeat (HR 1) of gp41, anάlor enhance the subsequent inhibition of conformational changes in gp41/gp120 necessary for the membrane fusion event. The altered carbohydrate structures may exert their beneficial effect on Enfuvirtide-mediated inhibition of the virion-cell membrane fusion event either directly or indirectly, via alteration of the tertiary or quaternary structure of the gp41/gp120 complex. It is also envisaged that the combined use of Bu-DNJ and Enfuvirtide will enable a dose of Enfurvirtide lower than the current dose of 90 mg/day to be used with therapeutic effect.

A wide variety of N-alkylated piperidine imino sugars suitable for use in the combinations of the invention are disclosed for example in US2004/0110795A1

Nojirimycin 1-Deoxynojirimycin α-Homonojirimycin (DNJ) (HNJ)

N-butylDNJ N-nonylDNJ N-methoxy-nonyl-DNJ (NB-DNJ or Zavesca™) (NN-DNJ)

Deoxygalactonojirimycin N-butylDGJ N-nonylDGJ (DGJ) (NB-DGJ) (NN-DGJ)

N-7-oxanonyl-6-MeDGJ N-nonyl-6-MeDGJ N-9-oxadecyl-6-MeDGJ ("UT-231 B")

However, the known alkovirs which appear to act independently of α-glucosidase inhibition may not have the required potency to treat or prevent viral infection (phase 2 trials with UT 231 -B (Λ/-7-oxanoyl-6-deoxy-DGJ) failed to demonstrate efficacy in non-responders in a Phase 2 study).

Castanospermine and its derivatives

As explained earlier, the indolizidine alkaloid castanospermine (1 ,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that modifies glycosylation by inhibiting α-glucosidase I. Castanospermine has been shown to exhibit potent antiviral activity against a range of different viruses, including HIV. In particular, the alkaloid has been shown to inhibit syncytium formation induced by the envelope glycoprotein of the human immunodeficiency virus and to inhibit viral replication. The decrease in syncytium formation in the presence of castanospermine can be attributed to inhibition of processing of the envelope precursor protein gp160, with resultant decreased cell surface expression of the mature envelope glycoprotein gp120. In addition, castanospermine may cause defects in steps involved in membrane fusion after binding of CD4 antigen. The antiviral effects of castanospermine may be due to modifications of the envelope glycoprotein that affect the ability of the virus to enter cells after attachment to the CD4 cell receptor.

Various castanospermine derivatives have been produced which exhibit improved pharmacokinetics, improved uptake, reduced toxicity and/or increased antiviral potency. For example, US5385911 , describes the synthesis and antiviral activity of a class of castanospermine esters while EP0297534 describes a class of castanospermine esters and glycosides. Any of the castanospermine analogues and derivatives described in these references may be used in the combinations of the invention.

Preferred are castanospermine esters (e.g. mono- or di-esters), glycosides, acyl derivatives or other derivatives thereof. Particularly preferred are castanospermine derivatives having formulae selected from the table below:

Generic formula 6-0-butanoylcastanospermine Castanospermine (e.g. EP0297534) (Bucast)

Particularly preferred is 6-0-butanoylcastanospermine (Bucast). BuCast is a 30-50-fold better inhibitor of HIV syncytia formation than castanospermine (Sunkara et a/., (1989) Anti-HIV activity of castanospermine analogs Lancet, i, 1206; Taylor et al,, (1991 ) 6-o-Butanoyl castanospermine (MDL 28,574) inhibits processing and growth of HlV. AIDS, 5, 693-698).

Functional considerations

Particularly preferred for use as alkaloids according to the invention are alkaloids falling into one or more of the following functional classes: (a) alkaloid PRR ligands (including NOD-protein ligands, TLR ligands and C-type lectin ligands); (b) non-metabolizable alkaloids (which may exhibit extended tissue residence durations, and so exhibit favourable pharmacokinetics); (c) immunomodulatory alkaloids (or cytokine stimulatory alkaloids), as herein defined; (d) glycosidase inhibitors (as herein described). Alkaloid PRR liqands for use according to the invention

Preferred alkaloids for use according to the invention are PRR ligands (as defined herein).

Such alkaloid PRR ligands may be readily identified by screening assays which detect: (a) binding to a PRR (for example, TLR, C-type lectin or NOD-protein); and/or (b) the stimulation of PRR (for example, TLR, C- type lectin or NOD-protein) signalling. In the former case, the assays may involve competitive binding assays using an isolated PRR and a known cognate PAMP ligand as test reagents. Such competitive binding assays are routine in the art, and those skilled in the art will readily be able to identify appropriate conditions and formats for such assays. In the latter case, assays for PRR (for example C-type lectin) signalling activity may involve the use of PRR (for example C-type lectin)-bearing immune cells (typically DCs) as test reagent. Those skilled in the art will readily be able to identify appropriate conditions and formats for such assays, including inter alia the nature and number of the dendritic cells, the relative concentrations of alkaloid and cells, the duration of stimulation with alkaloid and the methods used to detect signalling (for example by immunoassay for cytokine release).

The alkaloid PRR ligands of the invention may bind any PRR, including any TLR, C-type lectin or NOD- protein. Preferably, the alkaloids of the invention bind to PRRs displayed on/expressed by DCs, though they may bind to PRRs in, on or secreted by other cells including other cells of the innate immune system as well as to PRRs in, on or secreted by, for example, macrophages and T-cells.

NOD-protein ligands. The NOD-proteins (also known as the caterpillar family and NOD-LRR family) are cytosolic proteins that have a role in various innate and adaptive immune responses to cytosolic pathogens. Particularly preferred alkaloid NOD-protein ligands for use according to the invention are N0D1 and/or

N0D2 ligands. These latter proteins bind structures derived from peptidoglycan that are not TLR ligands.

NOD-protein PRRs comprise C-terminal leucine-rich repeats (LRRs), a central nucleotide-binding oligomerization domain (NOD), and N-terminal protein-protein interaction motifs, such as caspase recruitment domains (CARDs), pyrin domains or a TIR domain.

Toll-like receptor (TLR) ligands: The alkaloid PRR ligands of the invention may bind to any TLR receptor.

Thus, the alkaloid PRRs of the invention may bind to one or more of TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6,

TLR7, TLR8, TLR9, TLR10 and TLR11.

Preferably, the alkaloid TLR ligands for use according to the invention bind to:

(a) a TLR coupled with the MyD88 adaptor signalling pathway; and/or

(b) a TLR coupled with the TRIF adaptor signalling pathway; and/or

(c) a cell-surface TLR; and/or (d) an endosomal TLR (e.g. TLR7, TLR8 and/or TLR9);

(e) an intracellular TLR (e.g. TLR3). Particularly preferred are alkaloid TLR9 or TLR4 ligands.

Alkaloid C-type lectin ligands: The alkaloid PRR ligands of the invention may bind to any of the lectins described in Figdor et al. (2002) Nature Reviews Immunology 2: 77-84 (the disclosure of which relating to the identification of various C-type ligands being incorporated herein by reference).

Thus, the alkaloids of the invention may be ligands for type I and/or type Il C-type lectins. In particular, the alkaloids of the invention may be ligands for C-type lectins selected from:

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

(b) DEC-205; and/or

(c) Dectin 1 ; and/or

(d) Dectin 2; and/or (e) Langerin; and/or

(f) DC-SIGN; and/or

(g) BDCA-2; and/or (h) DCIR; and/or (i) DLEC; and/or G) CLEC; and/or

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

Biological activities of the immunomodulatory alkaloids of the invention

The alkaloids for use in the combinations of the invention are preferably cytokine stimulatory alkaloids capable of stimulating the activity of one or more cytokine(s) in a PRR-bearing cell. In preferred embodiments, the alkaloid may stimulate one or more Th1 cytokine(s) in a PRR-bearing cell, for example IL- 12 and/or IL-2.

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, the alkaloid-induced expression of IL-2 may directly potentiate a Th1 response and so increase the Th1 :Th2 response ratio. The alkaloid-induced expression 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).

The alkaloids of the invention may stimulate the expression of IL-12 in PRR-bearing cells (for example in dendritic cells and/or macrophages). 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 C04* Th1 cells and cytotoxic CDS+ cells which produce IFN-γ. It therefore increases T-cell invasion of tumours as well as the susceptibility of tumour cells to T-cell invasion.

Thus, without wishing to be bound by any theory, the immunomodulatory activity of the alkaloids for use in the combinations of the invention may arise from the stimulation of one or more cytokines (for example one or more Th1 cytokines, e.g. IL-12 and/or IL-2) in PRR-bearing cells (e.g. macrophages or dendritic cells). This leads to the stimulation of NK cells to produce IFN-γ and induces the development of CD4* Th1 cells. The induced TM cells then produce IFN- y and IL-2. The stimulated cytokine(s) (e.g. IL-12 and/or IL-2) then enhances further proliferation of Th1 cells and the differentiation of pathogen (e.g. tumour and virus) - specific CD8+ T cells. The cytokine(s) also stimulate the cytolytic activity of NK cells of the innate immune system.

Auxiliary antiviral agents for use with the combinations of the invention

In addition to the viral entry inhibitor and adjunctive agent selected from: (a) a glycosylate 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 combinations of the invention. This is particularly advantageous in the case where the invention is applied to the treatment of HlV 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 141 W94 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) Q) Viracept® nelfinavir NFV AG-1343 (Pfizer)

(k) Brecanavir™ GW640385 or VX-385 (GlaxoSmithKline)

(I) Darunavir™ TMC-114 (Tibotec)

Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs)

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 A2T 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)

(j) 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 lnterieukin-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-HlV Drugs

(a) lntegrase 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 combinations 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 combinations 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).

Co-therapeutic agents for use with the combinations of the invention

The combinations 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 combinations 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 combinations 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 combinations of the invention.

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); 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); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Irldoviridae (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, chikunguπya 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), Hepaclvirus (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, MA 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.

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 mode). 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).

Posoloqy

As explained herein, the invention contemplates the combined use of a viral entry inhibitor and an adjunctive agent selected from: (a) a glycosylation modulator; (b) an alkovir; and (c) a glycovir (e.g. a glucovir). However, as explained above, while the viral entry inhibitor and adjunctive agent are administered as part of the same overall treatment regimen, the posology of each of these components of the combinations of the invention may differ: for example, each may be administered at the same time or at different times. It will therefore be appreciated that the viral entry inhibitord and adjunctive agents (e.g. the imino sugars) of the invention may be administered sequentially (e.g. before or after) or simultaneously, either in the same pharmaceutical formulation (i.e. together), or in different pharmaceutical formulations (i.e. separately). Simultaneously in the same formulation is as a unitary formulation whereas simultaneously in different pharmaceutical formulations is non-unitary. The posologies of each of the two or more compounds/agents in a combination therapy may also differ with respect to the route of administration.

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

The amount of the adjunctive agent 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 adjunctive agent selected.

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

In general, the effective amount of the adjunctive agent 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 adjunctive agent, and can be taken one or more times per day. The adjunctive agent 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.

If Bu-DNJ is used as the imino sugar, it is preferably used at a dose of from 200 to 400 mg/day, preferably about 300 mg/day.

Enfuvirtide is preferably used at a dose of 80 to 100 mg/day and more preferably at a dose of about 90 mg/day. It is expected that if used with Bu-DNJ, the most clinically suitable doses of Enfuvirtide and Bu- DNJ to be used in combination would be determined within controlled clinical trials. However, an initial trial could be performed using the current doses authorised for both compounds. Therefore, initially a dose of 200 to 400 mg Bu-DNJ orally could be administered to patients also receiving a sub-cutaneous injection daily of about QO mg of Enfuvirtide. The dose of Bu-DNJ to be investigated in this novel clinical setting could however be as high as 3 g per day as previously used in the Phase Il efficacy trial, or even as high as 64 mg/kg/day as planned in the Phase I dose ranging trial.

Formulation

In embodiments where the combinations 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 one or more of the components of the combinations 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 adjunctive agent 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 adjunctive agent of the invention is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

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

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

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the sugars of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Injectable formulations may be preferred in embodiments where the viral entry inhibitor (e.g. fusion inhibitor) is an acid labile peptide.

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.

One or more of the components of the combinations of the invention may also be presented as liposome formulations. For example, the alkaloids of the combinations of the invention may be delivered as a liposome formulation (e.g. to increase uptake).

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

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

The viral entry inhibitor component (particularly in embodiments where the inhibitor is a membrane fusion inhibitor) may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally.

In such embodiments, the component 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, isopropano), 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 for use according to the invention will typically contain from about 0.5 to about 25% by weight of the adjunctive agent 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.

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

Exemplification

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

Example 1 : 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 a compound 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).

Plaque Assay: The materials and procedures were as described in Whitby et al. (2004) Antiviral Chemistry and Chemotherapy 15: 141-151. In brief, MDBK cells were seeded in 96 well plates and allowed to reach confluency. Monolayers were exposed to between 14 and 45 plaque forming units of BVDV and adsorption allowed to take place. Infected monolayers were then exposed to the test compound, medium added containing low gelling-point agarose and allowed to set. The plates were then incubated for 4 days post infection, fixed in 5% formalin and stained with 0.5% neutral red after removal of the agarose layer. Antiviral activity was measured by determination of plaque inhibition and expressed as IC50 values. Castanospermine, a known viral inhibitor, was used as a positive control.

Results:

The results show that the test compound of the invention exhibits good antiviral activity against BVDV. No cytotoxicity was noted. Example 2: Anti-influenza activity

In normal adults, infection with influenza A viruses can result in a range of clinical effects from asymptomatic infection to primary pneumonia that can progress rapidly to become fatal. Neonates and young children under 5 years of age have the highest rates of hospitalisation after acute viral infection.

Young children are more likely to develop lower respiratory tract disease with various complications. This is generally due to the low level of protective immunity in the very young. During influenza epidemics the death rate in the elderly increases significantly, the main culprit being influenza A strains. Influenza B virus, RSV and other respiratory viruses, including influenza A and B strains, are an increasing problem in transplant recipients and in other immunosuppressed patients.

While vaccination has proved successful in some circumstances, the approach requires constant change in immunogens to match the current wild-type virus. The benefits of a safe and effective therapy with small molecules has long been sought.

The ability of a compound of the invention:

H

to exert a direct anti-influenza A effect in vitro was therefore tested and activity demonstrated (as detailed below).

Plaque reduction assays were used to confirm anti-viral activity by monitoring the size and number of plaques in treated, infected cells (Hayden F. G., Cote K.M. and Douglas G. 1980. Plaque inhibition assay for drug susceptibility testing of influenza viruses. Antimicrobial Agents & Chemother. 17:865-870).

Briefly, confluent MDCK cells, seeded at 105 cells/well in 24-well culture plates used, infected at a MOI of 0.001 , that produced approximately 50-100 plaques per well. Virus (influenza A strain X31) was absorbed for 2hrs, at room temperature and removed. Infected cell monolayers were overlaid with 1ml of a 1:1agarose: serum free, double strength medium (supplemented with 0.6% BSA, 0.004% DEAE dextran and 2μg/ml. TPCK Trypsin) containing different concentrations of test compound in triplicate. Likewise, triplicate wells overlaid with compound-free medium, served as untreated controls. Plates were incubated (370C) for three days, fixed (10% formalin) and stained (3% methylene blue). Plaque number (% of control) was plotted versus compound concentration. Castanospermine, a known viral inhibitor, was used as a positive control.

The results show that the test compound of the invention exhibits antiviral activity against influenza A. No cytotoxicity was noted.

Example 3: Glucosidase inhibitory profile

Enzyme assays were carried out as described in Watson et al. (1997) Phytochemistry 46(2): 255-259. The test compound of the invention:

was compared with Celgosivir (a prodrug of castanospermine), HNJ, DNJ and NB-DNJ against a panel of different glycosidase enzymes.

All assays were carried out in triplicate, using water as a blank in place of the inhibitor. Reaction time was determined based on the length of time needed to give a final absorbance of 0.3 - 1.5 units. Linearity of the time course of the reaction was checked using a series of incubation times.

The following were combined in the well of a flat-bottomed 96-well (300 μl) microtitre plate:

10 μl enzyme solution

10 μl inhibitor solution/distilled water

50 μl substrate solution

The reaction mix was incubated at 250C for 5 to 15 minutes, and was stopped using 70 μl of glycine solution. Absorbance at 405 nm was measured immediately in a microtitre plate reader. Both compounds were initially tested at 1 mg ml"1, and IC50 and K; values were calculated if inhibition with 1 mg ml" solution was greater than 50%.

The table below shows the comparable % inhibition (at 0.8mM, 0.6mM for Celgosivir) for each compound tested.

The IC50 measured for the compound of the invention and Celgosivir against rice α-glucosidase is ~80μM whereas DNJ had an IC50 below 8μM. It should be noted that Celgosivir is a prodrug and releases the more potent and less selective glucosidase inhibitor castanospermine in the mammalian body.

Example 4: Inhibition of qlycosidase 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-nitropheπyl 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 pyrrolizidine compounds tested are shown in the table below.

The results (% inhibition) for these pyrrolizidine compounds (all at 1mg/ml) are shown in the table below:

The results show that the profile of inhibition for the compounds of the invention is quite different from that of castanospermine. None inhibits mannosidase significantly (see also further data below). Some of the compounds tested (e.g. 3,7-diep/-casuarine) do not significantly inhibit any of the enzymes tested.

The pyrroline and indolizidine 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 IC50 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 5: Differentia) inhibition of mannosidase and glucosidase

The glycosidase inhibitory profiles of swainsonine (4), casuaήne (8) and casuarine glucoside (9) with respect to a mannosidase and a glucosidase were compared. The results (all at <0.1mg/ml) are shown in the table below.

Example 6: Treatment of murine HSV-1 infection

Mice were 3-4 weeks old female BALB/c. Mice were inoculated with 104 p.f.u. HSV-1 (SC 16) using the neck skin method. This dose is sublethal but produces clinical symptoms, including inflammation (measured by increase in ear pinna thickness).

Mice were administered (100 ml i.p.) with one of two doses of casuarine (8) on day one and daily thereafter for 5 days. Group 1 received 15 mg/kg in PBS, group 2 received 150 mg/kg in PBS. A negative control group 3 were infected but received no casuarine. A positive control group 4 were administered with famciclovir (via drinking water spiked at 1 mg/ml for the same time period).

Mice were checked daily and samples were obtained from mice killed on selected days. The results are presented in Tables 6.1 - 6.3, below.

Table 6.1 : Weight (% change)

Table 6.2: Group mean weight (α)

Table 6.3: Ear pinna thickness (mm'2)

The results show the expected pattern of ear pinna thickness increase, peaking at day 4. Famvir almost completely negated the ear thickness response. Casuarine at both doses tested also produced a reduction in ear thickness. Example 7: Alkaloid mediated perturbation of glycoprotein processing and antiviral effects

The glycosidase inhibitory properties of per-O-acetyl-D-casuarine were analysed in an assay system that examines the expression of a highly glycosylated protein, HIV gp120, in insect cells. In this assay, because the glycan number is high, any inhibition of glycan trimming is easily seen as an increase in the molecular weight of the protein when assessed by SDS-PAGE and western blot.

Gp120 is expressed in insect cells using a recombinant baculovirus. The gp120 coding region has been inserted into the viral genome under the control of a strong, late promoter and high level expression of the protein occurs 24-48hrs after infection. In a typical experiment, recombinant virus is added to Spodoptera frugiperda (Sf9) cells at a multiplicity of infection of 1 and the supernatant harvested at 2 days post infection. Proteins in the supernatant are resolved by 10% SDS-PAGE, which is then western blotted with a gp120 specific serum. The normal pattern of expression produces an indistinct band at around 9OkDa, smaller than the equivalent protein from mammalian cells, due to the lack of complex sugar attachment in insect cells. Per-O-acetyl-D-casuarine was added to this experimental system in the media overlay following virus infection; no other experimental variables were altered.

Cells were routinely grown in suspension at 280C in Insect Express serum free media (BioWhittaker) supplemented with 2% foetal calf serum (Gibco). Six well dishes were seeded with 1 x 106 Sf9 cells per well and the cells were allowed to settle to form a 75% confluent monolayer for 1 hour at room temperature. The media was removed and 100 μl of a recombinant baculovirus stock previously titred at 3 x 107 pfu/ml was added for 1 hour at room temperature. The monolayers were overlaid with 2mls of media with or without MNL drug addition at 0.5mM final concentration and the plates incubated at 280C for 2 days. Two recombinant baculoviruses were used expressing two different clades of HIV gp120 (a clade C and a clade B).

Protein analysis Supernatants were harvested at two days post infection and 10μl mixed directly with loading dyes, heated and applied to a pre-cast SDS-10% polyacrylamide gel (BioRad). Following electrophoresis, the gel was transferred to a PVDF membrane and western blotted using antibodies appropriate for the C- or B-clade HIV gp120 proteins expressed. The blot was developed using chemiluminescence.

ELISA

Gp120 present in the supernatant of infected cell cultures was coated onto a lectin coated lmmulon Il plates and sera titred in two fold dilution. Primary serum binding was probed with appropriate conjugates (at 1 :1000) and the plate was washed extensively and incubated with 3,3',5,5'-Tetramethylbenzidine (TMB) chromagenic substrate (Europa Bioproducts). The reaction was stopped by addition of an equal volume of 0.5M HCI and absorbance was read at 420nm. Results

Per-O-acetyl-D-casuarine produced a particularly strong shift in MW consistent with blocking of glycan trimming enzymes (gel photograph not shown). To ensure this effect was repeatable and also true for a different HIV glycoprotein the addition of per-O-acetyl-D-casuarine was repeated (0.5mM final cone) with another recombinant baculovirus expressing a different clade (B) gp120. A clear shift in molecular weight is also seen in this assay (gel photograph not shown).

Dose response curve

The ability of per-O-acetyl-D-casuarine to shift the molecular weight of gp120 was assessed at different concentrations of drug from 0.2mM final concentration to zero. Cells were infected as before and then overlaid with drug loaded media and the gp120 present in the supernatant at 2 days post infection was identified by SDS-PAGE and Western blot. The data (gel photograph not shown) indicated saturation above 10OnM and essentially no detectable shift of molecular weight below 1OnM.

Effect on gp 120 conformation

The study was extended to include tests of the conformation of the per-O-acetyl-D-casuarine treated samples. Supernatant with the shifted MW protein were captured to microtitre plates and tested in ELISA with three sera - a polyclonal to assess overall conformation and 2 neutralising monoclonals, b12 which binds the CD4 binding site and 2G12 which binds the high mannose glycan cluster on one face of the molecule. Polyclonal Ab binding was diminished with per-O-acetyl-D-casuarine treated gp120 although this could be due to poorer capture by the lectin layer used as the first stage of the ELISA assay. However, significant binding remains. However, both b12 and 2 G12 binding was lost in the treated. sample.

Conclusions Per-O-acetyl-D-casuarine produced a significant shift in the molecular weight of a highly glycosylated protein (HIV gp120). The effect was repeatable and led to apparently specific loss of MAb binding sites. Insect cells add high mannose glycans attached to the Asn backbone via a core fucosylated structure which is then partly trimmed to the tri-man core. Mature glycoproteins from insect cells thus have a mix of long (man 9) and short (man 3) glycans. Blocking mannosidases in the cell should lead to only the longer form (man 9), a marginal increase in MW for large glycoproteins. However, the shift shown by per-O-acetyl-D-casuarine is substantial and the loss of 2G12 binding (which binds to mannose) suggests the drug acts in a more complex way than simple cellular mannosidase inhibition. The data would be consistent with blocking of cellular alpha glucosidase resulting in the maintenance of the primary glycosylated structure. The loss of neutralising antibody b12 binding is consistent with antiviral activity.

Example 8: Screening for suitable adjunctive antWIV alkaloids

Bucast, NB-DNJ (Zavesca™), per-O-acetyl-D-casuarine and per-O-butyl-casuarine ("the compounds") were each assessed for anti-HIV activity, their effects on gp120 and viral infectivity and this in several cellular assay systems. CeWs and viruses

The HIV-1 X4 molecular clone NL4.3 (Adachi et al., 1986) was obtained from the National Institute of Allergy and Infectious Disease AIDS reagent program. The transformed MT-4 T cell line and HUT-78 cells were obtained from ATCC (Manassas, VA, USA)). MT-4 cells were infected with HIV-1 NL4.3 in medium containing compounds at different concentrations. Cultures were incubated at 37 "C until an extensive cytopathic effect (CPE, or giant cell formation) was observed (4-5 days). The anti-HIV activity was determined in MT-4 cells using the MTT method (Pauwels et al., 1988). These results were confirmed by p- 24 viral Ag production, as measured by p-24 HIV-1 Ag ELISA (Perkin-Elmer). The mAb 2G12 was obtained from the MRC (Potter Bar, Hertfordshire, UK). HUT-78 cells were persistently infected with HIV-1 NL4.3, washed several times in medium before use, and incubated with different concentrations of the compounds for 2 days.

Sample preparation

Compounds were dissolved in DMSO or water and then diluted further with water and stored at room temperature. The compound n-butyldeoxynojirimycin (NB-DNJ) was obtained from Sigma.

Results

The compounds were tested for their anti-HIV activity in MT-4 cell line. Per-O-acetyl-D-casuarine was not active at the highest dose tested (up to 500 uM), celgosivir had an IC50 of 12 μM, per-O-butyl-casuarine had an IC50 of 40 μM and for NB-DNJ an IC50 of 80 μM was obtained.

On these cells 2G12 mAb staining was also performed and evaluated by flow cytometric analysis. The mean fluorescence intensity (MFI) values are shown in the table below. The MFI of uninfected MT-4 cells was 3.2 (negative control) and for HIV-infected MT-4 cells (untreated with compounds) this was 62.7 (positive control).

Compounds 2G12 staining (MFI)

500 μM 100 μM 20 μM 4 μM celgosivir 4.2 3.9 5.1 18.5 per-O-butyl-casuarine nd 5.5 65.1 58.4

Per-O-acetyl-D-casuarine 65.9 65.3 67.1 64.7

NB-DNJ 6.2 36.6 54.1 62.8

(Sigma)

From the values the percentages of inhibition of 2G12 mAb binding could be calculated and thus also their corresponding ICso values, as shown in the table below: Inhibition of 2G12 mAb staining (%) in MT-4 cells

Thus the compounds per-O-butyl-casuarine, NB-DNJ and especially celgosivir influenced the viral gp120 expression on infected cells. This difference in binding could be just due to the anti-HIV effects of the compounds but it could not be excluded that the compounds influenced also the gp120 on the cell membrane itself. To further examine this persistently HIV-1-infected HUT-78 cells, which express high levels of gp120 on their membrane and also produce high amount of virions, were studied.

In this experiment HIV-1-infected HUT-78 cells were incubated with different concentrations of compounds for 2 days and then 2G12 mAb staining was performed. In the table below the percentages of inhibition of 2G12mAb staining are shown. As can be seen, all compounds showed effects on gp120 processing on HIV- 1-infected cells after a 2 day period. Per-O-acetyl-D-casuarine (which did not show anti-HIV activity) was more potent than per-O-butyl-D-casuarine. The most potent inhibitor was celgosivir, although NB-DNJ also showed inhibitory effects.

Inhibition of 2G12 mAb staining (%) in persistently HIV-1 infected T cell line

% of inhibition of 2G12 mAb

The question remained whetehr the cells treated with these compounds produced virus particles in the same amounts as untreated cells and if this virus was less infectious then the untreated virus as it was shown that 2G12 mAb staining was clearly influenced.

For the following experiments H1V-1 -infected HUT-78 cells were incubated with different concentrations of the compounds for 2 days and then 2G12 mAb staining was performed. The table below shows the percentages of inhibition of 2G12mAb staining. As can be seen, all compounds showed effects on gp120 processing on HIV-1 -infected cells after a 2 day period.

Inhibition of 2G12 mAb staining (%) in persistently HIV-1 infected T cell line

% of inhibition of 2G12 mAb binding

500 μM 100 μM 20 μM 0.8 μM 0.16μM celgosivir nd nd 100 100 60 6 per-O-butyl-casuarine nd 80 0 0 nd nd

Per-O-acetyl-D-casuarine 100 60 0 nd nd nd

NB-DNJ 100 100 50 nd nd nd

(Sigma)

When the cells were harvested for 2G12mAb staining, the supernatant was also collected and p-24 Ag production was determined (pg/ml). The supernatant of untreated cells produced an average of 100000 pg/ml.

p-24 HIV-1 Aq production of HIV-1 -infected HUT-78 cells

p-24 HIV-1 Ag production

500 μM 100 μM 20 μM 4 μM 0.8 μM 0.16μM

10090 celgosivir nd nd 128743 101302 7 94787 per-O-butyl-casuarine nd 100512 15842 114331 nd nd

Per-O-acetyl-D-casuarine 55302 120057 120057 nd nd nd

NB-DNJ 124202 172965 101302 nd nd nd (Sigma) From these data it can be seen that although for some compounds even at 100% inhibition of 2G12 mAb binding, this did not influence at all the viral antigen production. Some of these virus preparations were then used in an experiment to infect MT-4 cells for the evaluation of the infectivity of the virus. For this purpose diluting viral p-24 Ag values were used as an input to infect these cells and no compound was added. The table below shows untreated virus or treated virus with celgosivir (20 μM), per-O-butyl-casuarine (100 μM), and NB-DNJ (500 μM) was used at different p-24 Ag values (from 2500 to 19.5 pg/ml) to infect MT-4 cells.

CPE (microscopic evaluation)

The scoring in cpe (syncytia formation) was also evaluated by p-24 viral Ag production 5 days after infection of the MT-4 cells (table below).

p-24 Aq production of H1V-1 -infected MT-4 cells p-24 Ag production (pg/ml)

2500 1250 625 312.5 156 78 39 19.5

untreated virus 553440 303550 322770 165978 122835 66877 29669 24714 celgosivir-treated 214461 152950 161066 114078 26038 5812 8969 2459

20 μM per-O-butyl- casuarine-treated 615380 421020 204849 199510 81187 67731 21937 16662

100 μM

NB-DNJ-treated 279762 167480 140420 102050 67921 18283 21554 28855

500 μM It can be seen that the compounds clearly influenced the infectivity of HIV-1 even after a treatment of only 2 days. The most profound effects were noticed with celgosivir and NB-DNJ. Also virus grown in the presence of per-O-butyl-casuarine became less infectious.

Conclusions

In these set of experiments it was demonstrated that the compounds did have some anti-HIV activity. However, control compounds such as celgosivir and NB-DNJ were in general more active. However, the compounds clearly inhibited anti-gp120 mAb (2G12) binding in a dose-dependent manner. This mAb has been shown to neutralise a wide spectrum of HIV-1 isolates and recognizes a cluster of α1-2 mannose residues on gp120, especially Asn295 and Asn392 (Scanlan et al., 2002). Moreover, when HIV-1 -infected cells were incubated with this class of compounds, effects on HIV-1 envelope gp120 processing was observed. Although these compounds did not influence viral replication and viral p-24 Ag production, the drugs influenced clearly the infectivity of the virus. These results are confirming data obtained with 6-0- butanoyl castanospermine , which also showed no effect on virus production, but the virions had decreased infectivity (Taylor et al., 1994).

Example 9: ICsn Shift Assay

Particular combinations of viral entry inhibitor (enfuvirtide, compound I) and adjunctive agent (imino sugar, compound II) can be assessed by an IC50 shift assay in which the IC50 for compound Il in the presence of varying doses of compound I is determined. Synergy was determined when the IC50 shifted down in the presence of sub-effective doses of Compound 1. Additivity was determined when the response to Compound Il and Compound I together resulted in an effect equivalent to the sum of the two compounds individually. Antagonistic effects were defined as those causing the IC50 to shift upwards, i.e. those where the response to the two compounds was less than the sum of the effect of the two compounds individually.

Any index of activity may be used in such assays, including the IC50 values determined in Examples 1 , 3, 4 and 7 (above).

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 combination of a viral entry inhibitor and an adjunctive agent selected from: (a) a glycosylation modulator; (b) an alkovir; and (c) a glycovir (e.g. a glucovir).
2. The combination of claim 1 wherein the adjunctive agent is an alkaloid is selected from the following structural classes:
(n) piperidine alkaloids;
(o) pyrroline alkaloids;
(p) pyrrolidine alkaloids;
(q) pyrrolizidine alkaloids;
(r) indolizidine alkaloids; and
(s) nortropane alkaloids.
3. The combination of any one of the preceding claims wherein the adjunctive agent is a polyhydroxylated alkaloid.
4. The combination of any one of the preceding claims wherein the adjunctive agent is an imino sugar.
5. The combination of any one of the preceding claims wherein the viral entry inhibitor is selected from: (a) an attachment inhibitor; (b) a co-receptor binding inhibitor; and (c) a membrane fusion inhibitor.
6. The combination of any one of the preceding claims further comprising one or more auxiliary antiviral agent(s).
7. The combination of claim 6 wherein the auxiliary antiviral agent is selected from one or more of: (a) protease inhibitors; (b) nucleoside/nucleotide reverse transcriptase inhibitors; (c) non-nucleoside reverse transcriptase inhibitors; (d) integrase inhibitors; (e) maturation inhibitors; and (f) cytokines or cytokine stimulatory factors.
8. The combination of any one of the preceding claims further comprising a co-therapeutic agent.
9. A combination according to any one of the preceding claims wherein the viral entry inhibitor and adjunctive agent are physically associated.
10. The combination of claim 9 wherein the viral entry inhibitor and adjunctive agent are: (a) in admixture (for example within the same unit dose); (b) chemically/physicochemically linked (for example by crosslinking, molecular agglomeration or binding to a common vehicle moiety); (c) chemically/physicochemically co-packaged (for example, disposed on or within lipid vesicles, particles (e.g. micro- or nanoparticles) or emulsion droplets); or (d) unmixed but co-packaged or co-presented (e.g. as part of an array of unit doses).
11. The combination of any one of claims 1 to 8 wherein the viral entry inhibitor and adjunctive agent are non- physically associated.
12. The combination of claim 11 wherein the combination comprises: (a) at least one of the viral entry inhibitor and adjunctive agent together with instructions for their extemporaneous association to form a physical association; or (b) at least one of the viral entry inhibitor and adjunctive agent together with instructions for combination therapy with the inhibitor and adjunctive agent; or (c) at least one of the viral entry inhibitor and adjunctive agent together with instructions for administration to a patient population in which either the viral entry inhibitor or adjunctive agent have been (or are being) administered; or (d) at least one of the viral entry inhibitor and adjunctive agent in an amount or in a form which is specifically adapted for use in combination.
13. The combination as defined in any one of the preceding claims: (a) in the form of a pharmaceutical pack, kit or patient pack; (b) in a pharmaceutical excipient; or (c) in unit dosage form.
14. A pharmaceutical composition comprising the combination as defined in any one of claims 1 to 12.
15. A combination according to any one of the preceding claims for use in therapy or prophylaxis (e.g. for use in antiviral therapy or prophylaxis).
16. A method for the treatment or prophylaxis of a viral infection in a mammal (e.g. in a human), which method comprises administering to the mammal a combination according to any one of claims 1 to 13 in an amount effective in treating or preventing viral infection.
17. Use of a combination as defined in any one of claims 1 to 12 for the manufacture of a medicament for use in the treatment or prophylaxis of viral infection.
18. A method for the treatment or prophylaxis of a viral infection in a mammal (e.g. a human), which method comprises administering to said mammal an effective amount of a viral entry inhibitor defined in any one of the preceding claims sequentially e.g. before or after, or simultaneously with an effective amount of an adjunctive agent as defined in any one of the preceding claims.
19. A method of combination antiviral therapy or prophylaxis in a mammal (e.g. a human), said method comprising administering to said mammal an effective amount of a viral entry inhibitor defined in any one of the preceding claims and an effective amount of an adjunctive agent as defined in any one of the preceding claims.
20. Use of a viral entry inhibitor as defined in any one of the preceding claims for the manufacture of a medicament for use in combination therapy with an adjunctive antiviral as defined in any one of the preceding claims.
21. Use of an adjunctive antiviral as defined in any one of the preceding claims for the manufacture of a medicament for use in combination therapy with a viral entry inhibitor as defined in any one of the preceding claims.
22. A method of enhancing or potentiating the antiviral activity of a viral entry inhibitor as defined in any one of the preceding claims in a patient suffering (or at risk from) from a viral infection, where the patient is being treated with an adjunctive agent as defined in any one of the preceding claims.
23. A method of enhancing or potentiating the antiviral activity of an adjunctive agent as defined in any one of the preceding claims in a patient suffering (or at risk from) from a viral infection, where the patient is being treated with a viral entry inhibitor as defined in any one of the preceding claims.
24. Use of a viral entry inhibitor as defined in any one of the preceding claims for the manufacture of a medicament for use in the treatment or prophylaxis of a viral infection in patient undergoing treatment with an adjunctive agent as defined in any one of the preceding claims.
25. Use of an adjunctive agent as defined in any one of the preceding claims for the manufacture of a medicament for use in the treatment or prophylaxis of a viral infection in patient undergoing treatment with a viral entry inhibitor as defined in any one of the preceding claims.
26. The invention of any one of the preceding claims wherein the viral infection involves infection with an enveloped virus.
27. The invention of claim 26 wherein the enveloped virus bears glycosylated envelope proteins.
28. The invention of claims 26 or claim 27 wherein the virus is selected from: 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, HCV, encephalitis viruses, yellow fever viruses); Coronoviridae (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); Birnavirldae; Hepadnaviridae (Hepatitis B virus); Parvoviήdae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (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).
29. A method for the treatment of a patient suffering from an infection caused by a virus bearing a glycosylated envelope protein by administering to said patient, simultaneously or sequentially, a glycosylation modulator and a membrane fusion inhibitor.
30. The invention of any one of the preceding claims wherein the glycosylation modulator is a glycosidase I inhibitor.
31. The invention of any one of the preceding claims wherein the glycosylation modulator is an imino sugar.
32. The invention of any one of the preceding claims wherein the glycosylation modulator is n- butyldeoxynojirimycin (Bu-DJ) or 6-0-butanoylcastanospermine (6-BuCS or Bucast).
33. The invention of claim 32 wherein the glycosylation modulator is Bu-DNJ used at a dose of from 200 to 400 mg/day.
34. The invention of claim 33 wherein the dose of Bu-DNJ is about 300 mg/day.
35. The invention of any one of the precding claims wherein the viral entry inhibitor is a membrane fusion inhibitor.
36. The invention of claim 35 wherein the membrane fusion inhibitor inhibits fusion events between a virion and a potential host cell.
37. The invention of any one of the preceding claims wherein the viral entry inhibitor is the membrane fusion inhibitor enfuvirtide.
38. The invention of claim 37 wherein the enfuvirtide is used at a dose of 80 to 100 mg/day.
39. The invention of claim 38 wherein the enfuvirtide is used at a dose of about 90 mg/day.
40. The invention of claim 37 wherein the enfuvirtide is used at a dose of less than about 90 mg/day.
41. The invention of any one of the preceding claims for use in the treatment of HIV-infected patients, for example in patients who have previously been treated or are undergoing treatment with other anti-HIV therapies.
42. The invention of any one of the preceding claims used in further combination with one or more other anti-HIV therapeutic(s).
43. The invention of claim 42 wherein the other anti-HIV therapeutic is selected from zidovudine, lamivudine, nelfinavir, indinavir and efavirenz.
44. The invention of any one of the preceding claims, wherein the adjunctive agent has the formula:
wherein R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof.
45. The invention of claim 44 wherein the adjunctive agent has the formula:
wherein R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof.
46. The invention of claim 44 or claim 45 wherein the adjunctive agent is an acyl derivative, for example being:
(a) peracylated; or
(b) acylated at C-3 hydroxy methyl; or
(c) acylated at C-6;
(d) acylated at C-3 hydroxymethyl and C-6.
47. The invention of claim 46 wherein the acyl derivative is alkanoyl or aroyl.
48. The invention of claim 47 wherein the acyl derivative is an alkanoyl selected from acetyl, propanoyl or butanoyl.
49. The invention of any one of claims 44 to 48 wherein R is a saccharide moiety, a glucoside or an arabinoside moiety.
50. The invention of claim 44 wherein the adjunctive agent is 1R,2R,3R,6S,7S,7aR)-3-(hydroxymethyl)-1 ,2,6,7- tetrahydroxypyrrolizidine (casuarine), wherein R is hydrogen and having the formula:
or a pharmaceutically acceptable salt or derivative thereof.
51. The invention of claim 44 wherein the adjunctive agent is a casuarine glycoside, or a pharmaceutically acceptable salt or derivative thereof.
52. The invention of claim 51 wherein the adjunctive agent is casuarine-6-α-D-glucoside of the formula:
or a pharmaceutically acceptable salt or derivative thereof.
53. The invention of claim 44 wherein the adjunctive agent Is 6-0-butanoylcasuarine, or a pharmaceutically acceptable salt or derivative thereof.
54. The invention of claim 44 wherein the adjunctive agent is selected from:
(a) 3,7-diepi-casuarιne;
(b) 7-ep/-casuarine;
(c) 3,6,7-triepi-casuanne;
(d) 6,7-d/'ep/-casuarine;
(e) 3-ep/-casuarine;
(f) 3,7-d/ep/-casuarine-6-α-D-glucoside;
(g) 7-ep/-casuarine-6-α-D-glucoside; (h) 3,6,7-fr/ep/-casuarine-6-α-D-glucoside; (i) 6,7-d/ep/-casuarine-6-α-D-glucoside; and (j) 3-ep/-casuarine-6-α-D-glucoside, or a pharmaceutically acceptable salt or derivative thereof.
55. The invention of any one of claims 1 to 43, wherein the adjunctive agent is Per-O-acetyl-D-casuarine having the formula:
CH-1
or an epimer, pharmaceutically acceptable salt or derivative thereof.
56. The invention of any one of claims 1 to 43 wherein the adjunctive agent is Per-O-butyl-casuarine having the formula:
or an epimer, pharmaceutically acceptable salt or derivative thereof.
57. The invention of any one of claims 1 to 43 wherein the adjunctive agent is a compound of the formula:
wherein R1 and R1 may be the same or different and are selected independently from linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl and aralkyl and wherein the optional substitution may be with one or more groups independently selected from: -OH; -F; -Cl; -Br; -I; -NH2; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl and aralkyl; aryl; heteroaryl; linear or branched alkoxy; aryloxy; aralkoxy; - (alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); -COO(aryl); -C(O)NH(alkyl); - C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH; and R2, R2', R3, R3', R4, R4', R5 and R5' may be the same or different and are selected independently from: -H; -OH; -F; -Cl; -Br; -I; -NHa; alkylamino; dialkylamino; linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, aralkyl, alkoxy, aryloxy and aralkoxy; aryl; heteroaryl; -(alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); - COO(aryl); -C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH; and wherein the optional substitution may be with one or more groups independently selected from -OH; -F; -Cl; -Br; -I; -NH2; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl, aralkyl, alkoxy, aryloxy and aralkoxy; aryl; heteroaryl; linear or branched -(alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); -COO(aryl); - C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH; or a pharmaceutically acceptable salt or derivative thereof.
58. The invention of claim 57 wherein R5 and/or R is hydroxyalkyl, for example Ci-6 hydroxyalkyl.
59. The invention of claim 58 wherein R5 and/or R5 is hydroxymethyl or hydroxyethyl.
60. The invention of claim 58 or claim 59 wherein at least one of R5 and/or R5 is -H.
61. The invention of any one of claims 57 to 60 wherein R2, R2', R3, R3', R4 and R4' are independently selected from -OH and -H, for example where R2 is -OH and R2' is -H; R3 is -OH and R3' is -H; and R4 is -OH and R4' is -H.
62. The invention of any one of claims 57 to 61 wherein R1 and R1 are independently selected from -H and Ci_i8 alkyl (for example, Ci_g alky], e.g. C-|.6 alkyl), C2-iβ alkenyl (for example, C2-9 alkenyl, e.g. C2-β alkenyl) and C2-18 alkynyl (for example, C2-9 alkynyl, e.g. C2-6 alkynyl).
63. The invention of claim 62 wherein R1 is -H and R1 is selected from CMS alkyl (for example, C1.9 alkyl, e.g. C1-6 alkyl), C2-18 alkenyl (for example, C2-9 alkenyl, e.g. C2.6 alkenyl) and C2-18 alkynyl (for example, C2-9 alkynyl, e.g. C2.6 alkynyl).
64. The invention of claim 63 wherein R1 is -H and R1' is methyl, ethyl, propyl or butyl.
65. The invention of any one of claims 1 to 43 or 57 to 64 wherein the compound has the formula:
wherein R1 is linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl and aralkyl and wherein the optional substitution may be with one or more groups independently selected from: -OH; -F; -Cl; -Br; -I; -NH2; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl and aralkyl; aryl; heteroaryl; linear or branched alkoxy; aryloxy; aralkoxy; -(alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); -COO(aryl); - C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH. and R2 is selected from -H; -OH; -F; -Cl; -Br; -I; -NH2; alkylamino; dialkylamino; linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyi, aralkyl, alkoxy, aryloxy and aralkoxy; aryl; heteroaryl; -(alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); - COO(aryl); -C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH; and wherein the optional substitution may be with one or more groups independently selected from -OH; -F; -Cl; -Br; -I; -NH2; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl, aralkyl, alkoxy, aryloxy and aralkoxy; aryl; heteroaryl; linear or branched -(alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); -COO(aryl); - C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH; or a pharmaceutically acceptable salt or derivative thereof.
66. The invention of claim 65 wherein R2 is hydroxyalkyl, for example C1.6 hydroxyalkyl.
67. The invention of any one of claims 1 to 43 or 57 to 66 wherein the compound has the formula:
wherein R1 is linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl and aralkyl and wherein the optional substitution may be with one or more groups independently selected from: -OH; -F; -Cl; -Br; -I; -NH2; alkylamino; dialkylamino; linear or branched alkyl, alkenyl, alkynyl and aralkyl; aryl; heteroaryl; linear or branched alkoxy; aryloxy; aralkoxy; -(alkylene)oxy(alkyl); -CN; -NO2; -COOH; -COO(alkyl); -COO(aryl); - C(O)NH(alkyl); -C(O)NH(aryl); sulfonyl; alkylsulfonyl; arylsulfonyl; sulfamoyl; alkylsulfamoyl; alkylthio; alkylsulfonamide; arylsulfonamide; -NHNH2; and -NHOH; or a pharmaceutically acceptable salt or derivative thereof.
68. The invention of any one of claims 65 to 67 wherein R1 is selected from CMS alkyl (for example, Ci_g alkyl, e.g. C-ι-6 alkyl), C2-18 alkenyl (for example, C2.g alkenyl, e.g. C2-6 alkenyl) and C2-is alkynyl (for example, C2.9 alkynyl, e.g. C2-6 alkynyl).
69. The invention of claim 68 wherein R1 is methyl, ethyl, propyl or butyl.
70. The invention of any one of claims 57 to 69 wherein the compound has a formula selected from:
wherein R1 and R2 are as defined in any one of claims 66 to69; or a pharmaceutically acceptable salt or derivative thereof.
71. The invention of any one of claims 57 to 69 wherein the compound is an N-substituted DNJ derivative having a formula selected from those set out in claim 70 except that the hydrogen atom on the N and the R1 group are transposed; or a pharmaceutically acceptable salt or derivative thereof.
72. The invention of any one of claims 57 to 69 wherein the compound is an N-substituted DNJ derivative having a formula selected from those set out in claim 70 except that the hydrogen atom on the N and the R2 group are transposed; or a pharmaceutically acceptable salt or derivative thereof.
73. The invention of any one of the preceding claims wherein the adjunctive agent has the formula:
for example:
or a pharmaceutically acceptable salt or derivative thereof.
74. The invention of any one of claims 1 to 43 wherein the adjunctive agent has a formula selected from: 1-Deoxynojirimycin α-Homonojirimycin (DNJ) (HNJ)
N-butylDNJ N-nonylDNJ N-methoxy-nonyl-DNJ (NB-DNJ or Zavesca™) (NN-DNJ)
Deoxygalactonojirimyciπ N-butylDGJ N-nonylDGJ (DGJ) (NB-DGJ) (NN-DGJ)
N-7-oxanonyl-6-MeDGJ N-nonyl-6-MeDGJ N-9-oxadecyl-6-MeDGJ ("UT-231 B")
75. The invention of any one of claims 1 to 43 wherein the adjunctive agent has a formula selected from:
6-O-butanoylcastanospermine Castanospermine (Bucast)
PCT/GB2006/000209 2005-01-21 2006-01-20 Antiviral drug combinations WO2006077427A3 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
GB0501352A GB0501352D0 (en) 2005-01-21 2005-01-21 Use of glycosylation modulators in combination with membrane fusion inhibitors for treatment of infections caused by viruses bearing glycosylated envelope
GB0501352.9 2005-01-21

Publications (2)

Publication Number Publication Date
WO2006077427A2 true true WO2006077427A2 (en) 2006-07-27
WO2006077427A3 true WO2006077427A3 (en) 2006-09-14

Family

ID=34259511

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2006/000209 WO2006077427A3 (en) 2005-01-21 2006-01-20 Antiviral drug combinations

Country Status (2)

Country Link
GB (1) GB0501352D0 (en)
WO (1) WO2006077427A3 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008009894A2 (en) * 2006-07-15 2008-01-24 Summit Corporation Plc Use of imino sugars in immunotherapy
WO2010144759A1 (en) 2009-06-12 2010-12-16 United Therapeutics Corporation Iminosugars and methods of treating bunyaviral and togaviral diseases
WO2011028779A1 (en) 2009-09-04 2011-03-10 United Therapeutics Corporation Iminosugars and methods of treating filoviral diseases
JP2012518648A (en) * 2009-02-24 2012-08-16 ユナイテッド セラピューティクス コーポレーション A method of treating an imino sugar and arenavirus infection
CN102659787A (en) * 2011-03-29 2012-09-12 中国科学院化学研究所 Fluoro polyhydroxy pyrrole pyrrolizidine, and preparation method and application thereof
JP2013503882A (en) * 2009-09-04 2013-02-04 ユナイテッド セラピューティクス コーポレイション A method of treating a poxvirus infection
JP2013503880A (en) * 2009-09-04 2013-02-04 ユナイテッド セラピューティクス コーポレイション A method of treatment of orthomyxovirus infection
US8450345B2 (en) 2009-02-23 2013-05-28 The Chancellor, Masters And Scholars Of The University Of Oxford Iminosugars and methods of treating viral diseases
CN103435619A (en) * 2013-09-04 2013-12-11 中国科学院化学研究所 Pochonicine analogue or pharmaceutically acceptable salts of pochonicine analogue and application of pochonicine analogue or pharmaceutically acceptable salts of pochonicine analogue
WO2014140680A1 (en) * 2013-03-15 2014-09-18 National University Of Singapore Dosing regimen for use of celgosivir as an antiviral therapeutic for dengue virus infection
CN106102464A (en) * 2013-05-02 2016-11-09 牛津大学之校长及学者 Glycolipid inhibition using iminosugars

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0295538A2 (en) * 1987-06-08 1988-12-21 Merrell Dow Pharmaceuticals Inc. Inhibitors of glycoprotein processing having anti retroviral activity
WO1996040191A1 (en) * 1995-06-07 1996-12-19 Trimeris, Inc. The treatment of hiv and other viral infections using combinatory therapy
WO2004064715A2 (en) * 2003-01-23 2004-08-05 M N L Pharma Limited Polyhydroxylated pyrrolizidine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0295538A2 (en) * 1987-06-08 1988-12-21 Merrell Dow Pharmaceuticals Inc. Inhibitors of glycoprotein processing having anti retroviral activity
WO1996040191A1 (en) * 1995-06-07 1996-12-19 Trimeris, Inc. The treatment of hiv and other viral infections using combinatory therapy
WO2004064715A2 (en) * 2003-01-23 2004-08-05 M N L Pharma Limited Polyhydroxylated pyrrolizidine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BALZARINI JAN ET AL: "Profile of resistance of human immunodeficiency virus to mannose-specific plant lectins" JOURNAL OF VIROLOGY, vol. 78, no. 19, October 2004 (2004-10), pages 10617-10627, XP002387019 ISSN: 0022-538X *
DE CLERCQ ERIK: "Toward improved anti-HIV chemotherapy: Therapeutic strategies for intervention with HIV infections" JOURNAL OF MEDICINAL CHEMISTRY, vol. 38, no. 14, 1995, pages 2491-2517, XP002387020 ISSN: 0022-2623 *

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008009894A2 (en) * 2006-07-15 2008-01-24 Summit Corporation Plc Use of imino sugars in immunotherapy
WO2008009894A3 (en) * 2006-07-15 2008-06-19 Summit Corp Plc Use of imino sugars in immunotherapy
US9579334B2 (en) 2009-02-23 2017-02-28 Emergent Virology Llc Iminosugars and methods of treating viral diseases
US9044470B2 (en) 2009-02-23 2015-06-02 United Therapeutics Corporation Iminosugars and methods of treating viral diseases
US8450345B2 (en) 2009-02-23 2013-05-28 The Chancellor, Masters And Scholars Of The University Of Oxford Iminosugars and methods of treating viral diseases
KR101787434B1 (en) * 2009-02-24 2017-11-15 유나이티드 세러퓨틱스 코오포레이션 Iminosugars and methods of treating arenaviral infections
JP2012518648A (en) * 2009-02-24 2012-08-16 ユナイテッド セラピューティクス コーポレーション A method of treating an imino sugar and arenavirus infection
EP2440205A4 (en) * 2009-06-12 2013-04-24 United Therapeutics Corp Iminosugars and methods of treating bunyaviral and togaviral diseases
CN102639133A (en) * 2009-06-12 2012-08-15 牛津大学之校长及学者 Iminosugars and methods of treating bunyaviral and togaviral diseases
CN102639133B (en) * 2009-06-12 2015-03-11 联合治疗公司 Iminosugars and methods of treating bunyaviral and togaviral diseases
JP2012530061A (en) * 2009-06-12 2012-11-29 ユナイテッド セラピューティクス コーポレイション Method of treating an imino sugar and Bunyaviridae and togaviruses disease
KR101463661B1 (en) * 2009-06-12 2014-11-19 유나이티드 세러퓨틱스 코오포레이션 Iminosugars and methods of treating bunyaviral and togaviral diseases
WO2010144759A1 (en) 2009-06-12 2010-12-16 United Therapeutics Corporation Iminosugars and methods of treating bunyaviral and togaviral diseases
EP2440205A1 (en) * 2009-06-12 2012-04-18 United Therapeutics Corporation Iminosugars and methods of treating bunyaviral and togaviral diseases
US8426445B2 (en) 2009-06-12 2013-04-23 United Therapeutics Corporation Iminosugars and methods of treating bunyaviral and togaviral diseases
US8748460B2 (en) 2009-06-12 2014-06-10 United Therapeutics Corporation Iminosugars and methods of treating togaviral diseases
WO2011028779A1 (en) 2009-09-04 2011-03-10 United Therapeutics Corporation Iminosugars and methods of treating filoviral diseases
JP2013503880A (en) * 2009-09-04 2013-02-04 ユナイテッド セラピューティクス コーポレイション A method of treatment of orthomyxovirus infection
CN102595895A (en) * 2009-09-04 2012-07-18 牛津大学之校长及学者 Iminosugars and methods of treating filoviral diseases
JP2013503882A (en) * 2009-09-04 2013-02-04 ユナイテッド セラピューティクス コーポレイション A method of treating a poxvirus infection
US20160243096A1 (en) * 2009-09-04 2016-08-25 Emergent Virology Llc Methods of treating orthomyxoviral infections
KR101459530B1 (en) * 2009-09-04 2014-11-07 유나이티드 세러퓨틱스 코오포레이션 Methods of treating poxviral infections
JP2013503881A (en) * 2009-09-04 2013-02-04 ユナイテッド セラピューティクス コーポレイション Iminosugars and methods of treating a filovirus disease
US20160243097A1 (en) * 2009-09-04 2016-08-25 Emergent Virology Llc Methods of treating poxviral infections
CN105748476A (en) * 2009-09-04 2016-07-13 联合治疗公司 Iminosugars And Methods Of Treating Filoviral Diseases
EP2473046A4 (en) * 2009-09-04 2013-04-24 United Therapeutics Corp Iminosugars and methods of treating filoviral diseases
EP2473046A1 (en) * 2009-09-04 2012-07-11 United Therapeutics Corporation Iminosugars and methods of treating filoviral diseases
CN102659787A (en) * 2011-03-29 2012-09-12 中国科学院化学研究所 Fluoro polyhydroxy pyrrole pyrrolizidine, and preparation method and application thereof
CN102659787B (en) * 2011-03-29 2015-04-01 中国科学院化学研究所 Fluoro polyhydroxy pyrrole pyrrolizidine, and preparation method and application thereof
WO2014140680A1 (en) * 2013-03-15 2014-09-18 National University Of Singapore Dosing regimen for use of celgosivir as an antiviral therapeutic for dengue virus infection
CN106102464A (en) * 2013-05-02 2016-11-09 牛津大学之校长及学者 Glycolipid inhibition using iminosugars
EP2991488A4 (en) * 2013-05-02 2016-12-21 The Chancellor Masters And Scholars Of The Univ Of Oxford Glycolipid inhibition using iminosugars
CN103435619B (en) * 2013-09-04 2015-08-05 中国科学院化学研究所 Pharmaceutically Pochonicine one kind of analog or pharmaceutically acceptable salts and its application
CN103435619A (en) * 2013-09-04 2013-12-11 中国科学院化学研究所 Pochonicine analogue or pharmaceutically acceptable salts of pochonicine analogue and application of pochonicine analogue or pharmaceutically acceptable salts of pochonicine analogue

Also Published As

Publication number Publication date Type
WO2006077427A3 (en) 2006-09-14 application
GB0501352D0 (en) 2005-03-02 grant

Similar Documents

Publication Publication Date Title
Wu et al. Antiviral effects of an iminosugar derivative on flavivirus infections
Liu et al. HIV entry inhibitors targeting gp41: from polypeptides to small-molecule compounds
US6689759B1 (en) Methods of Treating hepatitis virus infections with N-substituted-1,5-dideoxy-1,5-imino-d-glucitol compounds in combination therapy
US20050215544A1 (en) Methods of treating HIV infection
US20050215543A1 (en) Methods of treating HIV infection
US6458799B1 (en) Deazaguanine analog, preparation thereof and use thereof
Mastrolorenzo et al. Inhibitors of HIV-1 protease: current state of the art 10 years after their introduction. From antiretroviral drugs to antifungal, antibacterial and antitumor agents based on aspartic protease inhibitors
US5030638A (en) Method of antiviral enhancement
US6809083B1 (en) Use of N-substituted-1, 5-dideoxy-1, 5-imino-D-glucitol compounds for treating hepatitis virus infections
US20050239748A1 (en) Pharmaceutical salts of 3-O-(3&#39;,3&#39;-dimethylsuccinyl) betulinic acid
EP0286224A2 (en) Treatment of human viral infection by dsRNA combined with viral inhibitors
US20130165489A1 (en) Small Molecule Modulators of HIV-1 Capsid Stability and Methods Thereof
WO2010015816A2 (en) Treatment of lysosomal storage disorders and other proteostatic diseases
US6660749B2 (en) Inhibition of glycolipid biosynthesis
US6147095A (en) Method for improving the pharmacokinetics of tipranavir
WO1999059585A1 (en) Compositions for the treatment of hiv and other viral infections
US20090252785A1 (en) Endoplasmic reticulum targeting liposomes
US20080138351A1 (en) Liposome treatment of viral infections
Asano et al. N-Alkylated nitrogen-in-the-ring sugars: conformational basis of inhibition of glycosidases and HIV-1 replication
WO2005112930A1 (en) Combinations comprising a 4-isoquinolone derivative and anti-hiv agents
Martínez-Gutierrez et al. Statins reduce dengue virus production via decreased virion assembly
Hart et al. Glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin
Eggink et al. Inhibition of HIV-1 by fusion inhibitors
Watanabe et al. Dose-and schedule-dependent protective efficacy of celgosivir in a lethal mouse model for dengue virus infection informs dosing regimen for a proof of concept clinical trial
US20030027867A1 (en) Use of R-NSAID compounds for anti-HIV treatment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06700798

Country of ref document: EP

Kind code of ref document: A2

WWW Wipo information: withdrawn in national office

Ref document number: 6700798

Country of ref document: EP