WO2010116141A2

WO2010116141A2 - Drug combination for the treatment of proteostatic diseases

Info

Publication number: WO2010116141A2
Application number: PCT/GB2010/000710
Authority: WO
Inventors: Francis Xavier Wilson; Richard Storer; Akane Kawamura; Robert Nash; Alan Geoffrey Roach; Jonathon Mark Tinsley
Original assignee: Summit Corporation Plc
Priority date: 2009-04-09
Filing date: 2010-04-07
Publication date: 2010-10-14
Also published as: GB0906159D0; WO2010116141A3

Abstract

Combinations comprising: (a) an active-site-specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme); and (b) a non-active-site-specific pharmacoperone (non-ASSC) of said enzyme which: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co- chaperone of said enzyme are described The combinations have various uses in medicine, including the treatment of protein folding diseases (in particular lysosomal storage disorders).

Description

DRUG COMBINATION FOR THE TREATMENT OF PROTEOSTATIC DISEASES

Field of the Invention

This invention relates to combinations comprising certain compounds, in particular polyhydroxylated alkaloids or iminosugars, and to methods for the treatment of protein folding diseases (in particular lysosomal storage disorders) based on the use of these combinations. Also contemplated are bivalent pharmacoperones of a protein (e.g. an enzyme) comprising a first moiety which binds to a catalytic or active site of said protein and a second moiety which binds to a second, distinct and non-catalytic/active site of said protein.

Background of the Invention

Lysosomal storage disorders

Lysosomal storage disorders (LSDs) are a group of diseases which arise from abnormal metabolism of various substrates, including glycosphingolipids, glycogen, mucopolysaccharides and glycoproteins. More than fifty disorders have been identified that are caused by mutations in metabolic enzymes that are required for the degradation of such compounds. Many of them are neuronopathic and so may produce severe neurological impairment.

The metabolism of the substrates normally occurs in the lysosome and the process is regulated in a stepwise process by various degradative enzymes. Therefore, a deficiency in any one enzyme activity can perturb the entire process and result in the accumulation of particular substrates. Listed below are a number of lysosomal storage disorders and the corresponding defective enzymes:

Pompe disease: Acid alpha-glucosidase

Gaucher disease: Acid beta-glucosidase or glucocerebrosidase

Fabry disease: alpha-Galactosidase A

GMI-gangliosidosis: Acid beta-galactosidase

Tay-Sachs disease: beta-Hexosaminidase A

Sandhoff disease: beta-Hexosaminidase B Niemann-Pick disease: Acid sphingomyelinase

Krabbe disease: Galactocerebrosidase

Farber disease: Acid ceramidase

Metachromatic leukodystrophy: Arylsulfatase A

Hurler-Scheie disease: alpha-L-lduronidase

Hunter disease: lduronate-2-sulfatase

Sanfilippo disease A: Heparan N-sulfatase

Sanfilippo disease B: alpha-N-Acetylglucosaminidase

Sanfilippo disease C: Acetyl-CoA: alpha-glucosaminide N-acetyltransferase

Sanfilippo disease D: N-Acetylglucosamine-6-sulfate sulfatase

Morquio disease A: N-Acetylgalactosamine-6-sulfate sulfatase

Morquio disease B: Acid beta-galactosidase

Maroteaux-Lamy disease: Arylsulfatase B

Sly disease: beta-Glucύronidase alpha-Mannosidosis: Acid alpha-mannosidase beta-Mannosidosis: Acid beta-mannosidase

Fucosidosis: Acid alpha-L-fucosidase

Sialidosis: Sialidase

Schindler-Kanzaki disease: alpha-N-acetylgalactosaminidase

Enzyme replacement therapy (ERT) and bone marrow transplantation are currently being used to treat these disorders. Cell- and gene-based therapies are also under investigation. However, all of these treatment regimens have severe drawbacks: for example, ERT is limited by the inability of the enzymes to cross the blood/brain barrier and so is ineffective in ameliorating the neurological deficits commonly associated with LSDs.

There is therefore great interest in the development of small molecules for treating LSDs and pharmacoperones (including various iminosugar pharmacoperones) have emerged as an important class of drugs for the treatment of LSDs.

Various iminosugars are known to act to modify the underlying metabolic dysfunction in LSDs by: (a) inhibiting enzyme(s) involved in the biosynthesis of the accumulating substrate, thereby preventing pathological levels of accumulation (substrate reduction therapy). Here, the aim is to reduce the rate of biosynthesis of accumulating substrate to offset the catabolic defect, restoring the balance between the rate of biosynthesis and the rate of catabolism; and/or (b) promoting (or augmenting residual) endogenous enzymic activity by effecting proper folding and/or trafficking of a mutant enzyme by acting as molecular chaperones (pharmacoperones) in a treatment modality known as chaperone- mediated therapy (CMT).

Recent reviews of substrate reduction therapies based on the use of various iminosugars include Butters (2007) lminosugar inhibitors for substrate reduction therapy for the lysosomal glycosphingolipidoses, in Iminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons, pages 249-268 as well as in the Table set out in Chapter 14.8 thereof (the disclosure of which is hereby incorporated by reference).

Proteostatic diseases

Proteostasis (sometimes referred to as protein homeostasis) is the regulation of the concentration, conformation (tertiary structure), binding interactions (quaternary structure) and location of the individual proteins that constitute the proteome of an organism. Proteostasis is therefore essential for maintaining normal cellular function and so ultimately determines the health status of the organism as a whole.

Proteostasis is effected by numerous distinct but interacting regulated processes dubbed the proteostasis network. This comprises many diverse components, including transcription and translation factors, the protein quality control complex, degradative enzymes, organic and inorganic solutes, small molecule ligands, chaperones, cochaperones, folding enzymes, the ubuiquitin proteasome system (UPS) and constituents of the intra- and intercellular transport machinery. Proteosfatic mechanisms operating within the proteostasis network therefore include transcription, translation and posttranslational modification, quality control, the heat shock response (HSR), the unfolded protein response (UPR), as well as protein folding, trafficking, aggregation, disaggregation and degradation. These processes together control fundamental aspects of cell, tissue and organismal development and environmental adaptation, as well as the response to intrinsic and extrinsic challenges (such as aging, neoplasia, genetic abnormalities and infection).

While the role of transcription and translation in governing the concentration of any given protein has been intensively studied and is well-characterized, the mechanisms regulating protein folding, trafficking, aggregation, disaggregation and degradation are less well- understood. However, it is now known that a given protein does not exist in just one structure (its native state) but can assume many different conformations, some of which have biological functions and each of which may exhibit quite different types of interaction with the proteostatsis network in general (and degradative pathways in particular). Posttranslational modification, folding, trafficking, aggregation, disaggregation and degradation all contribute to regulating the relative concentrations of the different protein conformers. Small changes in the concentration of any one conformer can profoundly change its solution state, rate of degradation, trafficking and deposition.

Over the past two years there has been a growing recognition of the role of proteostatic deficiencies in a wide variety of diseases (see e.g. Balch et a/. (2008) Science 319: 916- 919 and Morimoto (2008) Genes & Development 22: 1427-1438). Such diseases are collectively referred to as proteostatic diseases: they include aggregative and misfolding proteostatic diseases. Aggregative proteostatic disease, typically associated with the accumulation of proteotoxic species, includes prion diseases and a wide range of neurodegenerative disorders (e.g. Parkinson's disease, Alzheimer's disease and Huntington's disease). Misfolding proteostatic diseases include lysosomal storage disorders, certain forms of diabetes, emphysema, cancer and cystic fibrosis.

As demonstrated by Mu et a/. (2008) Cell 134: 769-781 , pharmacological intervention at the level of the proteostasis network by the administration of proteostasis regulators constitutes a potentially new and extremely powerful approach to the treatment of a wide range of protein folding diseases.

Active-site-specific pharmacoperones/chaperones (ASSCs)

Certain small molecules can serve as molecular scaffolding and cause otherwise-misfolded mutant proteins to fold, and/or route correctly within the cell or its organelles. Molecules which can act in this way have been dubbed "chemical chaperones", "pharmaceutical chaperones" or "pharmacoperones". In particular, competitive inhibitors of the mutant enzymes implicated in various. lysosomal storage disorders can, at subinhibitory concentrations, act as "Active-Site-Specific pharmaCoperones/Chaperones" (ASSCs) by either inducing or stabilizing the proper conformation of the mutant enzyme by specific binding to the catalytic site. In this approach, the correctly folded mutant enzyme is secreted out of the ER where the ASSC (now in the presence of highly concentrated substrate and so at a subinhibitory concentration) is displaced to allow function of the enzyme (the dynamic exchange of ASSC as a competitive inhibitor and the enzyme's substrate being dependent on their relative concentrations). This area is reviewed, for example, by Fan (2007) lminosugars as active-site-specific chaperones for the treatment of lysosomal storage disorders, In lminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons), pages 225-247.

Various iminosugars have been identified as ASSCs and their specific binding to the catalytic active site of an enzyme implicated in lysosomal storage diseases exploited to form the basis of a new form of therapy dubbed active-site-specific chaperone therapy (see e.g. US 6,583,158, US 6,589,964 and US 6,599,919). ASSC therapy uses low concentrations of potent enzyme inhibitors to enhance the folding and activity of mutant proteins in specific LSDs. This approach was first tested in Fabry disease, where 1-deoxy- galactononjirimycin (DGJ), an inhibitor of alpha-galactosidase A, was used to enhance the residual alpha-galactosidase activity in cell lines from Fabry disease patients (see US 6,274,597 and US 6,583,158). The ASSC strategy has been extended to other lysosomal storage diseases, including Gaucher disease and GMI-gangliosidosis.

ASSC therapy is now currently under development for several LSDs, including Gaucher disease, and offers several advantages over ERT or substrate deprivation therapy. Most notably, since the active site inhibitors used in ASSC are specific for the disease-causing enzyme, the therapy is targeted to a single protein and metabolic pathway, unlike substrate deprivation therapy that inhibits an entire synthetic pathway.

Like substrate deprivation therapy, the small molecule inhibitors for ASSC have the potential of crossing the blood brain barrier and could be used to treat neurological LSD forms. Moreover, in addition to enhancing the activity of the deficient enzymes associated with the LSDs, the ASSCs have also been demonstrated to enhance the activity of the corresponding wild-type enzyme (see US 6,589,964) and so can be used adjunctively with enzyme replacement therapy in LSD patients.

However, ASSC therapy is complicated by the fact that therapeutic potential depends on a favourable ratio of inhibitory activity to chaperone activity: if the concentration of inhibitor required to promote proper folding approaches the inhibitory concentration then therapeutic utility is severely compromised. There have been some attempts to improve the chaperone:inhibitor ratio of various imino sugars by chemical means (see e.g. WO2004/037373), but such approaches are not generally applicable and have limited utility.

Summary of the Invention

The present inventors have now discovered that certain compounds (designated as non- active site-specific pharmacoperones, or non-ASSCs) can act as pharmacoperones of enzymes in a catalytic site-independent manner. Moreover, it has now been discovered that these non-ASSCs can act synergistically with ASSCs as chaperones for said enzymes. As a result, such that the problems associated with ASSC chaperone:inhibitor ratios are removed, a new class of pharmacoperone-based treatment for enzyme deficiencies arising from abnormal protein folding/processing/trafficking (including in particular LSDs) with an improved therapeutic index is provided.

Thus, according to the present invention there is provided a combination comprising: (a) an active-site-specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme); and (b) a non-active-site-specific pharmacoperone (non-ASSC) of said enzyme which: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme.

In another aspect, the invention provides a composition comprising an active-site-specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme) for use in combination therapy with a non-active-site-specific pharmacoperone (non-ASSC) of said enzyme, which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme.

In another aspect, the invention provides a composition comprising a non-active-site- specific pharmacoperone (non-ASSC) of an enzyme (e.g. a lysosomal enzyme), which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme, for use in combination therapy with an active-site-specific pharmacoperone (ASSC) of said enzyme.

The ASSC and non-ASSC may be physically associated. In such embodiments, the ASSC and non-ASSC may be: (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).

Alternatively, the ASSC and non-ASSC may be non-physically associated. In such embodiments, the combination may comprise: (a) at least one of the ASSC and non-ASSC together with instructions for their extemporaneous association to form a physical association; or (b) at least one of the ASSC and non-ASSC together with instructions for combination therapy with the ASSC and non-ASSC; or (c) at least one of the ASSC and non-ASSC together with instructions for administration to a patient population in which either the ASSC or non-ASSC has been (or are being) administered; or (d) at least one of the ASSC and non-ASSC in an amount or in a form which is specifically adapted for use as an ASSC/non-ASSC combination.

In another aspect, the invention provides a pharmaceutical composition comprising the combination of the invention.

The combination of the invention may take any form, for example being provided: (a) in the form of a pharmaceutical pack, kit or patient pack; (b) in a pharmaceutical excipient; or (c) in unit dosage form.

In another aspect, the invention contemplates the combination of the invention for use in therapy or prophylaxis (e.g. for use in the treatment of a lysosomal storage disorder). In another aspect, the invention contemplates a composition comprising an active-site- specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme) for the treatment of a subject undergoing treatment with a non-active-site-specific pharmacoperone (non-ASSC) of said enzyme, which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme.

In another aspect, the invention contemplates a composition comprising a non-active-site- specific pharmacoperone (non-ASSC) of an enzyme (e.g. a lysosomal enzyme), which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme, for the treatment of a subject undergoing treatment with an active-site-specific pharmacoperone (ASSC) of said enzyme.

The invention also contemplates a method for the treatment of a lysosomal storage disorder comprising the simultaneous, separate or sequential administration of an effective amount of: (a) a non-active-site-specific pharmacoperone (non-ASSC) of a lysosomal enzyme, which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a noncompetitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co- chaperone of said enzyme; and (b) an active-site-specific pharmacoperone (ASSC) of said lysosomal enzyme.

The combinations of the invention may also act in synergy with enzymes in enzyme replacement therapy, since the compounds may stabilize the replacement enzyme in situ (and so increase its half life) and/or relieve the inhibitory effects of substrate accumulation in the lysosome (so increasing the therapeutic activity of the replacement enzyme). This may provide the possibility of dose sparing of the replacement enzyme and/or the adoption of a more sustainable dosage regimen. The invention also contemplates a method of chaperone-mediated therapy of a disease arising from aberrant enzyme folding and/or processing comprising the administration of an effective amount of a first pharmacoperone which binds to the catalytic site of said enzyme and a second pharmacoperone which binds to said enzyme at a site remote from the catalytic site.

In such embodiments, preferred are first pharmacoperones which are competitive inhibitors of said enzyme and second pharmacoperones which are not competitive inhibitors of said enzyme.

Thus, the second pharmacoperone may binds to an allosteric site of said enzyme, or may be an activator of said enzyme or a non-competitive inhibitor of said enzyme.

Preferably, the first and second pharmacoperones do not compete for binding to said enzyme.

In such embodiments, the chaperone-mediated therapy may be the treatment of an LSD, for example an LSD as defined herein.

In such embodiments, the enzyme may be a lysosomal enzyme, for example selected from the enzymes listed herein.

In another embodiment, the invention provides a bivalent pharmacoperone of an enzyme comprising a first moiety which binds to a catalytic or active site of said enzyme and a second moiety which binds to a second, distinct and non-catalytic site of said enzyme.

In such embodiments, the second moiety may binds to amino acid residues which do not form part of the catalytic site of said enzyme. The second moiety may for example bind to amino acid residues which do not form part of the active site of said enzyme. For example, the second moiety may bind to an allosteric site of said enzyme or to a site on said enzyme which activates said enzyme.

The bivalent pharmacoperone of the invention may comprise a conjugate of a first and a second iminosugar. In such embodiments, the imino sugars may be selected from those described herein. The bivalent pharmacoperone of the invention find application in the treatment of a disease arising from aberrant enzyme folding and/or processing, for example an LSD as hereinbefore described.

In all aspects of the invention, the lysosomal enzyme may be selected from: (a) Acid alpha-glucosidase; (b) Acid beta-glucosidase; (c) glucocerebrosidase; (d) alpha- Galactosidase A; (e) Acid beta-galactosidase; (f) beta-Hexosaminidase A; (g) beta- Hexosaminidase B; (h) Acid sphingomyelinase; (i) Galactocerebrosidase; (j) Acid ceramidase; (k) Arylsulfatase A; (I) alpha-L-lduronidase; (m) lduronate-2-sulfatase; (n) Heparan N-sulfatase; (o) alpha-N-Acetylglucosaminidase; (p) Acetyl-CoA: alpha- glucosaminide N-acetyltransferase; (q) N-Acetylglucosamine-6-sulfate sulfatase; (r) N- Acetylgalactosamine-6-sulfate sulfatase; (s) Acid beta-galactosidase; (t) Arylsulfatase B; (u) beta-Glucuronidase; (v) Acid alpha-mannosidase; (w) Acid beta-mannosidase; (x) Acid alpha-L-fucosidase; (y) Sialidase; and (z) alpha-N-acetylgalactosaminidase.

The invention in all of its aspects finds utility in the treatment of lysosomal storage disorders (LSDs), for example in the treatment of an LSD is selected from: (a) Pompe disease (including infantile and late-onset forms); (b) Gaucher disease (including Type 1, Type 2 and Type 3 Gaucher disease); (c) Fabry disease; (d) GMI-gangliosidosis; (e) Tay- Sachs disease; (f) Sandhoff disease; (g) Niemann-Pick disease; (h) Krabbe disease:; (i) Farber disease; G) Metachromatic leukodystrophy; (k) Hurler-Scheie disease; (I) Hunter disease; (m) Sanfilippo disease A, B, C or D; (n) Morquio disease A or B; (o) Maroteaux- Lamy disease; (p) Sly disease; (q) alpha-Mannosidosis; (r) beta-Mannosidosis; (s) Fucosidosis; (t) Sialidosis; and (u) Schindler-Kanzaki disease.

The invention also contemplates adjunctive use of the combinations of the invention with . various adjunctive agents. The adjunctive agent may be selected from:

(a) a lysosomal enzyme; and/or

(b) an inhibitor of a lysosomal enzyme; and/or

(c) a cell expressing a lysosomal enzyme; and/or

(d) nucleic acid encoding a lysosomal enzyme.

In embodiments where the adjunctive agent is a lysosomal enzyme (for example, a lysosomal enzyme selected from (a)-(z), above), the lysosomal enzyme is preferably recombinant, for example being produced by the expression of heterologous DNA in a prokaryotic or eukaryotic host cell. In such embodiments, the lysosomal enzyme may have a modified primary amino acid sequence, for example containing N- and/or C-terminal tag sequences (for example, mannose-terminated recombinant glucocerebrosidase). Alternatively (or in addition), the lysosomal enzyme may be a truncated form of the wild type enzyme. It may be glycosylated, unglycosylated or deglycosylated.

In embodiments where the adjunctive agent is an inhibitor of the lysosomal enzyme, the inhibitor is preferably suitable for substrate reduction therapy of a lysosomal storage disorder, for example being selected from the inhibitors described in Butters (2007) lminosugar inhibitors for substrate reduction therapy for the lysosomal glycosphingolipidoses, In lminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons, pages 249-268 as well as in the Table set out in Chapter 14.8 thereof (the disclosure of which is hereby incorporated by reference).

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

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:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

The phrase "consisting essentially of is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention.

As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.

As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

The term "proteostatic disease" is a term of art used to define a set of diseases mediated, at least in part, by deficiencies in proteostasis. The term therefore covers aggregative and misfolding proteostatic diseases, including in particular neurodegenerative disorders (e.g. Parkinson's disease, Alzheimer's disease and Huntington's disease), lysosomal storage disorders, diabetes, emphysema, cancer and cystic fibrosis.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term "therapy".

Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term "prophylaxis".

In this context "subject" (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In preferred embodiments, the subject is a human.

As used herein, an effective amount or a therapeutically effective amount of a compound defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

As used herein, a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

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

As used herein, the term "combination", as applied to two or more compounds and/or agents (also referred to herein as the components), is intended to define material in which the two or more compounds/agents are associated. The terms "combined" and "combining" in this context are to be interpreted accordingly.

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

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

• compositions comprising material in which the two or more compounds/agents 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/agents 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/agents are co-packaged or co-presented (e.g. as part of an array of unit doses);

Examples of non-physically associated combined compounds/agents include:

• material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds/agents together with instructions for the extemporaneous association of the at least one compound/agent to form a physical association of the two or more compounds/agents;

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

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

• material comprising at least one of the two or more compounds/agents 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/agents.

As used herein, the term "combination therapy" is intended to define therapies which comprise the use of a combination of two or more compounds/agents (as defined above). Thus, references to "combination therapy", "combinations" and the use of compounds/agents "in combination" in this application may refer to compounds/agents that are administered as part of the same overall treatment regimen. As such, the posology of each of the two or more compounds/agents may differ: each may be administered at the same time or at different times. It will therefore be appreciated that the compounds/agents 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/agents in a combination therapy may also differ with respect to the route of administration.

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/agents, the individual compounds/agents 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/agents, the individual compounds/agents 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.

The combinations of the invention may produce a therapeutically efficacious (and in particular, a synergistic) effect relative to the therapeutic effect of the individual compounds/agents when administered separately.

The term iminosugar defines a saccharide analogue in which the ring oxygen is replaced by a nitrogen. The term is used herein sensu lato to include isoiminosugars, these being aza-carba analogues of sugars in which the C-1 carbon is replaced by nitrogen and the ring oxygen is replaced by a carbon atom, as well as azasugars in which an endocyclic carbon is replaced with a nitrogen atom. 1 -Azasugars (with the N in the anomeric position) in which the ring oxygen is substituted with a carbon atom are isoiminosugars (as herein defined), but 1-azasugars in which the ring oxygen remains unsubstituted (oxazines) or is substituted with a nitrogen atom (hydrazines) are also of particular importance. In all cases, one or more endocyclic carbon atoms may be substituted with a sulphur, oxygen or nitrogen atom.

As used herein, the term polyhydroxylated iminosugar defines a class of oxygenated iminosugars. Typically these have at least 2, 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

The term iminosugar acid defines mono- or bicyclic sugar acid analogues in which the ring oxygen is replaced by a nitrogen. The term N-acid ISA defines an iminosugar acid in which the carboxylic acid group is located on the ring nitrogen.

Preferred ISAs are selected from the following structural classes: piperidine (including (poly)hydroxypipecolic acids); pyrroline; pyrrolidine (including (poly)hydroxyprolines); pyrrolizidine; indolizidine and nortropane.

As used herein, the term polyhydroxylated as applied to iminosugar acids defines an ISA having at least 2 (preferably at least 3) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

As used herein, the term bicyclic polyhydroxylated iminosugar defines a class of highly oxygenated iminosugars having a double or fused ring nucleus (i.e. having two or more cyclic rings in which two or more atoms are common to two adjoining rings). Typically, such iminosugars have at least 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups on the ring system nucleus.

The term pharmacoperone is a term of art (from "pharmacological chaperone") used to define a class of biologically active small molecules (sometimes also referred to in the art as "chemical chaperones") that serve as molecular scaffolds, causing otherwise misfolded mutant proteins to fold and route correctly within the cell.

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

The term bioisostβre (or simply isostere) is a term of art used to define drug 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 hydfoxyl group with a fluorine atom is a commonly employed bioisosteric replacement. Sila-substitution (C/Si-exchange) is a relatively recent technique for producing isosteres. This approach involves the replacement of one or more specific carbon atoms in a compound with silicon (for a review, see Tacke and Zilch (1986) Endeavour, New Series 10: 191-197). The sila-substituted isosteres (silicon isosteres) may exhibit improved pharmacological properties, and may for example be better tolerated, have a longer half-life or exhibit increased potency (see for example Englebienne (2005) Med. Cherα, 1 (3): 215-226). In its broadest aspect, the present invention contemplates all bioisosteres (and specifically, all silicon bioisosteres) of the compounds of the invention.

In its broadest aspect, the present invention contemplates all optical isomers, racemic forms and diastereoisomers of the compounds described herein. Those skilled in the art will appreciate that, owing to the asymmetrically substituted carbon atoms present in the compounds of the invention, the compounds may be produced in optically active and racemic forms. If a chiral centre or another form of isomeric centre is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereoisomers, are intended to be covered herein. Compounds of the invention containing a chiral centre (or multiple chiral centres) may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. Thus, references to the compounds (e.g. iminosugars) of the present invention encompass the products as a mixture of diastereoisomers, as individual diastereoisomers, as a mixture of enantiomers as well as in the form of individual enantiomers.

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

The terms derivative and pharmaceutically acceptable derivative as applied to the compounds of the invention define compounds which are obtained (or obtainable) by chemical derivatization of the parent compound of.the invention. The pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with the tissues of humans without undue toxicity, irritation or allergic response (i.e. commensurate with a reasonable benefit/risk ratio). Preferred derivatives are those obtained (or obtainable) by alkylation, esterification or acylation of the parent compounds.

The pharmaceutically acceptable derivatives of the invention may retain some or all of the biological activities described herein. In some cases, the biological activity (e.g. chaperone activity) is increased by derivatization. The derivatives may act as pro-drugs, and one or more of the biological activities described herein (e.g. pharmacoperones activity) may arise only after in vivo processing. 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. Derivatization may also augment other biological activities of the compound, for example bioavailability and/or glycosidase inhibitory activity and/or glycosidase inhibitory profile. For example, derivatization may increase glycosidase inhibitory potency and/or specificity and/or CNS penetration (e.g. penetration of the blood-brain barrier).

The term pharmaceutically acceptable salt as applied to the iminosugars of the invention defines any non-toxic organic or inorganic acid addition salt of the free base which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and which are commensurate with a reasonable benefit/risk ratio. Suitable pharmaceutically acceptable salts are well known in the art. Examples are the salts with inorganic acids (for example hydrochloric, hydrobromic, sulphuric and phosphoric acids), organic carboxylic acids (for example acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic, 4- hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and mandelic acid) and organic sulfonic acids (for example methanesulfonic acid and p- toluenesulfonic acid).

These salts and the free base compounds can exist in either a hydrated or a substantially anhydrous form. Crystalline forms, including all polymorphic forms, of the iminosugars of the invention are also contemplated and in general the acid addition salts of the compounds 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.

Chaperones for use according to the invention

ASSCs for use according to the invention

Any of a wide variety of known ASSCs may be used according to the invention, including those selected from the pharmacoperones described by Fan (2007) Iminosugars as active- site-specific chaperones for the treatment of lysosomal storage disorders, In Iminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons, pages 225-247 as well as in the Table set out in Chapter 14.8 thereof (the disclosure of which is hereby incorporated by reference).

Suitable ASSCs for use according to the invention can be identified as described in Section 10.6 of Fan (2007) Iminosugars as active-site-specific chaperones for the treatment of lysosomal storage disorders, In Iminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons, pages 234-236 (the disclosure of which is hereby incorporated by reference). ASSCs for use according to the invention are preferably polyhydroxylated alkaloids or imino sugars (as described below).

Non-ASSCs for use according to the invention

Any non-ASSC may be used according to the invention. Suitable non-ASSCs may be identified, for example, by: (a) contacting a target enzyme with a test compound; (b) detecting an increase of wild-type conformation of the enzyme in the presence of the test compound; and (c) detecting the absence of competitive inhibition by the test compound on said enzyme in the presence of substrate.

Preferred non-ASSCs for use according to the invention are imino sugars or polyhydroxylated alkaloids, as described below.

Polyhvdroxylated alkaloids for use as non-ASSCs and/or ASSCs according to the invention

The ASSC and/or non-ASSC for use according to the invention may be polyhydroxylated alkaloids. Preferably, the polyhydroxylated alkaloid is a bicyclic polyhydroxylated alkaloid.

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 semisynthetic 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. As used herein, the term alkaloid also covers exocyclic amines in which the nitrogen is not present in the ring nucleus. Such exocyclic amines may be imino sugar analogues in which the ring nitrogen is absent and replaced with an exocyclic nitrogen. Such exocyclic amines may be piperidine or pyrrolidine alkaloid analogues in which the ring nitrogen is absent and replaced with an exocyclic nitrogen, so including piperidine analogues having the nucleus:

and pyrrolidine alkaloids having the nucleus:

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

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

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

As used herein, the term polyhydroxylated alkaloid defines a class of highly oxygenated alkaloids having at least 2,3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups on the ring system nucleus.

As used herein, the term bicyclic polyhydroxylated alkaloid defines a class of highly oxygenated alkaloids having a double or fused ring nucleus (i.e. having two or more cyclic rings in which two or more atoms are common to two adjoining rings). Typically, such alkaloids have at least 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups on the ring system nucleus.

As used herein, the term polyhydroxylated piperidine alkaloid defines a highly oxygenated alkaloid (e.g. having at least 2 (preferably at least 3) free hydroxyl groups on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated pyrrolidine alkaloid defines a highly oxygenated alkaloid (e.g. having at least 2 (preferably at least 3) free hydroxyl groups on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated pyrrolidine alkaloid defines a highly oxygenated alkaloid (e.g. having at least 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated indolizidine alkaloid defines a highly oxygenated alkaloid (e.g. having at least 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated quinolizidine alkaloid defines a highly oxygenated alkaloid (e.g. having at least 3, 4, 5, 6 or 7 (preferably 3, 4, 5 or 6) free hydroxyl groups on the ring system nucleus) that comprises the nucleus:

Yet other polyhydroxylated alkaloids for use according to the invention may comprise the nucleus:

The polyhydroxylated alkaloid for use according to the invention may be a calystegine. These are polyhdroxylated nor-tropane alkaloids which have been reported to inhibit β- glucosidases, β-xylosidases and α-galactosidases (Asano et al., 1997, Glycobiology 7: 1085-1088). The calystegines are common in foods belonging to the Solanaceae that includes potatoes and aubergines (egg plant). The calystegines have been shown to inhibit mammalian glycosidases including human, rat and bovine liver enzymes. Attaching sugars to the calystegines such as in 3-0-β-D-glucopyranoside of 1α,2β,3α,6α-tetrahydroxy-nor- tropane (Calystegine B₁) (Griffiths, et al., 1996, Tetrahedron Letters 37: 3207-3208) can alter the glycosidase inhibition to include α-glucosidases and β-galactosidases.

Exemplary calystegines for use according to the invention include the compounds calystegine A₃, calystegine B₁ and calystegine B₂ shown below:

Calystegine A₃calystegine Bi calystegines B₂

or pharmaceutically acceptable salts or derivatives (e.g. acyl derivatives) thereof.

Also suitable for use according to the invention are C-calystegines. These are pentahydroxycalystegines that possess the extra hydroxyl on the bridge as in calystegine B₁ and Λ/-methylcalystegines have also been reported from plants including Lycium chinense (Watson et al., 2001 , Phytochemistry 56, 265-295). Examples include compounds having the formulae shown below:

In other aspects, the alkaloid may be selected from:

(a) a piperidine alkaloid;

(b) a pyrroline alkaloid;

(c) a pyrrolidine alkaloid;

(d) a pyrrolizidine alkaloid;

(e) an indolizidine alkaloid;

(f) a quinolizidine alkaloid;

(g) a nortropane alkaloid (e.g. a calystegine); and (h) mixtures of any two or more of (a) to (g). In yet another aspect, the alkaloid may be:

(a) a glycoside (e.g. glucoside) derivative;

(b) a branched alkyl derivative; or

(c) a derivative in which one or more of the hydroxyl group(s) are masked or protected.

The alkaloid preferably has a molecular weight of 100 to 400 Daltons. Most preferred are alkaloids having a molecular weight of 150 to 300 Daltons (e.g. 200 to 250 Daltons).

In another aspect the pharmacoperone may be a polyhydroxylated piperidine alkaloid that comprises the nucleus:

In another aspect the pharmacoperone may be a polyhydroxylated pyrrolidine alkaloid that comprises the nucleus:

M r

In another aspect the pharmacoperone may be a polyhydroxylated indolizidine alkaloid that comprises the nucleus:

In another aspect the pharmacoperone may be a polyhydroxylated quinolizidine alkaloid that comprises the nucleus:

lminosugars for use as non-ASSCs and/or ASSCs according to the invention

The ASSC and/or non-ASSC for use according to the invention may be iminosugars, as hereinbefore defined and described below.

Thus, the compounds for use according to the invention may be selected from:

• iminosugars sensu stricto, being saccharide analogues in which the ring oxygen is replaced by a nitrogen; or

• isoiminosugars, being aza-carba analogues of sugars in which the C-1 carbon is replaced by nitrogen and the ring oxygen is replaced by a carbon atom; and

• azasugars in which an endocyclic carbon is replaced with a nitrogen atom.

In embodiments where the iminosugar for use according to the invention is an azasugar as defined above, then the iminosugar may be selected from:

• 1 -azasugars in which the N is in the anomeric position;

• oxazines in which the ring oxygen remains unsubstituted; and

• hydrazines in which the ring oxygen is substituted with a nitrogen atom.

In all of the above iminosugars, one or more endocydic carbon atoms may be substituted with a sulphur, oxygen or nitrogen atom.

The iminosugars for use according to the invention may be of Formula (1), (2) or (3) as defined in Section A(I) (above).

The iminosugars as defined above for use according to the invention may be of any structural class or subclass, including the classes described below:

(a) Principal structural iminosugar classes The compounds for use according to the invention may be an iminosugar (as herein defined). The iminosugars for use according to the invention may be of a structural class selected from:

(a) a piperidine;

(b) a pyrroline;

(c) a pyrrolidine;

(d) a pyrrolizidine;

(e) an indolizidine;

(f) a quinolizidine;

(g) a nortropane;

(h) ring-open iminosugars;

(i) 5,7 fused;

(j) an azepane;

(k) an azetidine;

(I) mixtures of any two or more of (a) to (k).

The iminosugars of any of the foregoing structural classes may be polyhydroxylated, as hereinbefore defined. As used herein, the term polyhydroxylated piperidine iminosugar defines an oxygenated iminosugar (e.g. having at least 2 (preferably at least 3) free hydroxyl groups (or alkyl groups with one or more OH substituents) on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated pyrrolidine iminosugar defines an oxygenated iminosugar (e.g. having at least 2 (preferably at least 3) free hydroxyl groups (or alkyl groups with one or more OH substituents) on the ring system nucleus) that comprises the nucleus:

O As used herein, the term polyhydroxylated pyrrolidine iminosugar defines an oxygenated iminosugar (e.g. having at least 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups (or alkyl groups with one or more OH substituents) on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated indolizidine iminosugar defines an oxygenated iminosugar (e.g. having at least 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) free hydroxyl groups (or alkyl groups with one or more OH substituents) on the ring system nucleus) that comprises the nucleus:

As used herein, the term polyhydroxylated quinolizidine iminosugar defines an oxygenated iminosugar (e.g. having at least 3, 4, 5, 6 or 7 (preferably 3, 4, 5 or 6) free hydroxyl groups (or alkyl groups with one or more OH substituents) on the ring system nucleus) that comprises the nucleus:

In each of the above iminosugar nuclei, it is to be understood that one or more endocyclic carbon atoms may be substituted with a sulphur, oxygen or nitrogen atom.

(O Piperidine iminosugars

Piperidine iminosugars comprise the nucleus:

Preferred are polyhydroxylated piperidine iminosugars as hereinbefore defined comprising the above nucleus and having at least 2 (preferably at least 3) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(ii) Pyrroline iminosugars

Pyrroline iminosugars comprise one of the following three nuclei:

Preferred are polyhydroxylated pyrroline iminosugars as hereinbefore defined having at least 2 hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(iii) Pyrrolidine iminosugars

Pyrrolidine iminosugars comprise the nucleus:

M r

Preferred are polyhydroxylated pyrrolidine iminosugars as hereinbefore defined comprising the above nucleus and having at least 2 (for example at least 3) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(iv) Pyrrolidine iminosugars

Pyrrolidine iminosugars comprise the nucleus:

Preferred are polyhydroxylated pyrrolizidine iminosugars as hereinbefore defined comprising the above nucleus and having at least 2, 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(v) lndolizidine iminosugars

Indolizidine iminosugars comprise the nucleus:

Preferred are polyhydroxylated indolizidine iminosugars as hereinbefore defined comprising the above nucleus and having at least 2, 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(vi) Quinolizidine iminosugars

Quinolizidine iminosugars comprise the nucleus:

Preferred are polyhydroxylated quinolizidine iminosugars as hereinbefore defined comprising the above nucleus and having at least 2, 3, 4, 5, 6 or 7 (preferably 3, 4, 5 or 6) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(vii) Nortropanes

Nortropane iminosugars comprise the nucleus:

wherein the dotted line represents a bridge containing 2 or 3 carbon atoms between any two different ring carbon atoms.

Preferred are polyhydroxylated nortropane iminosugars as hereinbefore defined comprising the above nucleus and having at least 3 (preferably at least 4) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

A preferred class of nortropane iminosugar for use according to the invention are calystegines. These are polyhdroxylated nortropanes which have been reported to inhibit β-glucosidases, β-xylosidases and α-galactosidases (Asano et al., 1997, Glycobiology 7: 1085-1088). The calystegines are common in foods belonging to the Solanaceae that includes potatoes and aubergines (egg plant). The calystegines have been shown to inhibit mammalian glycosidases including human, rat and bovine liver enzymes. Attaching sugars to the calystegines such as in 3-0-β-D-glucopyranoside of 1α,2β,3α,6α-tetrahydroxy-nor- tropane (Calystegine B₁) (Griffiths, et al., 1996, Tetrahedron Letters 37: 3207-3208) can alter the glycosidase inhibition to include α-glucosidases and β-galactosidases.

(viii) 5-7 fused

These iminosugars comprise the nucleus:

Preferred are polyhydroxylated 5-7 fused iminosugars as hereinbefore defined comprising the above nucleus and having at least 2, 3, 4, 5, 6 or 7 (preferably 3, 4 or 5) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

(ix) Azepanes

Azepane imino sugars comprise the nucleus:

Preferred are polyhydroxylated azepane iminosugars as hereinbefore defined comprising the above nucleus and having at least 2 (preferably at least 3 or 4) hydroxyl groups (or alkyl groups with one or more hydroxy substituent(s)) on the ring system nucleus.

In each of the above iminosugar nuclei described in subsections (i) to (ix), it is to be understood that one or more endocyclic carbon atoms may be substituted with a sulphur, oxygen or nitrogen atom.

It will also be appreciated that iminosugars comprising the various nuclei described in subsections (i) to (ix) comprise compounds having three, four or more rings.

(x) Ring-open iminosugars

Also considered are amino sugars acids formed by the opening of the imino ring such as compound P1 and P2 (found in Cucurbita spp.) and P3. Such compounds may also be the biological precursors of the iminosugar acids.

(b) Iminosugar structural subclasses

The principal structural classes described above can be further categorized into various subclasses, for example on the basis of the presence of various functional groups, as described below. The iminosugars for use according to the invention may therefore be further characterized on the basis of their structural subclass, for example being selected from:

(i) lminosugar acids

The iminosugar acids (ISAs) are mono- or bicyclic analogues of sugar acids in which the ring oxygen is replaced by a nitrogen. Although iminosugars are widely distributed in plants (Watson et a/. (2001) Phytochemistry 56: 265-295), the iminosugar acids are much less widely distributed.

Iminosugar acids can be classified structurally on the basis of the configuration of the N- heterocycle. Examples include piperidine, pyrroline, pyrrolidine, pyrrolidine, indolizidine and nortropanes iminosugar acids (see Figs. 1-7 of Watson et al. (2001) Phytochemistry 56: 265-295), the disclosure of which is incorporated herein by reference).

Particularly preferred are iminosugar acids selected from the following structural classes:

(a) piperidine ISAs (including (poly)hydroxypipecolic acids) ;

(b) pyrroline ISAs;

(c) pyrrolidine ISAs (including (poly)hydroxyprolines);

(d) pyrrolizidine ISAs;

(e) indolizidine ISAs; and

(f) nortropane ISAs.

The ISAs for use according to the invention may be N-acid ISAs (as hereinbefore defined).

ISA mixtures or combinations containing two or more different ISAs representative of one or more of the classes listed above may also be used.

Preferred are polyhydroxylated ISAs. Particularly preferred are ISAs having a small molecular weight, since these may exhibit desirable pharmacokinetics. Thus, the ISA may have a molecular weight of 100 to 400 Daltons, preferably 150 to 300 Daltons and most preferably 200 to 250 Daltons. Also preferred are ISAs, which are analogues of hydroxymethyl-substituted iminosugars in which one or more hydroxymethyl groups are replaced with carboxyl groups.

Exemplary piperidine iminosugar acids

The ISA of the invention may be a piperidine ISA having at least 3 free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus. Exemplary piperidine ISAs are hydroxypipecolic acids. Particularly preferred hydroxypipecolic acids are polyhydroxypipecolic acids having at least two (e.g. 3) free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.

Exemplary pyrrolidine iminosugar acids

The ISA of the invention may be a pyrrolidine ISAs having at least 2 (preferably at least 3) free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus. Preferred pyrrolidine ISAs are hydroxyprolines. Particularly preferred hydroxyprolines are polyhydroxyprolines having at least two (e.g. at least 3) free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.

Exemplary pyrrolidine iminosugar acids

The ISA of the invention may be a pyrrolidine ISA having at least 2 (preferably at least 3, 4 or 5) free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.

Exemplary indolizidine iminosugar acids

The ISA of the invention may be an indolizidine ISA having at least 2 (preferably at least 3, 4 or 5) free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.

Exemplary nortropane iminosugar acids

The ISA of the invention may be a nortropane ISA having at least 2 (preferably at least 3) free hydroxyl (or hydroxyalkyl) groups on the ring system nucleus.

(ii) 1 -Λ/-iminosugars (isoiminosugars) lsoimino sugars are carbohydrate mimics in which the anomeric carbon is replaced by a nitrogen atom and the ring oxygen is repaced by a carbon atom (for example, a methylene group in the case of monocyclic piperidine and pyrrolidine compounds).

(iii) lminosugar conjugates

Carbohydrates are often conjugated to other biomolecules in vivo, including lipids, proteins, nucleosides and phosphate groups. Thus, of particular interest as a subclass of the various principal classes of iminosugar described above are iminosugar conjugates. These include:

• Iminosugar-based glycopeptide analogues

• Iminosugar phosphonate analogues

• Iminosugar nucleotide analogues

• Iminosugar glycolipid analogues (e.g. C- or N-alkyl iminosugar derivatives)

(iv) Iminosugar C-glvcosides

Imino-analogues of glycosides in which an aglycone moiety is attached to the anomeric (C- 1 ) carbon via an O-glycosidic bond are of limited utility as drugs due to the lability of the N,O-acetal function. Replacement of the oxygen atom of the N,O-acetal by a methylene group yields iminosugar C-glycosides, which are stable analogues of glycoconjugates. The endocyclic nitrogen is preferably unsubstituted in such C-glycosides, so that the compounds may comprise a nucleus selected from those listed below:

lminosugars of this structural subclass are described by Compain (2007) In lminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons) pages 63-86 (the disclosure of which is hereby incorporated by reference). (v) N-substituted iminosugars

N-substitutecf iminosugars may be considered as analogues of the iminosugar C- glycosides described above in which the aglycone moiety is positioned on the endocyclic nitrogen rather than the "anomeric" C-1 carbon atom.

(vi) Imino-C-disaccharides and analogues

Imino-C-disaccharides and analogues for use according to the invention may fall into any one of the three structural subclasses described by Vogel et a/. (2007) In Iminosugars From Synthesis to Therapeutic Applications: Compain, Philippe / Martin, Olivier R. (eds.) ISBN-13: 978-0-470-03391-3 - John Wiley & Sons) pages 87-130 the disclosure of which is hereby incorporated herein by reference. For example, they may be: (a) linear (1→1)-C- linked; (b) linear (1— ► ω)-C-linked; or (c) branched (I→n)-C-Iinked (see Fig. 5.1 of Vogel et a/. (2007), op. cit.).

(vii) Iminosugar lactams

Iminosugar lactams for use according to the invention may for example comprise a nucleus selected from:

in which the =O group may be on both rings of the bicyclic nuclei.

In each of the above iminosugar lactam nuclei, it is to be understood that one or more endocyclic carbon atoms may be substituted with a sulphur, oxygen or nitrogen atom.

(viii) Branched iminosugars The iminosugars for use according to the invention may be a branched imino sugar. Branched iminosugars are as defined in sections (i) to (x) (above) but are distinguished by the presence of two non-H substituents (e.g. two alky! groups, two hydroxyalkyl groups, a hydroxy and hydroxyalkyl group or a hydroxy and alkyl group) on any one or more endocyclic carbon atom.

It will be appreciated that iminosugars with features characteristic of two or more of the foregoing subclasses (i) to (x) may also find application according to the invention.

General physicochemical considerations

The compounds for use as ASSCs and/or non-ASSCs according to the invention may have various physicochemical properties. They are preferably crystalline materials. Also preferred are compounds which are water soluble, or which are soluble in pharmaceutically acceptable excipients and formulations used in oral or i.v. administration (e.g. those described below). Also preferred are compounds which are subject to efficient passive or active transport to the desired site of action in vivo.

Preferred are iminosugars having a small molecular weight, since these may exhibit desirable pharmacokinetics. Thus, the iminosugar may have a molecular weight of 100 to 400 Daltons, preferably 150 to 300 Daltons and most preferably 200 to 250 Daltons.

Also preferred are non-metabolizable iminosugars. Such sugars may exhibit extended tissue residence durations, and so exhibit favourable pharmacokinetics.

Chemical synthesis I. General considerations

Generally applicable strategies for the synthesis of iminosugars and iminosugar libraries are described by La Ferla et a/. (2007) In "Iminosugars: From synthesis to therapeutic applications", Wiley ISBN 978-0-470-03391-3; Compain and Martin (Eds.) pages 25-61. These general techniques find application in the synthesis of a wide range of compounds for use according to the invention, including monocyclics, 1-N-iminosugars, bicyclic compounds and iminosugar conjugates. This disclosure is hereby incorporated herein by reference.

II. Synthesis of iminosugar C-glycosides

Generally applicable strategies for the synthesis of iminosugar C-glycosides are described by Compain (2007) In "Iminosugars: From synthesis to therapeutic applications", Wiley ISBN 978-0-470-03391-3; Compain and Martin (Eds.) pages 63-86. These general techniques find application in the synthesis of a wide range of iminosugar C-glycosides for use according to the invention and the disclosure is hereby incorporated herein by reference.

III. Synthesis of imino-C-disaccharides and analogues

Generally applicable strategies for the synthesis of imino-C-disaccharides and various analogues are described by Vogel et al. (2007) In "Iminosugars: From synthesis to therapeutic applications", Wiley ISBN 978-0-470-03391-3; Compain and Martin (Eds.) pages 87 -130 the disclosure of which is hereby incorporated herein by reference.

IV. Synthesis of polyhydroxylated iminosugars

The synthesis of polyhydroxylated iminosugars can be carried out by protecting or differentiating the reactivity of the oxygen functions. Bell et al. (1997) Tetrahedron Lett. 38(33): 5869-72 describe the synthesis of four diastereoisomers of casuarine from eight carbon sugar lactones by reduction of open chain azidodimesylates by Suzuki-Takaoka reduction to allow the formation of the pyrrolidine nucleus by bicyclisation (Bell et al. (1997) Tetrahedron Lett. 38(33): 5869-72).

Another approach is based on tandem [4+2]/[3+2] nitroalkene cycloadditions. It has been used for the synthesis of several pyrrolidine and indolizidines iminosugars with up to four contiguous stereogenic centres (see Denmark and Hurd (1999) Organic Lett. 1 (8): 1311- 14). The method was later extended by the same workers to the synthesis of (÷)-casuarine by the intermolecular [3+2] cycloaddition of a suitable substituted dipolarophile and a flexible, heavily substituted nitronate. WO2006/008493 (the content of which relating to synthetic schemes for producing iminosugars is hereby incorporated by reference) describes the synthesis of polyhydroxylated pyrrolizidine and indolizidine compounds without protecting all of the free hydroxyl groups, so achieving considerably shortened synthetic schemes. Moreover, the use of intermediates having free hydroxyl groups provides a mechanism for controlling the product distribution, stereospecificity and yield via complex formation at the free hydroxyl groups. According to WO2006/008493, polyhydroxylated bicyclic (for example pyrrolizidine, indolizidine or quinolizidine) iminosugars can be produced by cyclisation of a pyrrolidine or piperidine intermediate having three or more free hydroxyl groups. The application of a cyclisation step to an intermediate having three or more free hydroxyl groups eliminates the need for selective protection, deprotection and/or activation at these sites.

V. Synthesis of iminosugar acids

The ISAs described herein may be made by conventional methods. Methods of making heteroaromatic ring systems are well known in the art. In particular, methods of synthesis are discussed in Taylor et a/. (2005) Tetrahedron: 61(40) 9611-9617 and in Comprehensive Heterocyclic Chemistry, Vol. 1 (Eds.: AR Katritzky, CW Rees), Pergamon Press, Oxford, 1984 and Comprehensive Heterocyclic Chemistry II: A Review of the Literature 1982-1995 The Structure, Reactions, Synthesis, and Uses of Heterocyclic Compounds, Alan R. Katritzky (Editor), Charles W. Rees (Editor), E.F.V. Scriven (Editor), Pergamon Pr, June 1996. Other general resources which would aid synthesis of the compounds of interest include March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley-lnterscience; 5th edition (January 15, 2001). Some exemplary synthetic schemes for producing ISAs for use according to the invention are shown below:

Vl. Synthesis of nortropanes

Generally applicable strategies for the synthesis of nortropanes are described by Skaanderup and Madsen (2003) J. Org. Chem. 68(6): 2115-2122 the disclosure of which is hereby incorporated herein by reference.

VH. Synthesis of azepanes

Generally applicable strategies for the synthesis of azepanes are described by Li et al. (2007) Chem. Comm. (Cambridge, United Kingdom) (2): 183-185 the disclosure of which is hereby incorporated herein by reference.

VIII. Synthesis of pyrrolidines

Generally applicable strategies for the synthesis of pyrrolidines are described by Rountree et al. (2007) Tetrahedron Lett. 48: 4287-4291 and Behr and Guillerm (2007) Tetrahedron Lett. 48(13), 2369-2372 the disclosure of which is hereby incorporated herein by reference. IX. Synthesis of piperidines

Generally applicable strategies for the synthesis of piperidines are described by Mane et al. (2008) J. Org. Chem. 73 (8): 3284 -3287 and Rengasamy et al. (2008) J. Org. Chem. 73(7): 2898-2901 the disclosure of which is hereby incorporated herein by reference.

X. Synthesis of pyrrolidines

Generally applicable strategies for the synthesis of pyrrolizidines are described in Pyrrolidine Alkaloids, in The.Way of Synthesis, Tomas Hudlicky and Josephine W. Reed, 2007, Wiley, ISBN: 978-3-527-31444-7, pages 617-653 and by Van Ameijde et al. (2006) Tetrahedron: Asymm. 17: 2702-2713, the disclosure of which is hereby incorporated herein by reference.

XI. Synthesis of indolizidines

Generally applicable strategies for the synthesis of indolizidines are described in Abrams et al. (2008) J. Org. Chem. 73 (5): 1935 -1940 and Kumar et al. (2008) Org. Biomol. Chem. 6(4): 703-711 , the disclosure of which is hereby incorporated herein by reference.

XII. Synthesis of quinolizidines

Generally applicable strategies for the synthesis of quinolizidines are described in Pasniczek et al. (2007) J. Carbohydrate Chem. 26(3): 195-211 and Kumar et al. (2008) Org. Biomol. Chem. 6(4): 703-711 , the disclosure of which is hereby incorporated herein by reference.

XIII. Synthesis of 4-membered monocycles

Generally applicable strategies for the synthesis of 4-membered monocycles are described in Evans et al. (2008) J. Med. Chem. 51 (4): 948-956, the disclosure of which is hereby incorporated herein by reference.

XIV. Synthesis of 9-membered monocycles Generally applicable strategies for the synthesis of 9-membered monocycles are described in Leonard and Swann (1952) J. Am. Chem. Soc. 74: 4620-4, the disclosure of which is hereby incorporated herein by reference.

XV. Synthesis of 10-membered monocycles

Generally applicable strategies for the synthesis of 10-membered monocycles are described by Arata and Kobayashi (1972) Chem. Pharm. Bull. 20(2): 325-9, the disclosure of which is hereby incorporated herein by reference.

XVI. Synthesis of 4,6 fused tricyclics

Generally applicable strategies for the synthesis of 4,6 fused bicyclics are described in Pandey et al. (2006) Tetrahedron Lett. 47(45): 7923-7926, the disclosure of which is hereby incorporated herein by reference.

XVII. Synthesis of 4,7 fused bicyclics

Generally applicable strategies for the synthesis of 4,7 fused bicyclics are described in Alcaide and Saez (2005) Eur. J. Org. Chem. (Issue 8): 1680-1693, the disclosure of which is hereby incorporated herein by reference.

XVIH. Synthesis of 5,7 fused bicvclics

Generally applicable strategies for the synthesis of 5,7 fused bicyclics are described in Bande et al. (2007) Tetrahedron: Asymm. 18(10): 1176-1182, the disclosure of which is hereby incorporated herein by reference.

Purification from botanic sources

Botanic and microbial sources for a wide range of different iminosugars are described in Watson et al. (2001) Phytochemistry 56: 265-295. lminosugar acids also have a wide distribution in plants such as in Stevia, Gymnema, Citrus, Lycium species, leguminous spp.e.g. Aspalanthus linearis (Rooibos), Lotus species and Castanospermum australe (Fabaceae), Cucurbitaceae species and Andrographis paniculata (Acanthaceae). The distribution of iminosugar acids in microorganisms is not known but they are likely to be present.

Plant material from botanic sources such as Stevia species can be used as starting material for the isolation and purification of both iminosugars and iminosugar acids for use according to the invention. Microorganisms such as Bacillus, Streptomyces and Metarrhizium species can be used for isolation of iminosugars. The natural iminosugars and iminosugar acids of the invention are water-soluble and can be concentrated by using strongly acidic cation exchange resins to which they bind with the iminosugar acids then concentrated subsequently by binding them to strongly basic anion exchange resins. The iminosugars are not strongly retained on the anion exchange resins whereas the iminosugar acids are. Purification of the iminosugars and iminosugar acids can then be achieved by using a series of cation and anion exchange resins selected by those experienced in the art. Size exclusion methods can also be used to concentrate them. Thus, it will be appreciated that those skilled in the art can readily purify and isolate the iminosugar and iminosugar acids of the invention using standard techniques.

Medical uses of the invention

The invention finds general application in the treatment of any proteostatic disease. Accordingly, the compounds of the invention may be used for the treatment of both aggregative and misfolding proteostatic diseases, including prion diseases, various amyloidoses and neurodegenerative disorders (e.g. Parkinson's disease, Alzheimer's disease and Huntington's disease), lysosomal storage disorders, certain forms of diabetes, emphysema, cancer and cystic fibrosis. These medical uses are described in further detail below.

The folding and maintenance of proteins in a correctly folded, active (or native) form is essential to normal cellular function. The role of protein misfolding in a wide variety of human diseases is an emerging field of research and several previously unrelated diseases (including prion diseases, diabetes, cystic fibrosis, lysosomal storage diseases (LSDs), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyloidosis and cancer) are now known to involve proteotoxicity arising from misfolded protein. The proteotoxicity attendant on misfolding may arise from a variety of causes, including aggregation and deposition leading to tissue damage (e.g. in the amyloidoses) and inappropriate trafficking or clearance (e.g. in cystic fibrosis and the LSDs)₁ leading to enzymic deficits.

The combinations and bivalent pharmacoperones of the invention therefore find broad application in the treatment of protein misfolding diseases in general, including in particular the protein misfolding diseases described below:

Protein aggregation diseases

Amyloidoses

Certain proteins can assume a non-native, misfolded fi-pleated sheet conformation which accumulates as amyloid fibrils (sometimes referred to as amyloid deposits or plaques) in organs and/or tissues, causing disease. These diseases are collectively known as amyloidoses.

Amyloidoses can be classified clinically as primary, secondary, familial, systemic or isolated. Primary amyloidosis appears without any preceding disease, for example arising from immune cell dysfunction such as multiple myeloma and other immunocyte dyscrasias. Secondary amyloidosis is a sequela of an existing disorder (typically a chronic inflammatory disease). Familial amyloidosis (which includes neuropathic, cardiopathic and or nephropathic forms) arises from an inherited mutation and can now be identified by DNA tests. Systemic forms involve amyloid deposition in plural tissues and/or organs (although the brain is almost never directly involved in systemic amyloidosis), while isolated (or localized) amyloidosis involves a single organ, tissue type or system. Thus, recognized clinical forms include ocular amyloidosis and central nervous system amyloidosis.

Amyloidoses can also be classified according to the chemical type of the amyloid protein. The amyloidoses are referred to with a capital A (for amyloid) followed by an abbreviation for the fibril protein. Thus, AA amyloidosis is characterized by extracellular deposition of fibrils that are composed of fragments of serum amyloid A (SAA) protein, a major acute- phase reactant protein, produced predominantly by hepatocytes. Similarly, AL amyloidosis (also called primary amyloidosis or light chain amyloidosis) is characterized by extracellular deposition of fibrils that are composed of an immunoglobulin light chain or light chain fragment, while ATTR amyloidosis is characterized by extracellular deposition of fibrils consisting of the transport protein transthyretin (TTR). Aβ amyloidosis (which appears in Alzheimer's disease) is characterized by extracellular deposition of fibrils that are composed of β-protein precursor.

Symptoms, prognosis and clinical setting differ greatly between amyloid types. Although about 23 different proteins are known to form amyloid in humans, only a few are associated with clinically significant amyloidosis. The various amyloid proteins and the type of amyloidosis and clinical setting in which they are involved are shown in the table below:

Amyloidosis

Amyloid protein type Principal Clinical Setting(s) type

Immunoglobulin light

Plasma cell disorders chains

Familial amyloid polyneuropathies; senile

Transthyretin (TTR) cardiac amyloidosis

Amyloid A (AA) amyloidosis; Inflammation-

Systemic

Serum amyloid A associated amyloidosis; familial mediterranean fever β₂-microglobulin Dialysis-associated amyloidosis

Immunoglobulin heavy

Systemic amyloidosis chains

Fibrinogen alpha chain Familial systemic amyloidosis

Apolipoprotein Al Familial systemic amyloidosis

Hereditary

Apolipoprotein All Familial systemic amyloidosis

Lysozyme Familial systemic amyloidosis

Alzheimer's disease; Down's syndrome; β-protein precursor hereditary cerebral hemorrhage with amyloidosis (Dutch)

Creutzfeldt-Jakob disease; Gerstmann-

Central Prion protein (AScr or PrP- Straussler-Scheinker disease; fatal familial nervous 27) insomnia system hereditary cerebral hemorrhage with

Lrystatin L> amyloidosis (Icelandic)

ABri precursor protein Familial dementia (British)

ADan precursor protein Familial dementia (Danish)

Gelsolin Familial amyloidosis (Finnish)

Ocular Lactoferrin Familial corneal amyloidosis

Keratoepithelin Familial corneal dystrophies

Calcitonin Medullary thyroid carcinoma

Amylin Insulinoma; type 2 diabetes

Atrial natriuretic factor Atrial amyloidosis

Lucaiizθu

Prolactin Pituitary amyloid

Keratin Cutaneous amyloidosis

Medin Senile aortic amyloidosis Amyloid A (AA) amyloidosis is the most common form of systemic amyloidosis worldwide. It occurs in the course of a chronic inflammatory disease of either infectious or noninfectious aetiology, hereditary periodic fevers and with certain neoplasms such as Hodgkin disease and renal cell carcinoma.

Thus, the compounds of the invention find application in the treatment of any of the various amyloidoses described above (and in particular those listed in the above table).

Synucleinopathies

Synucleinopathies comprise a diverse group of neurodegenerative diseases characterized by the presence of lesions composed of aggregates of conformational and posttranslational modifications of α-synuclein in certain populations of neurons and glia. Abnormal filamentous aggregates of misfolded α-synuclein protein are the major components of Lewy bodies, dystrophic (Lewy) neurites, and the Papp-Lantos filaments in oligodendroglia and neurons in multiple system atrophy linked to degeneration of affected brain regions. In contrast to the extracellular amyloid plaques found in the brains of Alzheimer's patients, Lewy bodies are intracellular.

The synucleinopathies include Lewy body diseases (LBDs), dementia with Lewy bodies, multiple system atrophy (MSA), Hallervorden-Spatz disease, Parkinson's disease (PD), the Lewy body variant of Alzheimer's disease (LBVAD), neurodegeneration with brain iron accumulation type-1 (NBIA-1), pure autonomic failure, neuroaxonal dystrophy, amytrophic lateral sclerosis and Pick disease and various tauopathies.

Thus, the compounds of the invention find application in the treatment of Lewy body diseases (LBDs), dementia with Lewy bodies, multiple system atrophy (MSA), Hallervorden-Spatz disease, Parkinson's disease (PD), the Lewy body variant of Alzheimer's disease (LBVAD), neurodegeneration with brain iron accumulation type-1 (NBIA-1), pure autonomic failure, neuroaxonal dystrophy, amytrophic lateral sclerosis and Pick disease and various tauopathies.

Expanded CAG repeat diseases

Certain protein aggregation diseases stem from the expansion of CAG repeats in particular genes with the encoded proteins having corresponding polyglutamine tracts which lead to aggregation and accumulation in the nuclei and cytoplasm of neurons. Aggregated amino- terminal fragments of mutant huntingtin are toxic to neuronal cells and are thought to mediate neurodegeneration.

An example is Huntington's disease (HD). Huntington's disease (HD) is characterized by selective neuronal cell death primarily in the cortex and striatum. It is caused by a CAG repeat expansion in the first exon of the huntingtin gene, which encodes a large protein of unknown function. The CAG repeat is highly polymorphic and varies from 6 to 39 repeats in normal individuals and from 35 to 180 repeats in HD cases.

In addition to HD, CAG expansions have been found in at least seven other inherited neurodegenerative disorders, including for example spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, some forms of amyotrophic lateral sclerosis (ALS), dentatorubral pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia (SCA) types 1 , 2, 3, 6 and 7.

Thus, the compounds of the invention find application in the treatment of HD, SBMA, Kennedy's disease, ALS, DRPLA and SCA (e.g. types 1, 2, 3, 6 and 7).

Tauopathies

The tauopathies are a group of diverse dementias and movement disorders which have as a common pathological feature the presence of intracellular accumulations of abnormal filaments of tau protein. Examples include Down's Syndrome (DS), Corticobasal Degeneration (CBD), Frontotemporal Dementia with Parkinsonism linked to Chromosome 17 (FTDP17), Pick Disease (PiD) and Progressive Supranuclear Palsy (PSP).

Other aggregation diseases

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS). A growing body of evidence suggests that the familial form of ALS (fALS) is caused by destabilization of the native structure of SOD1 leading to aggregation.

Protein folding (conformational) diseases Factors that tilt the balance between correctly folded proteins and misfolded proteins are common causes of disease. The balance can be perturbed as the result of an age-related reduction in the efficiency of the quality control system and/or the acquisition or inheritance of mutations in the primary sequence of the encoding gene. Both lead to incorrect folding, lost activity, improper trafficking and/or misfolding outside the endoplasmic reticulum and cytoplasm.

Lysosomal storage diseases

All lysosomal storage disorders (LSDs) are single gene diseases in which a mutant lysosomal enzyme is aberrantly expressed, processed or translocated in a manner which eliminates or reduces enzyme activity resulting in defective lysosomal acid hydrolysis of endogenous macromolecules and their consequent accumulation. This accumulation leads to tissue enlargement together with a fundamental perturbation of cellular physiology (including ER and oxidative stress) which can lead to severe systemic symptoms (including neurological deficits).

Listed below are a number of lysosomal storage disorders and the corresponding defective enzymes:

Pompe disease: Acid alpha-glucosidase

Gaucher disease: Acid beta-glucosidase or glucocerebrosidase

Fabry disease: alpha-Galactosidase A

GMl-gangliosidosis: Acid beta-galactosidase

Tay-Sachs disease: beta-Hexosaminidase A

Sandhoff disease: beta-Hexosaminidase B

Niemann-Pick disease: Acid sphingomyelinase

Krabbe disease: Galactocerebrosidase

Farber disease: Acid ceramidase

Metachromatic leukodystrophy: Arylsulfatase A

Hurler-Scheie disease: alpha-L-lduronidase

Hunter disease: lduronate-2-sulfatase

Sanfilippo disease A: Heparan N-sulfatase

Sanfilippo disease B: alpha-N-Acetylglucosaminidase

Sanfilippo disease C: Acetyl-CoA: alpha-glucosaminide N-acetyltransferase Sanfilippo disease D: N-Acetylglucosamine-6-sulfate sulfatase

Morquio disease A: N-Acetylgalactosamine-6-sulfate sulfatase

Morquio disease B: Acid beta-galactosidase

Maroteaux-Lamy disease: Arylsulfatase B

Sly disease: beta-Glucuronidase alpha-Mannosidosis: Acid alpha-mannosidase beta-Mannosidosis: Acid beta-mannosidase

Fucosidosis: Acid alpha-L-fucosidase

Sialidosis: Sialidase

Schindler-Kanzaki disease: alpha-N-acetylgalactosaminidase

Thus, the compounds of the invention may be used for the treatment of an LSD selected from:

Pompe disease (including infantile and late-onset forms)

Gaucher disease (including Type 1 , Type 2 and Type 3 Gaucher disease)

Fabry disease

GMI-gangliosidosis

Tay-Sachs disease

Sandhoff disease

Niemann-Pick disease

Krabbe disease

Farber disease

Metachromatic leukodystrophy

Hurler-Scheie disease

Hunter disease

Sanfilippo disease A

Sanfilippo disease B

Sanfilippo disease C

Sanfilippo disease D

Morquio disease A

Morquio disease B

Maroteaux-Lamy disease

Sly disease alpha-Mannosidosis beta-Mannosidosis Fucosidosis

Sialidosis

Schindler-Kanzaki disease

In preferred embodiments, the lysosomal storage disease is selected from: (a) Pompe disease; (b) Gaucher disease; and (c) Fabry disease.

In particularly preferred embodiments, the lysosomal disease is selected from Type 1, Type 2 and Type 3 Gaucher disease.

Cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel important in creating sweat, digestive juices and mucus. Cystic fibrosis occurs when there is a mutation in the CFTR gene leading to reduced ion channel activity {via increased clearance of the misfolded CFTR proteins).

Thus, the compounds of the invention find application in the treatment of cystic fibrosis.

Emphysema

Misfolding/trafficking of alpha 1 anti-trypsin can cause emphysema, especially in childhood. Thus, the compounds of the invention can (at least partially) restore proper folding and/or trafficking of alpha 1 anti-trypsin and so find application in the treatment of emphysema, particularly childhood emphysema.

Endoplasmic reticulum stress-induced diseases

The endoplasmic reticulum (ER) fulfills multiple cellular functions, including protein folding and the transport of proteins destined for extracellular compartments. Many disorders cause accumulation of unfolded proteins in the ER, triggering the unfolded protein response (UPR). The UPR functions to increase folding capacity and retrograde transport of misfolded proteins into the cytosol for proteasome-dependent degradation. However, chronic or excessive ER stress triggers apoptosis. There is a growing recognition that ER stress underlies a wide range of different diseases, all caused (at least in part) by ER stress coupled with an aberrant UPR.

Diabetes, insulin resistance, obesity and metabolic syndrome

It has recently been recognized that aberrant UPR leads to ER stress and ultimately β-cell failure that contributes to the development of type Il diabetes, insulin resistance and metabolic syndrome.

Thus, the compounds of the invention find application in the treatment of insulin resistance. Insulin resistance is characterized by a reduced action of insulin in skeletal muscle, adipocytes and hepatocytes so that normal amounts of insulin become inadequate to produce a normal insulin response from the cells of these tissues. In adipocytes, insulin resistance results in hydrolysis of stored triglycerides, leading to elevated free fatty acids in the blood plasma. In muscle, insulin resistance reduces glucose uptake while in hepatocytes it reduces glucose storage. In both of the latter cases an elevation of blood glucose concentrations results. High plasma levels of insulin and glucose due to insulin resistance often progresses to metabolic syndrome and type 2 diabetes.

The invention finds application in the treatment of metabolic syndrome. The disorder is also known as (metabolic) syndrome X, insulin resistance syndrome, Reaven's syndrome and CHAOS.

The invention finds application in the treatment of diseases associated with metabolic syndrome, including for example: fatty liver (often progressing to non-alcoholic fatty liver disease), polycystic ovarian syndrome, hemochromatosis (iron overload) and acanthosis nigricans (dark skin patches).

The invention finds application in the treatment of Type 2 diabetes. Type 2 diabetes is a chronic disease that is characterised by persistently elevated blood glucose levels (hyperglycaemia). Insulin resistance together with impaired insulin secretion from the pancreatic β-cells characterizes the disease. The progression of insulin resistance to type 2 diabetes is marked by the development of hyperglycaemia after eating when pancreatic β-cells become unable to produce adequate insulin to maintain normal blood sugar levels (euglycemia)). The invention finds application in the treatment of Type 1 diabetes (or insulin dependent diabetes). Type 1 diabetes is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas, leading to a deficiency of insulin. The main cause of this beta cell loss is a T-cell mediated autoimmune attack. There is no known preventative measure that can be taken against type 1 diabetes, which comprises up to 10% of diabetes mellitus cases in North America and Europe. Most affected people are otherwise healthy and of a healthy weight when onset occurs. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages.

The invention also finds application in the treatment of insulin resistance, various forms of diabetes, metabolic syndrome, obesity, wasting syndromes (for example, cancer associated cachexia), myopathies, gastrointestinal disease, growth retardation, hypercholesterolemia, atherosclerosis and age-associated metabolic dysfunction.

The invention may also be used for the treatment of conditions associated with metabolic syndrome, obesity and/or diabetes, including for example hyperglycaemia, glucose intolerance, hyperinsulinaemia, glucosuria, metabolic acidosis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, macular degeneration, glomerulosclerosis, diabetic cardiomyopathy, insulin resistance, impaired glucose metabolism, arthritis, hypertension, hyperlipemia, osteoporosis, osteopenia, bone loss, . brittle bone syndromes, acute coronary syndrome, infertility, short bowel syndrome, chronic fatigue, eating disorders, intestinal motility dysfunction and sugar metabolism dysfunction.

Infectious disease and cancer

The compounds of the present invention can decreasing protein folding and or protein trafficking capacity, and since elevated enhanced folding and trafficking capacity is required for bacterial and viral replication and assembly as well as tumour cell growth and replication, the compounds of the invention find application in the treratment of infectious disease and cancer.

Age-onset proteotoxicity diseases

Age-associated decrease in cellular proteostasis capacity together with increasing protein damage leads to a plethora of age-onset diseases with a proteotoxic component. Thus, the compounds of the invention find application in the treatment of age-onset diseases, including for example various dementias and neurodegenerative diseases (e.g. AD, PD, ALS and HD), cancer, heart disease and autoimmune diseases (e.g. rheumatoid arthritis and diabetes).

Posology

The compounds 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 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 compound selected.

Moreover, the compounds of the invention can be used in conjunction with other agents known to be useful in the treatment of diseases or disorders arising from protein folding abnormalities (as described infra) and in such embodiments the dose may be adjusted accordingly.

In general, the effective amount of the compound administered will generally range from about 0.01 mg/kg to 500 mg/kg daily. A unit dosage may contain from 0.05 to 500 mg of the compound, and can be taken one or more times per day. The compound can be administered with a pharmaceutical carrier using conventional dosage unit forms either orally, parenterally, or topically, as described below.

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

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

Formulation

The compound for use according to the invention may take any form. It may be synthetic, purified or isolated from natural sources.

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

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

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

Tablets for oral use may include the compound for use according to 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 compound for use according to the invention is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

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

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

In another embodiment, the compounds of the invention are tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, lubricants intended to improve the flow of tablet granulations and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium, or zinc stearate, dyes, coloring agents, and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptably surfactant, suspending agent or emulsifying agent.

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

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

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

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

When used adjunctively, the pharmacoperones for use according to the invention may be formulated for use with one or more other drug(s). In particular, the compounds may be used in combination with lysosomal enzymes adjunctive to enzyme replacement therapy. Thus, adjunctive use may be reflected in a specific unit dosage designed to be compatible (or to synergize) with the other drug(s), or in formulations in which the compound is admixed with one or more enzymes. Adjunctive uses may also be reflected in the composition of the pharmaceutical kits of the invention, in which the compounds of the invention is co-packaged (e.g. as part of an array of unit doses) with the enzymes. Adjunctive use may also be reflected in information and/or instructions relating to the coadministration of the compound and/or enzyme.

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 : Identification of pharmacoperones for α-Mannosidase

Fresh solutions had been prepared a day or so earlier of 0.2M Mcllvane buffer at pH 4.5 and 5mM PNP-α-D- mannopyranoside (Sigma, N2127) in pH 4.5 buffer. Also prepared was a dilution of Jack bean α -D-mannosidase enzyme (Sigma, M7257, 22Units/mg, 6.2mg/ml.) at 0.6Units/ml in pH 4.5 buffer.

The incubation mixture consisted of 10 μl enzyme solution, 10 μl of 1 mg/ml aqueous inhibitor solution and 50 μl of 5mM substrate made up in buffer at the optimum pH for the enzyme. The reactions were stopped by addition of 70 μl 0.4M glycine (pH 10.4) during the exponential phase of the reaction, which had been determined at the beginning using uninhibited assays in which water replaced inhibitor. Final absorbances were read at 405 nm using a Versamax microplate reader (Molecular Devices). Assays were carried out in triplicate, and the values given are means of the three replicates per assay. Results were expressed as a percentage of uninhibited assays in which water replaced inhibitor.

Several polyhydroxylated alkaloids (imino sugars) were found to increase the activity of the enzyme by between 49% and 124% at the top concentration used (~0.8mM). The stimulation was so great in some cases that the absorbance values were above the linear range and so the compounds were repeated at 0.08mM and absorbance values were within range and still showed stimulation from 7% to 30% for the diluted samples.

An assay was set up in which one of the compounds showing strong stimulation was mixed with an equal concentration of swainsonine and compared with swainsonine alone and as compound 1 alone. The swainsonine plus the selected compound and the swainsonine alone both gave 100% inhibition whereas the compound alone gave 90% stimulation.

Stimulation of other glycosidase activities such as α-glucosidase, α-galactosidase and hexosaminidases was also noted by a range of other imino sugars without them being inhibitory to any glycosidase tested. Such compounds might therefore have utility in diseases where specific glycosidase activities are deficient, including lysosomal storage disorders (Pompe's disease, Sandhoffs and Fabry's for example). Many imino sugars have been observed by the inventors to increase the apparent activity of specific purified glycosidases. In the example given here we found that Jack Bean α- mannosidase activity (using p-nitrophenyl-α-D-mannopyranoside as the substrate) was greatly increased by certain imino sugars with a mannose configuration. These compounds did not cause inhibition of the mannosidase and swainsonine, a known inhibitor of this mannosidase, caused total inhibition of the promoted activity.

This study indicates that the catalytic site is free for binding of swainsonine and so we presume that the increased activity of the mannosidase is due to binding to another site on the enzyme. Swainsonine does not cause promotion of the mannosidase at any concentration tested.

Example 2: Identification of pharmacoperones for beta-glucocerebrosidase

I. Beta-glucocerebrosidase activity assay

Human Caucasian promyelocytic leukaemia cells (HL60, ECACC No. 98070106) were cultured using a standard sub-culture routine and lysed. The lysates were used as a source for wild type (wt) beta-glucocerebrosidase and used in an assay to determine the enzyme activity and conduct inhibition studies.

i) Cell lysate preparation

HL60 cells were cultured to confluency and washed twice with PBS. Cells were lysed by the addition of lysis buffer (citric phosphate buffer (pH5.2), 0.1% Triton X-100, 0.25% taucholate) at 10x10⁶ cells/ml and incubated at 25°C for 5 min. Lysates were cleared by centrifugation (400g, 25°C, 5 min) and protein concentration was determined by using QuantiPro BCA assay kit (Sigma-Aldrich). Lysates were stored in aliquots at -80⁰C.

ii) Beta-glucocerebrosidase activity assay

4-Methylumbelliferyl β-D-glucopyranoside (4MU-β-D-glc) (Sigma) was used as a substrate to measure beta-glucocerebrosidase activity in HL60 lysate. Enzyme assays were performed in 96-well microtitre plates. Thawed cell lysate and 0.5mM 4MU~β-D-glc in lysis buffer (50μl final reaction volume) were mixed and incubated at 37°C. The reaction was quenched with 150μl 0.5M sodium carbonate. The activity was measured by determining the rate of product (4MU) released using a fluorometer (OPTIMA, BMG) using excitation 360nm, emission 450nm filters. For detailed inhibition kinetic studies, various concentrations of iminosugars (1nM-1mM) and 4MU-β-D-Glc (100 μM - 4mM) were used.

II. Enzyme enhancement assay - cell based screening for chaperones

Lymphoblasts derived from Gaucher's patients can be used for the cell based screening assays. EBV transformed B-lymphocytes from Gaucher's patients such as cell lines homozygous for the N370S mutation (GM01873) and L444P mutation (GM08752) in beta- glucocerebrosidase, were obtained from Coriell Institute for Medical Research. Cells were cultured in RMP! 1640 (Sigma) supplemented with 15% FBS (PAA), 2mM L-glutamine and penicillin-streptomycin (PAA) as described in the culturing protocol.

Cells were seeded (8x10⁴ cells/well) and dosed (0.3-1 OOμM) in white 96-well plates (NUNC) to a final volume of 300μL, and incubated for 72hr at 37⁰C in a 5% CO₂ incubator. Cells (200μL) were transferred to 96-well Multiscreen harvester plates (Millipore) and harvested under vacuum. Cells were washed twice with PBS and lysed (and the enzyme reaction started) by the addition of 10OμL lysis buffer containing 5mM 4MU-β-D-glc. Cell ^■ debris was removed by filtering through and collecting the cleared lysates. Lysates were incubated at 37°C for a total time of 2 hrs. The enzyme reaction was quenched by addition of 150μl_ 0.5M sodium carbonate to 50μl of reaction mix. Fluorescence was measured as described above. QuantiPro BCA assay kit (Sigma) was used to determine the protein concentration in the cell lysates. Cell viability was measured using CellTiter-Glo® luminescent cell viability assay (Promega) on the remaining 100μl_ unlysed cells. All experiments were performed in triplicates. The fold beta-glucocerebrosidase enzyme activity was determined relative to the vehicle (water or 1% DMSO) control, and normalised against total protein amount per well.

III. Identification of non-active site chaperones of beta-glucocerebrosidases

Compounds that demonstrated a significant increase in cellular beta-glucocerebrosidase activity (protocol II) but showed no competitive inhibition of beta-glucocerebrosidase enzyme activity (protocol I) were considered to be non-active site chaperones. Compounds identified according to the methods describe above find utility in the treatment of Gaucher's disease.

Example 3: Identification of pharmacoperones for alpha-galactosidase

I. Alpha-galactosidase activity assay

Human Caucasian promyelocytic leukaemia cells (HL60, ECACC No. 98070106) were cultured using a standard sub-culture routine and lysed. The lysates were used as a source for wild type (wt) alpha-galactosidase and used in an assay to determine the enzyme activity and conduct inhibition studies.

i) Cell lysate preparation

Cell lysates were prepared as described above (Gaucher's l.i)

ii) Alpha-galactosidase activity assay

4-Methylumbelliferyl alpha-galactopyranoside (4MU-α-D-gal) (Sigma) was used as a substrate to measure alpha-galactosidase activity in HL60 lysate. Enzyme assays were performed in 96-well microtitre plates. Thawed cell lysate and 0.5mM 4MU-α-D-gal in citric phosphate buffer (pH 4.5) containing 0.1M N-acetylgalactosamine (50μl final reaction volume) were mixed and incubated at 37⁰C. The reaction was quenched with 150μl 0.5M sodium carbonate. The activity was measured by determining the rate of product (4MU) released using a fluorometer (OPTIMA, BMG) using excitation 360nm, emission 450nm filters. For detailed inhibition kinetic studies, various concentrations of iminosugars (1nM- 1mM) and 4MU-α-D-Gal (100 μM - 4mM) were used.

II. Enzyme enhancement assay - cell based screening for chaperones

Lymphoblasts derived from Fabry's patients can be used for the cell based screening assays. EBV transformed B-lymphocytes from Fabry's patient (GM04391) were obtained from Coriell Institute for Medical Research. Cells were cultured in RMPI 1640 (Sigma) supplemented with 15% FBS(PAA), 2mM L-glutamine and penicillin-streptomycin (PAA) as described in the culturing protocol.

Cells were seeded (8 x 10⁴ cells/well) and dosed (0.3-100μM) in white 96-well plates (NUNC) to a final volume of 300μL, and incubated for 72hr at 37°C in a 5% CO₂ incubator. Cells (200μL) were transferred to 96-well Multiscreen harvester plates (Millipore) and harvested under vacuum. Cells were washed twice with PBS and lysed (and the enzyme reaction started) by the addition of 100μL 5mM 4MU-α-D-gal in citric phosphate buffer (pH4.5) with 0.1% Triton X-100 and 0.1 M N-acetylgalactosamine (Sigma). Cell debris was removed by filtering through and collecting the cleared lysates, and the lysate was incubated at 37⁰C for 2 hrs The enzyme reaction was quenched by addition of 150μl_ 0.5M sodium carbonate to 50μl of reaction mix. Fluorescence was measured as described above. Cell viability was measured using CellTiter-Glo® luminescent cell viability assay (Promega) on the remaining 100μL unlysed cells. All experiments were performed in triplicates. The fold alpha-galactosidase enzyme activity was determined relative to the vehicle (water or 1% DMSO) control.

III. Identification of non-active site chaperones of alpha-galactosidase

Compounds that demonstrated a significant increase in cellular alpha-galactosidase activity (protocol II) but showed no competitive inhibition of alpha-galactosidase enzyme activity (protocol I) were considered to be non-active site chaperones.

Compounds identified according to the methods describe above find utility in the treatment of Fabry's disease.

Example 4: Identification of pharmacoperones for alpha-glucosidase

I. Alpha-glucosidase activity assay

Human Caucasian promyelocytic leukaemia cells (HL60, ECACC No. 98070106) were cultured using a standard sub-culture routine and lysed. The lysates were used as a source for wild type (wt) lysosomal alpha-glucosidase and used in an assay to determine the enzyme activity and conduct inhibition studies. i) Cell lysate preparation

Cell lysates were prepared as described above (Gaucher's l.i)

ϋ) Alpha-qlucosidase activity assay

4-methylumbelliferyl alpha-glucopyranoside (4MU-α-D-glc) (Sigma) was used as a substrate to measure alpha-glucosidase activity in HL60 lysate. Enzyme assays were performed in 96-well microtitre plates. Thawed cell lysate and 0.5mM 4MU-α-D-glc in citric phosphate buffer (pH 4.5) (50μl final reaction volume) were mixed and incubated at 37⁰C. The reaction was quenched with 150μl 0.5M sodium carbonate. The activity was measured by determining the rate of product (4MU) released using a fluorometer (OPTIMA, BMG) using excitation 360nm, emission 450nm filters. For detailed inhibition kinetic studies, various concentrations of iminosugars (1nM-1mM) and 4MU-α-D-Glc (100 μM - 4mM) were used.

Compounds that demonstrated an increase in cellular alpha-glucosidase activity (over 1.2 fold) (protocol II) but showed no direct inhibition of alpha-glucosidase enzyme activity (protocol I) were considered to be non-active site chaperones.

Compounds identified according to the methods describe above find utility in the treatment of Pompe's disease.

II. Enzyme enhancement assay - cell based screening for chaperones

Lymphoblasts derived from Pompe's patients can be used for the cell based screening assays. EBV transformed B-lymphocytes from Pompe's patient such as (GM013963) and (GM06314) were obtained from Coriell Institute for Medical Research. Cells were cultured in RMPI 1640 (Sigma) supplemented with 15% FBS(PAA), 2mM L-glutamine and penicillin- streptomycin (PAA) as described in the culturing protocol.

Cells were seeded (8 x 10⁴ cells/well) and dosed (0.3-1 OOμM) in white 96-well plates (NUNC) to a final volume of 300μL, and incubated for 72hr at 37°C in a 5% CO₂ incubator. Cells (200μL) were transferred to 96-well Multiscreen harvester plates (Millipore) and harvested under vacuum. Cells were washed twice with PBS and lysed (and the enzyme reaction started) by the addition of 100μL 5mM 4MU-α-D-glc in citric phosphate buffer (pH4.5) with 0.1% Triton X-100 (Sigma). Cell debris was removed by filtering through and collecting the cleared lysates, and the lysate was incubated at 37⁰C for 2 hrs. The enzyme reaction was quenched by addition of 150μl_ 0.5M sodium carbonate to 50μl of reaction mix. Fluorescence was measured as described above. Cell viability was measured using CellTiter-Glo® luminescent cell viability assay (Promega) on the remaining 100μL unlysed cells. All experiments were performed in triplicates. The fold alpha-glucosidase enzyme activity was determined relative to the vehicle (water or 1% DMSO) control.

III. Identification of non-active site chaperones of alpha-glucosidase

Compounds that demonstrated a significant increase in cellular alpha-glucosidase activity (protocol II) but showed no competitive inhibition of alpha-glucosidase enzyme activity (protocol I) were considered to be non-ASSCs.

Example 5 Demonstration of the synergistic effect of ASSC/non-ASSC compounds for alpha-galactosidase

Fabry's patient cells were seeded (8 x 10⁴ cells/well) and co-dosed with a matrix of different ASSC/non-ASSC concentrations (0-100μM) in white 96-well plates (NUNC) to a final volume of 300μL (up to 1% DMSO), and incubated for 72 at 37°C in a 5% CO₂ incubator. The cells were processed as described in Examople 3 Il above, and the fold lysosomal enzyme activity was determined relative to the vehicle control (water or 1% DMSO). The dose response for the fold increase in lysosomal enzyme activity in cells dosed with ASSC only was compared with the dose response obtained for the ASSC/non-ASSC co-dosed cells.

An example of synergistic effect observed in ASSC/non-ASSC co-dosing experiments is shown in Figure 1. Fabry's patient lymphoblast with N215S α-galactosidase mutation was co-dosed with 1-deoxy-galactonojirimycin (DGJ), a known ASSC of α-galactosidase A, and a non-ASSC identified in the Fabry's cell based screen (Example 3 III). The intracellular α- galactosidase A activity observed with DGJ is further enhanced in the presence of the non- ASSC in a dose-dependent manner, shifting the dose-response curve to the lower DGJ concentration and increasing the maximal cellular response. The following compound demonstrates synergy in such an assay:

(2R,3R,4R)-1-butyl-2-(hydroxymethyl)piperidine-3,4-diol

Example 6 Demonstration of the synergistic effect of ASSC/non-ASSC compounds for beta-glucocerebrosidase

Gaucher's patient cells were seeded (8 x 10⁴ cells/well) and co-dosed with a matrix of different ASSC/non-ASSC concentrations (0-100μM) in white 96-well plates (NUNC) to a final volume of 300μL (up to 1% DMSO), and incubated for 72 at 37°C in a 5% CO₂ incubator. The cells were processed as described in Example 2 above, and the fold lysosomal enzyme activity was determined relative to the vehicle control (water or 1 % DMSO). The dose response for the fold increase in lysosomal enzyme activity in cells dosed with ASSC only was compared with the dose response obtained for the ASSC/non- ASSC co-dosed cells.

An example of synergistic effect observed in ASSC/non-ASSC co-dosing experiments is shown in Figure 2. Gaucher's patient lymphoblast (N370S) was co-dosed with isofagomine (IFG), a known ASSC of beta-glucocerebrosidase, and a non-ASSC identified in the Gaucher's cell based screen (Example 2 111). The intracellular beta-glucocerebrosidase activity observed with IFG is further enhanced in the presence of the non-ASSC in a dose- dependent manner.

The following compound demonstrates synergy in such an assay: 2-((2S,4S)-4-azido-2-(hydroxymethyl)pyrrolidin-1-yl)ethanol

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

1. A combination comprising: (a) an active-site-specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme); and (b) a non-active-site-specific pharmacoperone (non-ASSC) of said enzyme which: (i) does not bind to a catalytic site of said enzyme; or (H) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co- chaperone of said enzyme.

2. A composition comprising an an active-site-specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme) for use in combination therapy with a non-active-site- specific pharmacoperone (non-ASSC) of said enzyme, which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme.

3. A composition comprising a non-active-site-specific pharmacoperone (non-ASSC) of an enzyme (e.g. a lysosomal enzyme), which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme, for use in combination therapy with an active-site-specific pharmacoperone (ASSC) of said enzyme.

4. The combination of claim 1 wherein the ASSC and non-ASSC are physically associated.

5. The combination of claim 4 wherein the ASSC and non-ASSC are: (a) in admixture (for example within the same unit dose); (b) chemically/physicochemically linked (for example by conjugation, 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).

6. The combination of claim 1 wherein the ASSC and non-ASSC are non-physically associated.

7. The combination of claim 6 wherein the combination comprises: (a) at least one of the ASSC and non-ASSC together with instructions for their extemporaneous association to form a physical association; or (b) at least one of the ASSC and non-ASSC together with instructions for combination therapy with the ASSC and non-ASSC; or (c) at least one of the ASSC and non-ASSC together with instructions for administration to a patient population in which either the ASSC or non-ASSC has been (or are being) administered; or (d) at least one of the ASSC and non-ASSC in an amount or in a form which is specifically adapted for use as an ASSC/non-ASSC combination.

8. A pharmaceutical composition comprising the combination as defined in any one of the preceding claims.

9. The combination of any one of the preceding claims which is: (a) in the form of a pharmaceutical pack, kit or patient pack; (b) in a pharmaceutical excipient; or (c) in unit dosage form.

10. A combination according to any one of the preceding claims for use in therapy or prophylaxis (e.g. for use in the treatment of a lysosomal storage disorder).

11. A composition comprising an an active-site-specific pharmacoperone (ASSC) of an enzyme (e.g. a lysosomal enzyme) for the treatment of a subject undergoing treatment with a non-active-site-specific pharmacoperone (non-ASSC) of said enzyme, which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme.

12. A composition comprising a non-active-site-specific pharmacoperone (non-ASSC) of an enzyme (e.g. a lysosomal enzyme), which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (ii) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme, for the treatment of a subject undergoing treatment with an active-site-specific pharmacoperone (ASSC) of said enzyme.

13. A method for the treatment of a protein folding disorder (e.g. a lysosomal storage disorder) comprising the simultaneous, separate or sequential administration of an effective amount of: (a) a non-active-site-specific pharmacoperone (non-ASSC) of an enzyme (e.g. a lysosomal enzyme), which non-ASSC: (i) does not bind to a catalytic site of said enzyme; or (U) is not a competitive inhibitor of said enzyme; or (iii) is an activator of said enzyme; or (iv) is a non-competitive inhibitor of said enzyme; or (v) binds (e.g. specifically) to an allosteric site of said enzyme; or (vi) does not bind to said enzyme but binds to a chaperone or co-chaperone of said enzyme; and (b) an active-site-specific pharmacoperone (ASSC) of said enzyme.

14. The combination, composition or method of any one of the preceding claims, wherein the lysosomal enzyme is selected from: (a) Acid alpha-glucosidase; (b) Acid beta- glucosidase; (c) glucocerebrosidase; (d) alpha-Galactosidase A; (e) Acid beta- galactosidase; (f) beta-Hexosaminidase A; (g) beta-Hexosaminidase B; (h) Acid sphingomyelinase; (i) Galactocerebrosidase; (j) Acid ceramidase; (k) Arylsulfatase A; (I) alpha-L-lduronidase; (m) Iduronate-2-sulfatase; (n) Heparan N-sulfatase; (o) alpha-N- Acetylglucosaminidase; (p> Acetyl-CoA: alpha-glucosaminide N-acetyltransferase; (q) N- Acetylglucosamine-6-sulfate sulfatase; (r) N-Acetylgalactosamine-6-sulfate sulfatase; (s) Acid beta-galactosidase; (t) Arytsulfatase B; (u) beta-Glucuronidase; (v) Acid alpha- mannosidase; (w) Acid beta-mannosidase; (x) Acid alpha-L-fucosidase; (y) Sialidase; and (z) alpha-N-acetylgalactosaminidase.

15. The combination or composition of any of the preceding claims for use in the treatment of a lysosomal storage disorder (LSD).

16. The combination or composition of claim 15 wherein the LSD is selected from: (a) Pompe disease (including infantile and late-onset forms); (b) Gaucher disease (including Type 1 , Type 2 and Type 3 Gaucher disease); (c) Fabry disease; (d) GMI-gangliosidosis; (e) Tay-Sachs disease; (f) Sandhoff disease; (g) Niemann-Pick disease; (h) Krabbe disease:; (i) Farber disease; 0) Metachromatic leukodystrophy; (k) Hurler-Scheie disease; (I) Hunter disease; (m) Sanfilippo disease A, B, C or D; (n) Morquio disease A or B; (o) Maroteaux-Lamy disease; (p) Sly disease; (q) alpha-Mannosidosis; (r) beta-Mannosidosis; (s) Fucosidosis; (t) Sialidosis; and (u) Schindler-Kanzaki disease.

17. A method of chaperone-mediated therapy of a disease arising from aberrant protein folding and/or processing comprising the administration of an effective amount of a first pharmacoperone which binds to an active site (e.g. a catalytic site or an effector, receptor or ligand-binding site) of said protein and a second pharmacoperone which binds to said protein at a site remote from said active site.

18. The method of claim 17 wherein the protein is an enzyme and the first pharmacoperone binds to a catalytic or active site of said enzyme and the second pharmacoperone binds to said enzyme at a site remote from the catalytic or active site.

19. The method of claim 18 wherein the first pharmacoperone is a competitive inhibitor of said enzyme and the second pharmacoperone is not a competitive inhibitor of said enzyme.

20. The method of claim 18 or claim 19 wherein the second pharmacoperone binds to an allosteric site of said enzyme.

21. The method of any one of claims 18 to 20 wherein the second pharmacoperone is an activator of said enzyme.

22. The method of any one of claims 18 to 21 wherein the second pharmacoperone is a non-competitive inhibitor of said enzyme.

23. The method of any one of claims 18 to 22 wherein the first and second pharmacoperones do not compete for binding to said enzyme.

24. The method of any one of claims 18 to 23 wherein the disease is an LSD, for example an LSD as defined in claim 16.

25. The method of any one of claims 18 to 24 wherein the enzyme is a lysosomal enzyme, for example selected from the enzymes listed in claim 14.

26. A composition comprising a pharmacoperone for use in a method as defined in any one of claims 17 to 25.

27. A bivalent pharmacoperone of an enzyme comprising a first moiety which binds to a catalytic or active site of said enzyme and a second moiety which binds to a second, distinct and non-catalytic site of said enzyme.

28. The bivalent pharmacoperone of claim 27 wherein the second moiety binds to amino acid residues which do not form part of the catalytic site of said enzyme.

29. The bivalent pharmacoperone of claim 27 or claim 28 wherein the second moiety binds to amino acid residues which do not form part of the active site of said enzyme.

30. The bivalent pharmacoperone of any one of claims 27 to 29 wherein the second moiety binds to an allosteric site of said enzyme.

31. The bivalent pharmacoperone of any one of claims 27 to 30 wherein the second moiety binds to a site on said enzyme which activates said enzyme.

32. The bivalent pharmacoperone of any one of claims 27 to 31 wherein the enzyme is selected from: (a) Acid alpha-glucosidase; (b) Acid beta-glucosidase; (c) glucocerebrosidase; (d) alpha-Galactosidase A; (e) Acid beta-galactosidase; (f) beta- Hexosaminidase A; (g) beta-Hexosaminidase B; (h) Acid sphingomyelinase; (i) Galactocerebrosidase; Q) Acid ceramidase; (k) Arylsulfatase A; (I) alpha-L-lduronidase; (m) lduronate-2-sulfatase; (n) Heparan N-sulfatase; (o) alpha-N-Acetylglucosaminidase; (p) Acetyl-CoA: alpha-glucosaminide N-acetyltransferase; (q) N-Acetylglucosamine-6-sulfate sulfatase; (r) N-Acetylgalactosamine-6-sulfate sulfatase; (s) Acid beta-galactosidase; (t) Arylsulfatase B; (u) beta-Glucuronidase; (v) Acid alpha-mannosidase; (w) Acid beta- mannosidase; (x) Acid alpha-L-fucosidase; (y) Sialidase; and (z) a(pha-N- acetylgalactosaminidase.

33. The bivalent pharmacoperone of any one of claims 27 to 32 which is a conjugate of a first and a second iminosugar.

34. The bivalent pharmacoperone of any one of claims 27 to 33 for the treatment of a disease arising from aberrant enzyme folding and/or processing, for example a proteostatic disease (e.g. Parkinson's disease, Alzheimer's disease or Huntington's disease), LSD, diabetes, emphysema, cancer or cystic fibrosis.

35. The bivalent pharmacoperone of claim 34 for the treatment of an LSD as defined in claim 16.