WO2019211205A1

WO2019211205A1 - Pharmaceutical compositions for use in the treatment of lysosomal diseases

Info

Publication number: WO2019211205A1
Application number: PCT/EP2019/060802
Authority: WO
Inventors: Stéphane DEMOTZ; Julie CHAROLLAIS-THOENIG; Brigitte Rigat; Don Joseph MAHURAN
Original assignee: Dorphan S.A.; The Hospital For Sick Children
Priority date: 2018-04-30
Filing date: 2019-04-26
Publication date: 2019-11-07

Abstract

The present invention relates to pharmaceutical compositions comprising a compound of general formula (I), pharmaceutically acceptable salts thereof and a beta-galactosidase inhibitor. The invention also relates to the use of said pharmaceutical compositions in the treatment and/or prevention of lysosomal storage diseases.

Description

PHARMACEUTICAL COMPOSITIONS FOR USE IN THE TREATMENT OF LYSOSOMAL DISEASES

TECHNICAL FIELD

BACKGROUND OF THE INVENTION

Lysosomal storage diseases constitute a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function, usually as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins or

mucopolysaccharides.

Morquio disease type B, also called mucopolysaccharidosis IV type B (MPSIVB), and GM1 -gangliosidosis are lysosomal storage diseases. They are caused by deficiencies of the beta- galactosidase (Brunetti-Pierri and Scaglia 2008, Caciotti et al. 2011, Sandhoff and Harzer 2013). In the absence of beta-galactosidase activity, keratan sulfate, GMl-gangliosides and

glycoproteins are not properly degraded and accumulate in the lysosomes, causing cell dysfunction and, ultimately, the diseases. In most cases, these two diseases appear early in life and in many cases the patients succumb before reaching adulthood. Morquio disease type B affects mainly the development of peripheral organs, such as skeleton and heart, with a limited involvement of the central nervous system, and GM1 -gangliosidosis mainly affects brain development.

Different therapies have been exploited for the treatment of lysosomal storage diseases. The enzyme replacement therapy has been successfully exploited for the treatment of lysosomal storage diseases affecting mainly peripheral organs readily accessible to protein administered in the circulation. Notable examples of successful enzyme replacement drugs are Fabrazyme for the treatment of Fabry disease, Cerezyme, for the treatment of Gaucher disease and Naglazyme for the treatment of mucopolysaccharidosis VI.

Hematopoietic stem cell transplantation has been successful in severe cases of Hurler syndrome. This medical procedure, however, was not found to alleviate symptoms in other mucopolysaccharidoses and, in addition, constitutes a high-risk medical procedure (Noh and Lee 2014).

Substrate reduction therapy has been exploited to treat Gaucher disease with the drugs miglustat and eliglustat. The therapy consists in limiting the synthesis of the cell constituent, which is no longer degraded due to glucocerebrosidase deficiency. It follows that accumulation of the un-degraded cell constituent is slowed down, delaying onset and severity of the disease. Ceramidase inhibitors were also described as a method to treat various lysosomal storage diseases by impeding synthesis of the substrates that accumulate due to particular lysosomal enzyme deficiencies (WO 2016/210116 Al LYSOSOMAL THERAPEUTICS INC [US]).

The pharmacological chaperone-based therapy is another considered therapeutic strategy that exploits the capacity of certain small molecules to rescue mutated lysosomal enzymes from degradation and to favour their transport to the lysosomes. In 2009, Phase II clinical trials with the experimental drug afegostat, also known as isofagomine, a pharmacological chaperone of glucocerebrosidase, have failed to demonstrate sufficient therapeutic efficacy for the treatment of certain forms of Gaucher disease, and led to the termination of its development (Nash et al 2011). Other glucocerebrosidase inhibitors were developed and evaluated for their potential use for the treatment of Gaucher disease (WO 2004/037373 A2 SCRIPPS RESEARCH INST [US], US 2009/075960 Al MAHURAN DON J [CA] et AL). Van Breemen M. J. et al:“Potential artefacts in proteome analysis of plasma of Gaucher patients due to protease abnormalities” CLINICA CHIMICA ACTA, ELSEVIER BV, AMSTERDAM, NL, vol. 396, no. 1-2, 1 October 2008, pages 26-32, XP024340849, investigated plasma proteome of type I Gaucher disease patients by 2D gel electrophoresis (2DGE) but the latter should be interpreted cautiously given the abnormal high levels of proteases associated with this disorder.

Migalastat, also called l-deoxygalactonojirimycin, a pharmacological chaperone of alpha-galactosidase, was successfully developed for the treatment of Fabry disease. It was also attempted to treat other lysosomal storage diseases, in particular GM1 -gangliosidosis and Morquio disease type B, by the use of beta-galactosidase inhibitors, acting as pharmacological chaperones (WO 2016/174131 Al DORPHAN SA [CH]).

As of today, no curative treatment is available to fight lysosomal storage diseases related to beta-galactosidase enzyme dysfunction, such as GM1 -gangliosidosis and Morquio disease type B. Patients are only offered palliative care. This situation means that there is a significant unmet medical need for an efficient treatment of lysosomal storage diseases related to beta- galactosidase enzyme dysfunction. In particular, an efficient pharmaceutical composition having limited toxic effects and capable to pass the blood brain barrier is still awaited.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising a compound of general formula (I):

and/or pharmaceutically acceptable salts thereof, wherein:

Ri is selected from the group comprising: -0-CH₂-R^a; C6-C10 aryl; C1-C12 alkyl optionally substituted with 1, 2, or 3 substituents independently selected from -CH3 and -NH-C(0)-CH₃; and C2-C6 heterocycloalkyl optionally substituted with 1, 2, or 3 substituents independently selected from -C(0)0R^b and -C(0)-NHR^b;

R2 is C1-C₆ alkyl optionally substituted with 1, 2, or 3 substituents independently selected from -CH3 and C₆-C1₀ aryl;

R3 is H;

R₄ is C1-C6 alkyl optionally substituted with 1, 2, or 3 substituents independently selected from the group comprising: -CH3; C6-C10 aryl optionally substituted with one or more halogen; -C(O)- CH2F; -C(0)H; -NH-CR^CNH2 whereby R^c is =NH; 5-10 membered heteroaryl; -C(0)C=N+=N+; and C6-C10 aryl substituted with -0-C(R^b)3;

or R3 and R₄ together with the nitrogen atom to which they are attached represent pyrrolidine substituted with -C(0)-0-R^b;

R^ais C₆-C1₀ aryl;

R^bis Ci-Ce alkyl;

X is C or S=0; in combination with a beta-galactosidase inhibitor or a pharmacological chaperone or inhibitor of glucocerebrosidase selected from the group consisting of: N-butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C- methyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5-C-alkyl-imino-L-ribitol, N- substituted 5-amino- l-hydroxymethyl-cyclopentanetriols, N-

(dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomcthylidcnc)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and a pharmaceutically acceptable carrier, diluent or excipient; wherein said compound of general formula (I) is selected from the group consisting of:

The invention also provides said pharmaceutical compositions for use as a medicament, in particular for use in the treatment and/or prevention of lysosomal diseases selected from the group comprising GMl-gangliosodosis and mucopolysaccharidosis IVB/Morquio disease type B.

The invention also provides said pharmaceutical compositions for use as a medicament, in particular, for use in the treatment and/or prevention of Gaucher disease.

Furthermore, the invention also encompasses a kit comprising a first container and a second container, wherein the first container contains a first composition comprising (i) the compound of general formula (I) and/or pharmaceutically acceptable salts thereof as defined above; and (ii) a pharmaceutically acceptable carrier, diluent or excipient; and the second container contains a second composition comprising (i) a beta-galactosidase inhibitor or a pharmacological chaperone or inhibitor of glucocerebrosidase selected from the group consisting of:

N-butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C- methyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5-C-alkyl-imino-L-ribitol, N- substituted 5-amino- l-hydroxymethyl-cyclopentanetriols, N-

(dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomethylidene)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and ii) a pharmaceutically acceptable carrier, diluent or excipient; for use in the treatment and/or prevention of lysosomal diseases selected from the group comprising GM1 -gangliosidosis, mucopolysaccharidosis IVB/Morquio disease type B and Gaucher disease, in a subject, and preferably the kit further comprises instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The figure presents the fold enhancement of beta-galactosidase activity in treated cells relative to untreated cells. The compound of formula (b): MDL-28170 (circles) and as control, PD 150606 (squares), an inhibitor of calpain acting through its calmodulin domain, which formula is not covered by the general formula (I).

Figure 2. The figure presents the fold enhancement of beta-galactosidase activity in treated patient cells relative to untreated cells. Panel A: CA-074 Me alone (triangles and continuous line) and CA-074 alone (circles and dotted line); Panel B: CA-074 Me in the presence of N-nonyl-deoxygalactonojirimycin at 1 mM (triangles and continuous line) and CA- 074 in the presence of N-nonyl-deoxygalactonojirimycin at 1 mM (circles and dotted line).

Figure 3. Beta-galactosidase maturation in 5a-C-pentyl 4-epi-isofagomine and compound (b): MDL-28l70-treated GM1 -gangliosidosis patient cells. Zrh #4 fibroblasts (from a GM1- gangliosidosis patient bearing the p.G76E/p.R20lH mutations) were treated for 5 days with the indicated compounds. 5a-C-pentyl 4-epi-isofagomine was used at 2 mM and MDL-28170 at 50 mM. Cell lysates were then analysed by Western blotting using an anti-beta-galactosidase antibody and an anti-GAPDH (glyceraldehyde 3 -phosphate dehydrogenase) antibody. The position of molecular weight markers is indicated on the left in kDa. Figure 4. shows the synergic expression of mature beta-galactosidase induced by E64d and N-butyl-deoxygalactonojirimycin combination. GM05335 (upper two panels;

p.Q255H/p.K578R GLB1 mutations) and Zurich #4 (lower two panels; p.G76E/p.R20lH GLB1 mutations) fibroblasts from GM1 -gangliosidosis patients were treated with E64d at 10 mM (E), N-butyl-deoxygalactonojirimycin at 250 mM, the two compounds combined (E + L) or left untreated (U) for 5 and 7 days, respectively. The cells were then washed with phosphate buffer saline, lysed in deionized water, lyophilized and suspended in sample buffer. Samples (80 mg) were electrophoresed, transferred onto nitrocellulose membrane, probed with anti-beta- galactosidase (upper panels of each cell line) and anti-glyceraldehyde 3 -phosphate

dehydrogenase (lower panels of each cell line) antibodies, and finally revealed by enhanced chemiluminescence. C: lysate from untreated GM02456 fibroblasts (100 mg), from a healthy donor. MW: molecular weight markers. The size of the markers are in kDa.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising a compound of general formula (I)

and/or pharmaceutically acceptable salts thereof, wherein

R2 is C1-C6 alkyl optionally substituted with 1, 2, or 3 substituents independently selected from -CH3 and C6-C10 aryl;

R3 is H;

R₄ is C1-C6 alkyl optionally substituted with 1, 2, or 3 substituents independently selected from the group comprising: -CH3; C6-C10 aryl optionally substituted with one or more halogen; -C(O)- CH2F; -C(0)H; -NH-CR^CNH₂ whereby R^c is =NH; 5-10 membered heteroaryl; -C(0)C=N+=N+; and C6-C10 aryl substituted with -0-C(R^b)₃;

R^ais C₆-C1₀ aryl;

R^bis Ci-Ce alkyl;

X is C or S=0; in combination with a beta-galactosidase inhibitor and/or a pharmacological chaperone or inhibitor of glucocerebrosidase selected from the group consisting of:

(dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomcthylidcnc)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and a pharmaceutically acceptable carrier, diluent or excipient; wherein, said compound of general formula (I) is selected from the group consisting of:

(g), and

Preferably, a beta-galactosidase inhibitor is selected from the group consisting of N- butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C-methyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5-C-alkyl-imino-L-ribitol, N-substituted 5- amino- 1 -hydroxymethyl-cyclopentanetriols, N-(dansylamino)hexylaminocarbonylpentyl- 1,5- dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi-beta-valienamine, 5N,6S-(N'- butyliminomethylidene)-6-thio-l-deoxygalactonojirimycin, N-nonyl-deoxygalactonojirimycin; and a pharmaceutically acceptable carrier, diluent or excipient.

More preferably, the beta-galactosidase inhibitor is N-butyl-deoxygalactonojirimycin or 5a-C- pentyl-4-epi-isofagomine. Even more preferably, the beta-galactosidase inhibitor is N-butyl- deoxy galactonoj irimycin.

Preferably, the pharmacological chaperone or inhibitor of glucocerebrosidase is selected from the group consisting of N-butyldeoxynojirimycin, ambroxol and isofagomine; together with a pharmaceutically acceptable carrier, diluent or excipient. More preferably, the pharmacological chaperone or inhibitor of glucocerebrosidase is N-butyldeoxynojirimycin.

The following definitions are supplied in order to facilitate the understanding of the present invention.

As used herein, the term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

As used herein, the singular form "a”, "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the term“chaperone” refers to small molecular weight compounds which bind to proteins to support their folding and/or stabilization in their native conformation, or favour them to adopt or restore a conformation resembling the physiologically native one.

According to the present invention, the terms“pharmacological chaperone” also refers to “inhibitors of glucocerebrosidase” selected from the group consisting of N- butyldeoxynojirimycin, ambroxol and isofagomine. In other words, in the present disclosure the terms“pharmacological chaperone” stand for or is equivalent to“inhibitors of

glucocerebrosidase”. b-Glucocerebrosidase (also called acid b-glucosidase, D-glucosyl-N-acylsphingosine

glucohydrolase, or GCase) is an enzyme with glucosylceramidase activity that is needed to cleave, by hydrolysis, the beta-glucosidic linkage of the chemical glucocerebroside, an intermediate in glycolipid metabolism that is abundant in cell membranes (particularly skin cells). It is localized in the lysosome, where it remains associated with the lysosomal membrane. Glucocerebrosidase (GC) is the lysosomal enzyme deficient in Gaucher disease. Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the

glucocerebrosidase gene.

As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other

embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

The term“an effective amount” refers to an amount necessary to obtain a physiological effect. The physiological effect may be achieved by one application dose or by repeated applications. The dosage administered may, of course, vary depending upon known factors, such as the physiological characteristics of the particular composition; the age, health and weight of the subject; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired and can be adjusted by a person skilled in the art.

It is usually accepted that“synergy” occurs when the combined action of two or more agents is greater than the sum of their individual effects. In other words, synergy is said to occur when the combined action of two or more agents is greater than could have been predicted based on the performance of the agents when used alone.

The term "carrier" refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. According to the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.

Possible carrier substances for parenteral administration are e.g. sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or

polyoxyethylene/polyoxy- propylene copolymers.

The term "excipient" when used herein is intended to indicate all substances which may be present in a composition described herein and which are not active ingredients such as, e.g., carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.

As used herein, the term“pharmaceutical composition” refers to a composition wherein compounds of general formula (I), pharmaceutically acceptable salts thereof, and a beta- galactosidase inhibitor, also often referred to as a pharmacologic chaperone, are administered in combination or co-administered, either concomitantly or subsequently.

The following paragraphs provide definitions of the various chemical moieties that make up the compounds according to the invention and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

“C1-C12 alkyl” refers to monovalent straight-chained and branched alkyl groups having 1 to 12 carbon atoms, such as C1-C₃ alkyl, C1-C4 alkyl or C1-C₆ alkyl. Examples of straight chain alkyl groups include, but are not limited to, those with from 1 to 12 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl groups, n-heptyl, n-octyl, n-nonyl and n-decyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec- butyl, tert-butyl, isopentyl, and 2,2-dimethylpropyl groups. Alkyl groups may be substituted or unsubstituted. Representative substituted alkyl groups may be substituted one or more times with for example, methyl, amino, oxo, hydroxy, cyano, carboxy, nitro, thio, alkoxy, F, Cl, Br, I, cycloalkyl, aryl, heterocyclyl and heteroaryl groups.

“C2-C₆ heterocycloalkyl” refers to non-aromatic ring groups containing 2 or more carbon atoms, and one or more heteroatom such as, but not limited to, N, O and S. Such examples are but not limited to, heterocyclopropyl, heterocyclobutyl, cyclopentyl, cyclohexyl. In some embodiments, the heterocycloalkyl contains 1, 2 or 3 heteroatoms.

The term“aryl” refers to cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups include monocyclic, bicyclic and polycyclic ring systems. Thus, aryl groups include, but are not limited to, CY. aryl, (such as phenyl, benzyl, tolyl, xylyl, benzyliden, benzoyl), C₆-Cis aryl, and C6-C10 aryl (such as azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, naphthyl groups). The term "aryl" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (such as naphtyl, indanyl, tetrahydronaphthyl, and the like). Groups such as phenyl and naphtyl are preferred. Aryl groups may be substituted or unsubstituted. Representative substituted aryl groups may be mono-substituted or substituted more than once. Aryl groups may be fused to 5-10 membered heteroaryl containing one or more heteroatom such as, but not limited to, N, O and S.

According to the invention, the groups identified above may be substituted or unsubstituted. In general, the term "substituted" refers to a functional group, as defined below, in which one or more bonds to a hydrogen atom are replaced by a bond to a non-hydrogen atom. Substituted groups also include groups, in which one or more bonds to a hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. In some embodiments, substituted groups have 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include, but are not limited to, halogens (i.e. F, Cl, Br and I), hydroxyls, alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);

carboxyls; esters; ethers; urethanes; oximes; hydroxylamines; alkoxyamines; thiols; sulfides such as alkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl and heterocyclylalkyl sulfide groups;

sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides;

hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates;

isothiocyanates; cyanates; thiocyanates; imines; nitriles; alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocyclyl, heterocyclylalkyl and cycloalkyl groups.

The invention also relates to salts of the compounds of general formula (I), pure or mixed, stereoisomers, hydrates, solvates, solid forms, chemical modified compounds, and/or mixtures thereof. Preferably, these salts are pharmaceutically acceptable that are produced from acidic inorganic or organic compounds. As used herein, the phrase“pharmaceutically acceptable salt” refers to a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable.

Compounds of general formula (I) and pharmaceutically acceptable salts thereof, are stable in several solid forms. All the solid forms of the compounds, according to the invention, include amorphous, polymorphous, mono- and polycrystalline forms. They can also exist in non-solvated or solvated form, for example with pharmaceutically acceptable solvents such as water

(hydrates) or ethanol.

The compound of general formula (I) is selected from the group consisting of:

In some embodiments, the compound of general formula (I) is

In a preferred embodiment, the compound of general formula (I) is:

In yet another preferred embodiment, the compound of general formula (I) is:

Compounds of general formula (I), such as (a), (b), (c), (d), (e), (f), (g) and (h) are cathepsin B and/or L inhibitors as set forth in Table 1 below.

Table 1:

As shown in the examples, the beta-galactosidase activity measured in GM1- gangliosidosis patient cells is enhanced in presence of increasing concentrations of different compounds of general formula (I), such as (a), (b), (e), (g) and (d) that are specific cathepsin B and/or L inhibitors (Example 1, Tables 2, 3 and 4).

The beta-galactosidase activity is enhanced in presence of increasing concentrations of compound (b) but not in presence of PD 150606, a selective calpain inhibitor (Example 2, Figure 1). Similarly, BML-244, a cathepsin K inhibitor, does not enhance the beta-galactosidase activity (Example 3, Table 5). The chemical formulae of PD150606 and BML-244 are not covered by the general formula (I).

Furthermore, the beta-galactosidase activity is enhanced in mucopolysaccharidosis IVB patient cells in presence of increasing concentrations of compound (a) or (b) (Example 4, Tables 6 and

V).

Surprisingly, pharmaceutical compositions of the invention comprising a compound of general formula (I), and/or pharmaceutically acceptable salts thereof, and a beta-galactosidase inhibitor selected from the group comprising N-butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C- methyl 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5- C-alkyl-imino-L-ribitol, N-substituted 5-amino- l-hydroxymethyl-cyclopentanetriols, N- (dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomethylidene)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin and derivatives thereof, have been found to enhance in a synergistic manner the beta-galactosidase activity in GM1 -gangliosidosis and

mucopolysaccharidosis IVB patient cells (see Tables 8 to 13).

In some embodiments, the beta-galactosidase inhibitor is N-butyl-deoxygalactonojirimycin, or 5a-C-methyl 4-epi-isofagomine, or 5a-C-pentyl 4-epi-isofagomine, or N-nonyl- deoxy galactonoj irimycin.

In some other embodiments, the present invention relates to pharmaceutical compositions comprising compound (a) and N-butyl-deoxy galactonoj irimycin, or compound (b) and N-butyl- deoxygalactonoj irimycin, or compound (c) and N-butyl-deoxygalactonojirimycin, or compound (d) and N-butyl-deoxygalactonojirimycin, or compound (e) and N-butyl- deoxygalactonoj irimycin, or compound (f) and N-butyl-deoxy galactonoj irimycin, or compound (g) and N-butyl-deoxygalactonojirimycin, or compound (h) and N-butyl- deoxygalactonoj irimycin, or compound (a) and 5a-C-pentyl 4-epi-isofagomine, or compound (b) and 5a-C-pentyl 4-epi-isofagomine, or compound (c) and 5a-C-pentyl 4-epi-isofagomine, or compound (d) and 5a-C-pentyl 4-epi-isofagomine, or compound (e) and 5a-C-pentyl 4-epi- isofagomine, or compound (f) and 5a-C-pentyl 4-epi-isofagomine, or compound (g) and 5a-C- pentyl 4-epi-isofagomine, or compound (h) and 5a-C-pentyl 4-epi-isofagomine.

As shown in Example 5, a synergistic enhancement of the beta-galactosidase activity of 21 -fold has been observed in presence of a pharmaceutical composition comprising compound (b) and 5a-C-methyl 4-epi-isofagomine. In contrast, the beta-galactosidase activity is enhanced by 6.6- fold in presence of 5a-C-methyl 4-epi-isofagomine alone, or by 1.5-fold in presence of compound (b) alone (Table 8).

Furthermore, a synergistic enhancement of the beta-galactosidase activity of 21 -fold has been observed in presence of a pharmaceutical composition comprising compound (b) and N-butyl- deoxygalactonoj irimycin (see Example 6, Table 9). A synergy was also observed in presence of a pharmaceutical composition comprising compound (c) and N-nonyl-deoxygalactonojirimycin (see Example 6, Figure 2).

Besides, a synergistic enhancement of the beta-galactosidase activity of 17-, 19-, 29- and 16-fold has been observed with the GM1 -gangliosidosis patient cell lines GM05335, Zrh #2, Zrh #4 and Zrh #7 in presence of a pharmaceutical composition comprising compound (a) and N-butyl- deoxygalactonojirimycin, respectively (see Example 7, Table 14).

In addition, it has been observed that pharmaceutical compositions of the present invention increase the amount of mature beta-galactosidase protein. As shown on Figure 3, a combination of compound (b) and 5a-C-pentyl 4-epi-isofagomine further increases the abundance of the mature form of beta-galactosidase, while leaving the precursor unchanged (Example 8, Figure 3).

Further, it has been observed that pharmaceutical compositions of the present invention increase the amount of mature beta-galactosidase protein. As shown on Figure 4, a combination of compound (a) and N-butyl-deoxygalactonojirimycin further increases the abundance of the mature form of beta-galactosidase, while the amount of the precursor is not substantially modified (see Example 8, Figure 4).

The present invention further relates to pharmaceutical compositions comprising a compound of general formula (I) as defined above, and/or pharmaceutically acceptable salts thereof, in combination with a beta-galactosidase inhibitor or a pharmacological chaperone or inhibitor of glucocerebrosidase selected from the group consisting of:

N-butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C- methyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(F)-ribitol, 5-C-alkyl-imino-F-ribitol, N- substituted 5-amino- l-hydroxymethyl-cyclopentanetriols, N-

(dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomcthylidcnc)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and a pharmaceutically acceptable carrier, diluent or excipient, for use as a medicament.

In particular, compounds of general formula (I) are selected from the group consisting of compounds (a), (b), (c), (d), (e), (f), (g), (h).

More preferably, compounds of general formula (I) are selected from the group consisting of compounds (a), (b), (c), (d), (e), (f), (g), (h) and the beta-galactosidase inhibitor is N-butyl- deoxygalactonojirimycin or 5a-C-pentyl 4-epi-isofagomine.

In another embodiment of the invention, compounds of general formula (I) are selected from the group consisting of compounds (a), (b), (c), (d), (e), (f), (g), (h) and the pharmacological chaperone or inhibitor of glucocerebrosidase is selected from the group consisting of N- butyldeoxynojirimycin, ambroxol and isofagomine.

The present invention also relates to pharmaceutical compositions comprising a compound of general formula (I) as defined above, and/or pharmaceutically acceptable salts thereof, and a beta-galactosidase inhibitor selected from the group comprising N-butyl- deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C-methyl 4-epi- isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5-C-alkyl-imino-L-ribitol, N-substituted 5- amino- 1 -hydroxymethyl-cyclopentanetriols, N-(dansylamino)hexylaminocarbonylpentyl- 1,5- dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi-beta-valienamine, 5N,6S-(N'- butyliminomethylidene)-6-thio-l-deoxygalactonojirimycin, N-nonyl-deoxygalactonojirimycin and derivatives thereof, and a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment and/or prevention of lysosomal diseases selected from the group comprising GM1- gangliosodosis, and mucopolysaccharidosis IVB/Morquio disease type B.

More preferably, compounds of general formula (I) are selected from the group comprising compounds (a), (b), (c), (d), (e), (f), (g), (h) and the beta-galactosidase inhibitor N-butyl- deoxygalactonojirimycin or 5a-C-pentyl 4-epi-isofagomine.

In another embodiment, the invention also relates to pharmaceutical compositions comprising a compound of general formula (I) as defined above, and/or pharmaceutically acceptable salts thereof, and a pharmacological chaperone or inhibitor of glucocerebrosidase selected from the group consisting of N-butyldeoxynojirimycin, ambroxol and isofagomine, together with a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment and/or prevention of Gaucher disease.

As used herein,“N-butyldeoxynojirimycin” is a compound of formula (2R,3R,4R,5S)-l-butyl-2- (hydroxymethyl)piperidine-3,4,5-triol.

As used herein,“ambroxol” is a compound of formula 4-[(2-amino-3,5- dibromophenyl)methylamino]cyclohexan- 1 -ol.

As used herein,“isofagomine” is a compound of formula (3R,4R,5R)-5- (hydroxymethyl)piperidine-3,4-diol. More preferably, compounds of general formula (I) are selected from the group consisting of compounds (a), (b), (c), (d), (e), (f), (g), (h) and the pharmacological chaperone or inhibitor of glucocerebrosidase is N-butyldeoxynojirimycin.

As shown in the Example 9, a pharmaceutical composition comprising compound (b) and isofagomine induces recovery of the beta-galactosidase activity by 3.1 -fold (Table 15).

The pharmaceutical composition of the invention is formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. The excipient of the composition can be any pharmaceutically acceptable excipient, including specific carriers able to target specific cells or tissues. As stated earlier, possible pharmaceutical compositions include those suitable for oral, rectal, topical, transdermal, buccal, sublingual, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. For these formulations, conventional excipients can be used according to techniques well known by those skilled in the art. The compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions, which can optionally be prepared immediately before use from solid or lyophilized form. For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations, such as syrups, elixirs, and concentrated drops. Non-toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents, which impart cohesive qualities to powdered materials, are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablets. Disintegrants include starches, clays, celluloses, algins, gums and cross-linked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion of the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant and compounds, such as talc or stearic acid are most commonly used as lubricants. For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide. For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols

(carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate. In a preferred embodiment, the pharmaceutical composition of the invention is suitable for parenteral administration.

Pharmaceutical composition according to the invention may be formulated to substantially release the active drug immediately upon administration or at any predetermined time or a time period after administration.

In a particular embodiment, the pharmaceutical composition according to the invention comprises 0.1 mg to 5 g of the compound of the invention. Preferably, pharmaceutical composition according to the invention comprises 1 mg to 2 g of the compound of the invention. Even more preferably, pharmaceutical composition according to the invention comprises 10 mg to 1 g of the compound of the invention.

In general, the pharmaceutical composition may be administered by parenteral injection (e. g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e. g. by aspiration or nebulization), orally, sublingually, or topically, through the skin or through the rectum.

As specified, in another embodiment, the pharmaceutical composition of the present invention is in a form suitable for oral administration. For example, the composition may be in the form of tablets, ordinary capsules, gelatin capsules or syrup for oral administration. These gelatin capsule, ordinary capsule and tablet forms can contain excipients conventionally used in pharmaceutical formulation, such as adjuvants or binders like starches, gums and gelatin, adjuvants like calcium phosphate, disintegrating agents like cornstarch or alginic acids, a lubricant like magnesium stearate, sweeteners or flavourings. Solutions or suspensions can be prepared in aqueous or non-aqueous media by the addition of pharmacologically compatible solvents. These include glycols, polyglycols, propylene glycols, polyglycol ether, DMSO and ethanol.

Pharmaceutical compositions according to the invention can comprise one or more compounds of the invention in association with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above. A further object of the present invention relates to a kit comprising a first container and a second container, wherein the first container contains a first composition comprising (i) a compound of general formula (I):

and/or pharmaceutically acceptable salts thereof, wherein said compound of general formula (I) is selected from the group consisting of:

; and (ii) a pharmaceutically acceptable carrier, diluent or excipient; and the second container contains a second composition comprising (i) a beta-galactosidase inhibitor or a pharmacological chaperone or inhibitor of glucocerebrosidase selected from the group consisting of:

N-butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C- methyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5-C-alkyl-imino-L-ribitol, N- substituted 5-amino- l-hydroxymethyl-cyclopentanetriols, N- (dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomethylidene)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and ii) a pharmaceutically acceptable carrier, diluent or excipient;

for use in the treatment and/or prevention of lysosomal diseases selected from the group comprising GM1 -gangliosidosis, mucopolysaccharidosis IVB/Morquio disease type B and Gaucher disease, in a subject, and preferably or optionally the kit further comprises instructions for use.

Preferably, the compound of general formula (I) of said kit is:

In one embodiment, the beta-galactosidase inhibitor of said kit is N-butyl- deoxygalactonojirimycin or 5a-C-pentyl-4-epi-isofagomine.

According to another embodiment, the pharmacological chaperone or inhibitor of

glucocerebrosidase of said kit is selected from the group consisting of N-butyldeoxynojirimycin, ambroxol and isofagomine. Administration of the first composition and/or the second composition may be oral, intravenous, sub-cutaneous, intra-muscular or intra-peritoneal.

Preferably, the first composition and/or the second composition may be administered

concomitantly, or simultaneously, separately or sequentially depending on the intended use envisioned by a skilled in the art.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following

Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Example 1: Enhancement of beta-galactosidase activity in GM1 -gangliosidosis patient cells induced by cathepsin B and/or cathepsin L inhibitors of general formula (I)

Different cell lines from GM1 -gangliosidosis or mucopolysaccharidosis IVB patients were treated for 5 days with the indicated concentrations of cathepsin B and/or cathepsin L inhibitors, as disclosed in the tables below.

Cathepsin B and/or cathepsin L inhibitors that were used in the experiment are E64d, MDL- 28170, leupeptin, Z-FY(tBu)-DMK, and Z-FA-FMK. Nafamostat, which inhibits serine proteases and kallikrein, is not a compound of general formula (I).

The cells were then washed with phosphate buffer saline and lysed in citrate buffer 50 mM pH 4.3 with 1% Triton X-100. Beta-galactosidase activity assays were then conducted using 4- methylumbelliferyl beta-D-galactopyranoside as a substrate. The results present the fold increase of beta-galactosidase activity relative to the activity in untreated cells. Each measure was conducted in quadruplicates. The standard deviations were <15% of the mean.

Table 2: beta-galactosidase activity measured in Zrh #3 cell line (p.R20lH/p.H28lY)

The beta-galactosidase activity is enhanced in presence of increasing concentrations of E64d. Table 3: beta-galactosidase activity measured in Zrh #4 (p.G76E/p.R20lH) cell line

Beta-galactosidase activity is enhanced in presence of increasing concentrations of different compounds of general formula (I). In contrast, increasing concentrations of nafamostat, an inhibitor of serine proteases and kallikrein, which is not a compound of general formula (I), does not enhance beta-galactosidase activity.

Table 4: beta-galactosidase activity measured in Zrh #7 cell line (p. R20lC/p.H28lY)

Beta-galactosidase activity is enhanced in presence of increasing concentrations of different compounds of general formula (I). In contrast, increasing amounts of nafamostat (inhibitor of serine proteases and kallikrein), which is not a compound of general formula (I), does not enhance beta-galactosidase activity.

Example 2: Enhancement beta-galactosidase activity in GM1 -gangliosidosis patient cells induced by the calpain and cathepsin B inhibitor MDL-28170

14771 cells (a fibroblast line from a GM1 -gangliosidosis patient bearing the p.R20lH/IVSl4- 2A>G mutations in the GLB1 gene) were treated with graded concentrations of the calpain and cathepsin B inhibitor MDL-28170 (N-benzyloxycarbonylvalylphenylalaninal) or the uncompetitive calmodulin domain-specific calpain inhibitor PD 150606 [(2Z)-3-(4-iodophenyl)- 2-mercapto-2-propenoic acid, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid]. After 5 days of culture, the cells were washed with phosphate buffer saline and lysed in sodium citrate 50 mM pH 4.3 containing 1% Triton X-100. Beta-galactosidase activity was then determined by incubating lysate samples in the presence of the fluorogenic beta-galactosidase substrate 4- methylumbelliferyl beta-D-galactopyranoside at 1 mM. After 2 hours at 37°C, the enzymatic reactions were stopped by the addition of sodium carbonate 0.4 M pH 11.6. Fluorescence, as readout of beta-galactosidase activity, was measured at 445 nm using 365 nm as the excitation wavelength. Figure 1 presents the fold enhancement of beta-galactosidase activity in treated cells relative to untreated cells. MDL-28170 (circles) and PD 150606 (squares).

Example 3: Beta-galactosidase activity in GM1 -gangliosidosis patient cells in presence of a cathepsin K inhibitor (BML-244)

Table 5: beta-galactosidase activity in Zrh #2 (p.H28lY/-) and GM05335 (p.Q255H/p.K578R; from the Coriell Institute, Camden, NJ) cell lines treated with various concentrations of BML- 244

Beta-galactosidase activity is not enhanced in presence of increasing concentrations of BML- 244, a cathepsin K inhibitor, which is not a compound of general formula (I), in two different GM1 -gangliosidosis patient cell lines, the Zrh #2 and GM05335 cell lines.

Example 4: Enhancement of beta-galactosidase activity in mucopolysaccharidosis IVB patient cells induced by cathepsin B and/or cathepsin L inhibitors of general formula (I)

Table 6: beta-galactosidase activity in the mucopolysaccharidosis IVB GM01602

(p.W273L/p.R482H; from the Coriell Institute, Camden, NJ) patient cell line treated with compound (a):E64d or (b):MDL28l70

Beta-galactosidase activity is enhanced in presence of increasing concentrations of different compounds of general formula (I).

Table 7: beta-galactosidase activity in the GMl-gangliosidosis/mucopolysaccharidosis IVB

GM03251 (p.W273L/p.W509C; from the Coriell Institute, Camden, NJ) patient cell line treated with compound (a): E64d

Beta-galactosidase activity is enhanced in presence of increasing concentrations of E64d.

Example 5: Synergistic enhancement of beta-galactosidase activity in GM1 -gangliosidosis patient cells induced by combinations of compounds of general formula (I) and beta- galactosidase inhibitors

The following patient cell lines were used: Zrh #2 fibroblasts (p.H28lY/-); GM05335 fibroblasts (p.Q255H/p.K578R; from the Coriell Institute, Camden, NJ); Zrh #7 fibroblasts

(p.R20lC/p.H28lY) and Zrh #4 fibroblasts (p.G76E/p.R20lH).

The cell lines were treated for 5 days with the indicated compounds in the tables below. The cells were then washed with phosphate buffer saline and lysed in citrate buffer 50 mM pH 4.3 with 1% Triton X-100. Beta-galactosidase activity assays were then conducted using 4- methylumbelliferyl beta-D-galactopyranoside as a substrate. The results present the fold increase of beta-galactosidase activity relative to the activity in untreated cells. Each measure was conducted in quadruplicates. The standard deviations were <15% of the mean.

Combination of (b): MDL-28170 and 5a-C-methyl 4-epi-isofagomine

5a-C-methyl 4-epi-isofagomine was used at 100 mM or 25 mM and MDL-28170 at 10 mM.

Table 8: beta-galactosidase activity in GM1 -gangliosidosis Zrh #2 (p.H28lY/-) patient cells treated with compound (b): MDL-28170 and/or 5a-C-methyl 4-epi-isofagomine

MDL-28170 and 5a-C-methyl 4-epi-isofagomine combinations synergistically enhance beta- galactosidase activity in Zrh #2 patient cells.

Combination of (b): MDL-28170 and N-butyl-deoxygalactonojirimycin

N-butyl-deoxygalactonojirimycin and MDL-28170 were used at 250 and 10 mM, respectively.

Table 9: beta-galactosidase activity in GM1 -gangliosidosis GM05335 (p.Q255H/p.K578R; from the Coriell Institute, Camden, NJ) patient cells treated with compound (b): MDL-28170 and/or N -buty l-deoxy galactonoj irimy cin

MDL-28170 and N-buty l-deoxy galactonoj irimy cin synergistically enhance beta-galactosidase activity in GM05335 patient cells.

Combination of (a): E64d and 5a-C-pentyl 4-epi-isofagomine

5a-C-pentyl 4-epi-isofagomine was used at 2 mM and E64d at various concentrations specified in Table 10 for each patient cell line.

Table 10: beta-galactosidase activity in various GM1 -gangliosidosis patient cells treated with compound (a): E64d and/or 5a-C-pentyl 4-epi-isofagomine

E64d and 5a-C-pentyl 4-epi-isofagomine synergistically enhance beta-galactosidase activity in various GM1 -gangliosidosis patient cells.

Combination of (b): MDL-28170 and 5a-C-pentyl 4-epi-isofagomine

MDL-28170 and 5a-C-pentyl 4-epi-isofagomine were used at 50 mM and 2 mM, respectively.

Table 11: beta-galactosidase activity in various GM1 -gangliosidosis patient cells treated with compound (b): MDL-28170 and/or 5a-C-pentyl 4-epi-isofagomine

MDL-28170 and 5a-C- pentyl 4-epi-isofagomine synergistically enhance beta-galactosidase activity in various GM1 -gangliosidosis patient cells. Combination of (d): Z-Phe-Ala fluoromethyl ketone and 5a-C-pentyl 4-epi-isofagomine

Z-Phe-Ala fluoromethyl ketone and 5a-C-pentyl 4-epi-isofagomine were used at 4 mM and 2 mM, respectively.

Table 12: beta-galactosidase activity in GM1 -gangliosidosis Zrh #7 (p.R20lC/p.H28lY) patient cells treated with compound (d): Z-Phe-Ala fluoromethyl ketone and/or 5a-C-pentyl 4-epi- isofagomine

Z-Phe-Ala fluoromethyl ketone and 5a-C-pentyl 4-epi-isofagomine, synergistically enhance beta-galactosidase activity in Zrh #7 patient cells.

Combination of (f): l-napthalenesulfonyl-Ile-Trp-aldehyde and 5a-C-pentyl 4-epi-isofagomine l-napthalenesulfonyl-Ile-Trp-aldehyde and 5a-C-pentyl 4-epi-isofagomine were used at 10 mM and 2 mM, respectively.

Table 13: beta-galactosidase activity in GM1 -gangliosidosis GM05335 (p.Q255H/p.K578R; from the Coriell Institute, Camden, NJ) patient cells treated with compound (f): 1- napthalenesulfonyl-Ile-Trp-aldehyde and/or 5a-C-pentyl 4-epi-isofagomine

l-napthalenesulfonyl-Ile-Trp-aldehyde and 5a-C-pentyl 4-epi-isofagomine synergistically enhance beta-galactosidase activity in GM05335 patient cells.

Example 6: Synergistic enhancement of beta-galactosidase activity in GM1 -gangliosidosis patient cells by combination of N-nonyl-deoxygalactonojirimycin and the cell-permeable cathepsin B-specific inhibitor CA-074 Me

14771 cells (a fibroblast line from a GM1 -gangliosidosis patient bearing the p.R20lH/IVSl4- 2A>G mutations in the GLB1 gene) were treated with graded concentrations of the cell- permeable and cathepsin B-specific CA-074 Me compound [(L-3-trans- (propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester] or its cell- impermeable derivative CA-074 [(L-3-trans-(propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl- L-proline] in the presence or absence of a constant concentration of the beta-galactosidase pharmacological chaperone compound N-nonyl-deoxygalactonojirimycin at 1 mM. After 5 days of culture, the cells were washed with phosphate buffer saline and lysed in sodium citrate 50 mM pH 4.3 containing 1% Triton X-100. Beta-galactosidase activity was then determined by incubating lysate samples in the presence of the fluorogenic beta-galactosidase substrate 4- methylumbelliferyl beta-D-galactopyranoside at 1 mM. After 2 hours at 37°C, the enzymatic reactions were stopped by the addition of sodium carbonate 0.4 M pH 11.6. Fluorescence, as readout of beta-galactosidase activity, was measured at 445 nm using 365 nm as the excitation wavelength.

Figure 2 presents the fold enhancement of beta-galactosidase activity in treated patient cells relative to untreated cells. Panel A: CA-074 Me alone (triangles and continuous line) and CA- 074 alone (circles and dotted line); Panel B: CA-074 Me in the presence of N-nonyl- deoxygalactonojirimycin at 1 mM (triangles and continuous line) and CA-074 in the presence of N-nonyl-deoxygalactonojirimycin at 1 mM (circles and dotted line).

Example 7: Enhancement of beta-galactosidase activity in GM1 -gangliosidosis patient cells induced by E64d and N-butyl deoxygalactonojirimycin combination GM1 -gangliosidosis GM05335 (p.Q255H/p.K578R), Zrh #2 (p.H28lY/-), Zrh #4

(p.G76E/p.R20lH) and Zhr #7 (p.R20lC/p.H28lY) patient cells were treated for 5 days with the indicated compounds. E64d and N-butyl-deoxygalactonojirimycin were used at 10 and 250 mM, respectively. The cells were then washed with phosphate buffer saline and lysed in citrate buffer 50 mM pH 4.3 with 1% Triton X-100. Beta-galactosidase activity assays were then conducted using 4-methylumbelliferyl beta-D-galactopyranoside as a substrate. The results present the fold increase of beta-galactosidase activity relative to the activity in untreated cells. Each measure was conducted in quadruplicates. The standard deviations were <15% of the mean.

Table 14: beta-galactosidase activity measured in GM05335 (p.Q255H/p.K578R), Zrh #2 (p.H28lY/-), Zrh #4 (p.G76E/p.R20lH) and Zrh #7 (p.R20lC/p.H28lY) patient cells

E64d and N-butyl deoxygalactonojirimycin synergistically enhance beta-galactosidase activity in GM05335 (p.Q255H/p.K578R), Zrh #2 (p.H28lY/-), Zrh #4 (p.G76E/p.R20lH) and Zrh #7 (p.R20lC/p.H28lY) patient cells.

Example 8: Beta-galactosidase maturation in 5a-C-pentyl 4-epi-isofagomine and MDL-28170 and in N-butyl-deoxygalactonojirimycin and E64d treated GM1 -gangliosidosis patient cells

The effect of a compound of general formula (I) on the maturation of beta-galactosidase was explored in Zrh #4 fibroblasts (from a GM1 -gangliosidosis patient bearing the p.G76E/p.R20lH mutations). Cells were treated for 5 days with the indicated compounds: 5a-C-pentyl 4-epi- isofagomine was used at 2 mM and MDL-28170 at 50 mM. Cell lysates were then analysed by Western blotting using an anti-beta-galactosidase antibody and an anti-GAPDH (glyceraldehyde 3 -phosphate dehydrogenase) antibody. The position of molecular weight markers is indicated on the left in kDa (Figure 3).

It is assumed that maturation of the beta-galactosidase (i.e. appearance of a low molecular weight form of beta-galactosidase, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting) reflects transport of the enzyme to the lysosomes. Whereas the beta-galactosidase inhibitor 5a-C-pentyl 4-epi-isofagomine enhances maturation of the deficient beta-galactosidase in patient cells, MDL-28170 increases the abundance of the precursor form of beta-galactosidase.

Combination of MDL-28170 and 5a-C-pentyl 4-epi-isofagomine_further increases the abundance of the mature form of beta-galactosidase, while leaving the precursor unchanged (Figure 3).

Furthermore, the effect of another compound of general formula (I) on the maturation of beta- galactosidase was also explored in GM05335 fibroblasts (from a GM1 -gangliosidosis patient bearing the p.Q255H/p.K578R mutations) and Zrh #4 fibroblasts (from a GM1 -gangliosidosis patient bearing the p.G76E/p.R20lH mutations). GM05335 (expressing the p.Q255H/p.K578R GLB1 mutations) and Zurich #4 cells (expressing the p.G76E/p.R20lH GLB1 mutations) fibroblasts from GM1 -gangliosidosis patients, were treated for 5 and 7 days, respectively, with the indicated compounds: N-butyl-deoxygalactonojirimycin was used at 250 mM and E64d at 10 mM. The cells were then washed with phosphate buffer saline and frozen at -80°C in deionized water. The resulting cell lysate was lyophilized and then resuspended in sample buffer. Cell lysates were then analyzed by Western blotting using an anti-beta-galactosidase antibody and an anti-GAPDH (glyceraldehyde 3 -phosphate dehydrogenase) antibody. The position of molecular weight markers is indicated on the right in kDa (Figure 4).

These analyses revealed that in untreated cells, the precursor and mature forms of beta- galactosidase were detected in low quantities. Treatment of the cells with E64d increased expression of beta-galactosidase to a limited extent. By contrast, treatment of the cells with N- butyl deoxygalactonojirimycin increased the level of the mature form of beta-galactosidase, in particular in GM05335 cells. Remarkably, when E64d and N-butyl deoxygalactonojirimycin were combined to treat the cells, expression of the mature form of beta-galactosidase was substantially increased. These results paralleled the results of the enzymatic activity tests, which also showed that beta-galactosidase activity in cells was synergistically increased by a treatment combining E64d and N-butyl deoxygalactonojirimycin. In other words, the beta-galactosidase inhibitor N-butyl-deoxygalactonojirimycin enhances maturation of the deficient beta-galactosidase in patient cells, whereas E64d increases to a limited and variable extent the amount of mature beta-galactosidase (no mature beta- galactosidase detected in GM05335 cells treated with E64d and some beta-galactosidase detected in Zrh #4 cells treated with E64d). By contrast, the abundance of the mature form of beta- galactosidase is synergistically increased in patient cells treated with combination of N-butyl- deoxygalactonojirimycin and E64d (see Figure 4).

Example 9: Enhancement of beta-glucosidase activity in Gaucher disease patient cells induced by a combination of MDL-28170 and isofagomine

Gaucher disease GM01607 (bearing the p.N370S and p.V394L mutations in the glucocerebrosidase gene; from the Coriell Institute, Camden, NJ) patient cells were treated for 5 days with the indicated compounds. Isofagomine and MDL-28170 were used at 20 and 10 mM, respectively. The cells were then washed with phosphate buffer saline and lysed in citrate buffer 100 mM pH 5.2 with 2.5 mg/ml of taurocholic acid and 0.1% Triton X-100. Beta-glucosidase activity assays were then conducted using 4-methylumbelliferyl beta-D-glucopyranoside as a substrate. The results present the fold increase of beta-glucosidase activity in treated cells relative to the activity in untreated cells. Each measure was conducted in quadruplicates. The standard deviations were <15% of the mean.

Table 15: beta-glucosidase activity measured in Gaucher disease GM01607 patient cells

A 3.1 -fold enhancement of beta-glucosidase activity is obtained when MDL-28170 and isofagomine are combined.

Claims

1. A pharmaceutical composition comprising a compound of general formula (I)

and/or pharmaceutically acceptable salts thereof, wherein

R3 is H;

R^ais C₆-C1₀ aryl;

R^bis C1-C6 alkyl;

(dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomcthylidcnc)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and a pharmaceutically acceptable carrier, diluent or excipient, characterized in that, said compound of general formula (I) is selected from the group consisting of:

2. The pharmaceutical composition according to claim 1, wherein the compound of general formula (I) is:

3. The pharmaceutical composition according to any one of claims 1-2, wherein the compound of general formula (I) is:

4. The pharmaceutical composition according to any one of claims 1-2, wherein the compound of general formula (I) is:

5. The pharmaceutical composition according to any one of claims 1-4, wherein the beta- galactosidase inhibitor is selected from the group consisting of:

N-butyl-deoxygalactonojirimycin, 4-epi-isofagomine, 5a-C-pentyl 4-epi-isofagomine, 5a-C- methyl 4-epi-isofagomine, l,5-dideoxy-l,5-imino-(L)-ribitol, 5-C-alkyl-imino-L-ribitol, N- substituted 5-amino- l-hydroxymethyl-cyclopentanetriols, N- (dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomcthylidcnc)-6-thio- 1 -dcoxygalactonoj irimycin and N- nonyl-deoxygalactonoj irimycin.

6. The pharmaceutical composition according to claim 5, wherein the beta-galactosidase inhibitor is N-butyl-deoxygalactonojirimycin or 5a-C-pentyl-4-epi-isofagomine.

7. The pharmaceutical composition according to claim 5 or 6, wherein the beta- galactosidase inhibitor is N-butyl-deoxygalactonojirimycin.

8. The pharmaceutical composition according to any one of claims 1-4, wherein the pharmacological chaperone or inhibitor of glucocerebrosidase is selected from the group consisting of N-butyldeoxynoj irimycin, ambroxol and isofagomine.

9. The pharmaceutical composition according to any one of claims 1-7, for use in the treatment and/or prevention of lysosomal diseases selected from the group comprising GM1- gangliosidosis and mucopolysaccharidosis IVB/Morquio disease type B.

10. The pharmaceutical composition according to claim 8, for use in the treatment and/or prevention of Gaucher disease.

11. The pharmaceutical composition according to any one of the preceding claims, for use as a medicament.

12. A kit comprising a first container and a second container, wherein

the first container contains a first composition comprising (i) a compound of general formula (I)

ĭ44

(dansylamino)hexylaminocarbonylpentyl- 1 ,5-dideoxy- 1 ,5-imino-D-galactitol, N-octyl-4-epi- beta-valienamine, 5N,6S-(N'-butyliminomcthylidcnc)-6-thio- 1 -deoxygalactonojirimycin, N- nonyl-deoxygalactonojirimycin, N-butyldeoxynojirimycin, ambroxol, isofagomine; and ii) a pharmaceutically acceptable carrier, diluent or excipient,

for use in the treatment and/or prevention of lysosomal diseases selected from the group comprising GM1 -gangliosidosis, mucopolysaccharidosis IVB/Morquio disease type B and Gaucher disease, in a subject, and preferably the kit further comprises instructions for use.

13. The kit for use according to claim 12, wherein the compound of general formula (I) is:

14. The kit for use according to any one of claims 12-13, wherein the beta-galactosidase inhibitor is N-butyl-deoxygalactonojirimycin or 5a-C-pentyl-4-epi-isofagomine.

15. The kit for use according to any one of claims 12-13, wherein the pharmacological chaperone or inhibitor of glucocerebrosidase is selected from the group consisting of N- butyldeoxynojirimycin, ambroxol and isofagomine.