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WO2009141627A1 - Treating niemann-pick disease type c and related disorders by elevating intracellular calcium - Google Patents

Treating niemann-pick disease type c and related disorders by elevating intracellular calcium Download PDF

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WO2009141627A1
WO2009141627A1 PCT/GB2009/001295 GB2009001295W WO2009141627A1 WO 2009141627 A1 WO2009141627 A1 WO 2009141627A1 GB 2009001295 W GB2009001295 W GB 2009001295W WO 2009141627 A1 WO2009141627 A1 WO 2009141627A1
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dideoxy
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Frances Mary Platt
Emyr Lloyd-Evans
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Oxford University Innovation Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/221—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having an amino group, e.g. acetylcholine, acetylcarnitine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia

Definitions

  • the present invention relates to the treatment of diseases which have a Niemann- Pick disease type C (NPC) like cellular phenotype including, but not limited to, Niemann- Pick disease type C.
  • NPC Niemann- Pick disease type C
  • NPC Niemann-Pick disease type C
  • Le/Lys neurodegenerative late endosomal/lysosomal lipid storage disorder. It is caused by mutations in the NPCl gene that encodes a transmembrane protein of the Le/Lys. NPCl has been suggested to facilitate endocytic transport and lysosomal lipid efflux. Additionally, a clinically identical disease (also called NPC) can result from mutation of an unrelated gene, NPC2, that encodes a soluble protein of the Le/Lys.
  • NPC2 is a protein that transports cholesterol from inner lysosomal vesicles to the limiting membrane of the lysosome.
  • NPC disease An unusual feature of NPC disease is the broad range of lipid species that accumulate, including cholesterol, sphingomyelin, glycosphingolipids (GSLs) and unusually sphingosine.
  • GSLs glycosphingolipids
  • lipid storage causes the neurodegenerative phenotype is also not fully understood.
  • NPC Niemann-Pick disease type C
  • SLOS Smith-Lemli-Opitz Syndrome
  • NPC2 Niemann-Pick disease type C2
  • the present invention relates to the findings presented herein that NPCl cells have a large reduction in the late endosomal/lysosomal calcium store.
  • the first measurable event in the pathogenic cascade was sphingosine storage that in turn caused calcium depletion in the late endosome/lysosome. This then led to defective endocytic transport/fusion and the secondary storage of cholesterol, sphingomyelin and glycosphingolipids.
  • the unique calcium phenotype, caused by sphingosine storage represents a novel target for therapeutic intervention for the treatment of diseases having an NPC-like cellular phenotype.
  • NPC Niemann-Pick disease type C
  • SLOS Smith-Lemli- Opitz Syndrome
  • the present invention provides a compound which elevates intracellular calcium for use in the treatment of a disease which has a Niemann-Pick disease type C (NPC) like cellular phenotype.
  • NPC Niemann-Pick disease type C
  • the invention also provides a method of treating a disease which has a NPC like cellular phenotype, which method comprises administering to a patient in need of such treatment an effective amount of a compound which elevates intracellular calcium.
  • the invention also provides a pharmaceutical composition for use in treating a disease which has a NPC like cellular phenotype, comprising a pharmaceutically acceptable carrier or diluent and a compound which elevates intracellular calcium.
  • the invention also provides the use of a compound which elevates intracellular calcium in the manufacture of a medicament for the treatment of a disease which has a NPC like cellular phenotype.
  • the invention also provides an agent for the treatment of a disease which has a
  • NPC like cellular phenotype comprising a compound which elevates intracellular calcium.
  • pathogens which are capable of blocking phagosome-lysosome fusion target the Niemann-Pick Disease type C (NPC) cellular pathway in order to promote their survival in late endosomes.
  • NPC Niemann-Pick Disease type C
  • Infections caused by such pathogens, including the infectious disease tuberculosis are also therefore "diseases which have a NPC like cellular phenotype" that can be treated in accordance with the present invention.
  • Examples 11 to 13 hereinbelow support that therapies which are effective in treating NPC disease, including administration of agents that elevate intracellular calcium and compounds that inhibit sphingolipid biosynthesis, are effective in promoting clearance of such pathogens from infected cells.
  • the disease which has a Niemann-Pick disease type C (NPC) like cellular phenotype is a pathogenic infection, which pathogenic infection is capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
  • the pathogenic infection is tuberculosis.
  • the invention provides a compound which reduces sphingosine storage for use in the treatment of Niemann-Pick disease type C.
  • the invention also provides a method of treating Niemann-Pick disease type C, which method comprises administering to a patient in need of such treatment an effective amount of a compound which reduces sphingosine storage.
  • the invention also provides a pharmaceutical composition for use in treating Niemann-Pick disease type C, comprising a pharmaceutically acceptable carrier or diluent and a compound which reduces sphingosine storage.
  • a pharmaceutical composition for use in treating Niemann-Pick disease type C comprising a pharmaceutically acceptable carrier or diluent and a compound which reduces sphingosine storage.
  • the invention also provides the use of a compound which reduces sphingosine storage in the manufacture of a medicament for the treatment of Niemann-Pick disease type C.
  • the invention also provides an agent for the treatment of Niemann-Pick disease type C, comprising a compound which reduces sphingosine storage.
  • FIGURE 1 shows six graphs, A to F, in which:
  • A is a graph of ER calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with lOO ⁇ M ryanodine in wild type cells (black line) and NPCl cells (grey line).
  • B is a graph of ER calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with l ⁇ M thapsigargin in wild type cells (black line) and NPCl cells (grey line).
  • C is a graph of mitochondrial calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with 2 ⁇ M CCCP in wild type cells (black line) and NPCl cells (grey line).
  • D is a graph of Le/Lys calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with 200 ⁇ M GPN in wild type cells (black line) and NPCl cells (grey line).
  • E is a graph of Le/Lys calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with 50OnM bafilomycin Al in wild type cells (black line) and NPCl cells (grey line).
  • F is a graph of Le/Lys calcium release (y axis) using 200 ⁇ M GPN in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes, in normal cells, after 5 min pre-incubation with 1 ⁇ M sphinganine (line 1), 0.1 ⁇ M sphingosine (line 3), 1 ⁇ M sphingosine (line 4) and after no preincubation (control; line 2).
  • Figure 2 shows: A (upper panel / first row): micrographs of RAW macrophages treated with Ul 8666a over a 24h timecourse, leading to a blockage in transport of BODIPY labelled lactosylceramide (BODIPY-LacCer) from late endosomes (punctate appearance) to the Golgi (perinuclear crescent staining) at 2h post-treatment, persisting up to 24h.
  • BODIPY-LacCer lactosylceramide
  • A lower panel / second row: micrographs of RAW macrophages treated with Ul 8666a over a 24h timecourse, showing that cholesterol levels are normal for the first 4h of treatment but are elevated after 8h and 24h.
  • B is a bar chart showing the GSL level (y axis) in units of pmol GSL / ⁇ g protein in normal cells (left hand bars) and NPCl cells (right hand bars) after 1 hour (white bars), 4 hours (dark grey bars) and 24 hours (light grey bars) of treatment with l ⁇ M thapsigargin and without thapsigargin treatment (control; black bars).
  • A micrographs I to IV: show that cholesterol localization (filipin) returns to normal at Ih post thapsigargin treatment.
  • B is a bar chart showing cholesterol levels (y axis) in units of ⁇ g cholesterol / ⁇ g protein in RA25 cells (left hand bars) and CT43 cells (right hand bars) after 1 hour (dark grey bars), 4 hours (light grey bars) and 24 hours (white bars) of treatment with l ⁇ M thapsigargin and without thapsigargin treatment (control; black bars).
  • micrographs I to FV show the levels and localization of neutral lipids using nile red; correct localization of neutral lipids to the perinuclear ER is observed, illustrating that the stored cholesterol in NPCl cells can be delivered to the ER for utilization following elevation of cytosolic calcium.
  • B is bar chart showing the levels of cholesterol esters (y axis) in units of ⁇ g cholesterol esters / ⁇ g protein in RA25 cells (left hand bars) and CT43 cells (right hand bars) after 1 hour (dark grey bars) and 4 hours (white bars) of treatment with l ⁇ M thapsigargin and without thapsigargin treatment (control; black bars).
  • Figure 4 consists of micrographs of BODIP Y-LacCer transport in wild- type (first row / upper panel) and NPCl null glial cells (second row / lower panel) which are untreated (fist column), treated with l ⁇ M thapsigargin (second column), 30 ⁇ M curcumin (third column) and 1OnM l ⁇ , 25-dihydroxyvitamin D 3 (l ⁇ ,25(OH) 2 VD 3 ) (fourth column).
  • micrographs I to IV show that curcumin-fed mice (right hand column, micrographs ⁇ and rV) had superficial improvements in coat condition and also improved gait compared to ataxic untreated NPC mice (left hand column, micrographs I and IH).
  • B graph of mouse weight (y axis) in units of grams versus time (x axis) in units of weeks.
  • the data points represented by solid black squares are for NPCl knock-out mice which were fed a diet of pelleted mouse chow without curcumin from weaning at 3 weeks of age until death.
  • the data points represented by hollow black circles are for NPCl knockout mice which were fed a diet of pelleted mouse chow with curcumin amounting to a dosage of 150mg/kg/day from weaning at 3 weeks of age until death.
  • C bar chart of mouse exploration activity in units of rearing/activity per minute (y axis) versus age (x axis) in units of weeks.
  • the black bars are for NPCl knock-out mice which were fed a diet of pelleted mouse chow with curcumin amounting to a dosage of 150mg/kg/day from weaning at 3 weeks of age until death.
  • the white bars are for NPCl knock-out mice which were fed a diet of pelleted mouse chow without curcumin from weaning at 3 weeks of age until death.
  • the grey bars are for wild type NPCl mice which were fed a diet of pelleted mouse chow without curcumin from weaning at 3 weeks of age until death.
  • Figure 6 shows images of BODIPY-LacCer transport in wild-type (first row / upper left panel) and SLOS murine embryonic fibroblast cells (first row / upper right panel & second row both panels) which are untreated (fist column), or grown in lipoprotein deficient serum (LPDS) (second column) with 30 ⁇ M curcumin (second column / lower panel).
  • Figure 6 also show micrographs of GPN mediated lysosomal calcium release in units of arbitrary fluorescence (right hand side 4 panels) of wild-type (upper row) and SLOS (lower row) fibroblasts grown in foetal calf serum (left hand side column) or LPDS (right hand side column).
  • Figure 7 shows a graph of lysotracker fluorescence absorption (indicative of lysosomal storage) (y axis) in fluorescence units versus time (x axis) in units of hours for NPCl -null CHO cells either without (“npcl”) or with ("4hs", “8hs", “16hs”) treatment with 30 ⁇ M curcumin for the time indicated.
  • the two micrographs (labelled “CHO” and “curcumin”) show NPCl -null CHO cell lysotracker staining ("CHO") and 24-hour 30 ⁇ M curcumin- treated NPCl -null CHO cell lysotracker staining ("curcumin”).
  • Figure 8 is a graph of mouse weight (y axis) in units of grams versus age (x axis) in units of weeks.
  • the data points represented by triangles are for untreated NPCl knock-out mice.
  • the data points represented by circles are for NPCl knock-out mice fed a diet supplemented with 150 mg/kg/day curcumin.
  • the data points represented by diamonds are for NPCl knock-out mice treated with a combination of curcumin and NB-DNJ.
  • Figure 9 is a graph of fluorescence (indicative of intracellular calcium levels) (y axis) in fluorescence units versus time (x axis) in units of minutes for wild-type (upper, black trace), NPCl -null (middle, light grey trace) and 15OnM myriocin-treated NPCl (lower, dark grey trace) CHO cells labelled with Calcium Green 1-AM and Fura Red- AM.
  • Figure 10 shows: A: Four micrographs, showing lysotracker-stained wild type CHO cells (CHO WT),
  • NPC-I null CHO cells (CHO NPCl), NPCl cells treated with 15OnM ISP-I (myriocin) for 3 days (NPCl+ISP-1 3 DAYS) and for 5 days (NPCl+ISP-1 5 DAYS).
  • FIG. B A bar chart of fluorescence absorption (indicative of lysosomal storage) (y axis) for various CHO cells (x axis): either wild type (WT), NPCl -null (NPCl), NPCl treated with 15OnM ISP-I (myriocin) for 3 days (NPCl+ISP-1 3 DAYS) or NPCl treated with 15OnM ISP-I (myriocin) for 5 days (NPCl+ISP-1 5 DAYS).
  • Figure 11 consists of micrographs which show that live Mycobacterium bovis (BCG) and mycolic acid lipids (M. A.) from Mycobacterium tuberculosis (Tb) induce an NPCl phenotype in RAW mouse macrophages.
  • BCG Mycobacterium bovis
  • M. A. mycolic acid lipids
  • Tb Mycobacterium tuberculosis
  • Figure 12 comprises micrographs which show that live BCG infection depletes lysosomal calcium, and that this can be overcome using a calcium agonist (curcumin) to clear the infection.
  • Fig. 2A contains a graph showing the concentration of intralysosomal calcium in ⁇ M (y axis) in control RAW cells (left hand bar), cells infected overnight with live BCG (middle bar) and cells infected with heat-killed BCG (right hand bar).
  • Figure 13 consists of micrographs of RAW macrophages which show that depletion of sphingolipids using an inhibitor of sphingo lipid biosynthesis (miglustat, NB-DNJ) reverses the accumulation of sphingomyelin induced by BCG and Tb secreted lipids.
  • NB-DNJ an inhibitor of sphingo lipid biosynthesis
  • Ci -20 alkyl group is an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical. Typically it is C 1-I o alkyl, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl, or Ci -6 alkyl, for example methyl, ethyl, propyl, butyl, pentyl or hexyl, or C M alkyl, for example methyl, ethyl, i-propyl, n-propyl, t- butyl, s-butyl or n-butyl.
  • alkyl group When an alkyl group is substituted it typically bears one or more substituents selected from substituted or unsubstituted Ci -2O alkyl, substituted or unsubstituted aryl (as defined herein), cyano, amino, C MO alkylamino, di(Ci-i 0 )alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, Ci -20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e.
  • alkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups.
  • alkaryl as used herein, pertains to a Ci -20 alkyl group in which at least one hydrogen atom has been replaced with an aryl group.
  • a substituted Ci -2O alkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
  • a C 3-25 cycloalkyl group is an unsubstituted or substituted alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound, which moiety has from 3 to 25 carbon atoms (unless otherwise specified), including from 3 to 25 ring atoms.
  • cycloalkyl includes the sub-classes cycloalkyenyl and cycloalkynyl.
  • Examples of groups OfC 3-25 cycloalkyl groups include C 3-20 cycloalkyl, C 3-15 cycloalkyl, C 3-10 cycloalkyl, C 3-7 cycloalkyl.
  • a C 3-25 cycloalkyl group When a C 3-25 cycloalkyl group is substituted it typically bears one or more substituents selected from C 1-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, Ci -20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e.
  • thiol -SH
  • Ci-I 0 alkylthio arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl.
  • a substituted C 3-25 cycloalkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
  • Examples OfC 3-25 cycloalkyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds, which C 3-25 cycloalkyl groups are unsubstituted or substituted as defined above: cyclopropane (C 3 ), cyclobutane (C 4 ), cyclopentane (C 5 ), cyclohexane (C 6 ), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ), methylcyclohexane (C 7 ), dimethylcyclohexane (C 8 ), menthane
  • unsaturated monocyclic hydrocarbon compounds cyclopropene (C 3 ), cyclobutene (C 4 ), cyclopentene (C 5 ), cyclohexene (C 6 ), methylcyclopropene (C 4 ), dimethylcyclopropene (C 5 ), methylcyclobutene (C 5 ), dimethylcyclobutene (C 6 ), methylcyclopentene (C 6 ), dimethylcyclopentene (C 7 ), methylcyclohexene (C 7 ), dimethylcyclohexene (C 8 ); saturated polycyclic hydrocarbon compounds: thujane (C 10 ), carane (C 1O ), pinane (Ci 0 ), bomane (C 1O ), norcarane (C 7 ), no ⁇ inane (C 7 ), norbornane (C 7 ), adamantane (C] 0 ), decalin (decahydronaphthalene) (C 10 );
  • unsaturated polycyclic hydrocarbon compounds camphene (C 10 ), limonene (C 10 ), pinene (C 10 ),
  • polycyclic hydrocarbon compounds having an aromatic ring indene (C 9 ), indane (e.g., 2,3-dihydro-lH-indene) (C 9 ), tetraline
  • a C 3-2O heterocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms.
  • each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
  • C 3-20 heterocyclyl group When a C 3-20 heterocyclyl group is substituted it typically bears one or more substituents selected from C 1-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, Ci -I0 alkylamino, di(Ci -10 )alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C 1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e.
  • thiol -SH
  • C 1-I0 alkylthio arylthio
  • phosphoric acid phosphate ester
  • phosphonic acid phosphonate ester
  • sulfonyl Typically a substituted C 3-20 heterocyclyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
  • groups of heterocyclyl groups include C 3-2 oheterocyclyl, C 5-20 heterocyclyl, C 3-15 heterocyclyl, C 5-15 heterocyclyl, C 3- i 2 heterocyclyl, C 5-12 heterocyclyl, C 3- ioheterocyclyl, C 5- i 0 heterocyclyl, C 3-7 heterocyclyl, C ⁇ heterocyclyl, and Cs- ⁇ heterocyclyl.
  • Examples of (non-aromatic) monocyclic C 3-20 heterocyclyl groups include, but are not limited to, those derived from: N 1 : aziridine (C 3 ), azetidine (C 4 ), pyrrolidine (tetrahydropyrrole) (C 5 ), pyrroline
  • O 1 oxirane (C 3 ), oxetane (C 4 ), oxolane (tetrahydrofuran) (C 5 ), oxole (dihydrofuran) (C 5 ), oxane (tetrahydropyran) (C 6 ), dihydropyran (C 6 ), pyran (C 6 ), oxepin (C 7 ); Si : thiirane (C 3 ), thietane (C 4 ), thiolane (tetrahydrothiophene) (C 5 ), thiane
  • N 2 imidazolidine (C 5 ), pyrazolidine (diazolidine) (C 5 ), imidazoline (C 5 ), pyrazoline (dihydropyrazole) (C 5 ), piperazine (C 6 );
  • N 1 Oi tetrahydrooxazole (C 5 ), dihydrooxazole (C 5 ), tetrahydroisoxazole (C 5 ), dihydroisoxazole (C 5 ), mo ⁇ holine (C 6 ), tetrahydrooxazine (C 6 ), dihydrooxazine (C 6 ), oxazine (C 6 );
  • NiS 1 thiazoline (C 5 ), thiazolidine (C 5 ), thiomorpholine (C 6 ); N 2 O 1 : oxadiazine (C 6 );
  • N 1 OiSi oxathiazine (C 6 ).
  • substituted (non-aromatic) monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C 5 ), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C 6 ), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
  • C 3-20 heterocyclyl includes groups derived from heterocyclic compounds of the following structure:
  • R 80 , R 81 , R 82 , R 83 and R 84 which are the same or different, are independently selected from H, Ci -6 alkyl, OH, acyloxy, SH, Ci -6 alkoxy, aryloxy, amino, Ci-I 0 alkylamino, di(Ci.io)alkylamino, amido, acylamido and a group derived from a second group of the following structure:
  • C 3-20 heterocyclyl includes groups of the following structure:
  • each of the ring carbon atoms is independently unsubstituted or substituted with Ci -6 alkyl, OH, acyloxy, SH, Ci -6 alkoxy, aryloxy, amino, C] -I0 alkylamino, di(Ci-io)alkylamino, amido and acylamido.
  • C 3-20 heterocyclyl also includes groups in which two heterocyclic rings are linked by an oxygen atom.
  • C 3-20 heterocyclyl includes disaccharide groups, in which two monosaccharide heterocyclic rings are linked with an oxygen atom.
  • C 3-20 heterocyclyl includes groups of the following formula (m):
  • each R m which is the same or different, is independently selected from Ci -6 alkyl, OH, acyloxy, SH, C 1-6 alkoxy, aryloxy, amino, Ci-I 0 alkylamino, di(Ci-i 0 )alkylamino, amido and acylamido.
  • disaccharide group is one example of a substituted C 3-20 heterocyclic group:
  • C 3-20 heterocyclyl groups which are also aryl groups are described below as heteroaryl groups.
  • An aryl group is a substituted or unsubstituted, monocyclic or bicyclic aromatic group which typically contains from 6 to 14 carbon atoms, preferably from 6 to 10 carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl and indanyl groups. An aryl group is unsubstituted or substituted.
  • aryl group as defined above When an aryl group as defined above is substituted it typically bears one or more substituents selected from Ci-C 6 alkyl which is unsubstituted (to form an aralkyl group), aryl which is unsubstituted, cyano, amino, C 1-10 alkylamino, di(C 1-10 )alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, halo, carboxy, ester, acyl, acyloxy, C 1-20 alkoxy, aryloxy, haloalkyl, sulfhydryl (i.e.
  • a substituted aryl group may be substituted in two positions with a single C 1-6 alkylene group, or with a bidentate group represented by the formula -X-Ci -6 alkylene, or -X-Ci -6 alkylene-X-, wherein X is selected from O, S and NR, and wherein R is H, aryl or Ci -6 alkyl.
  • a substituted aryl group may be an aryl group fused with a cycloalkyl group or with a heterocyclyl group.
  • aralkyl as used herein, pertains to an aryl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with a Ci- 6 alkyl group.
  • examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), and cumenyl (or cumyl, from cumene), and duryl (from durene).
  • the ring atoms of an aryl group may include one or more heteroatoms (as in a heteroaryl group).
  • Such an aryl group (a heteroaryl group) is a substituted or unsubstituted mono- or bicyclic heteroaromatic group which typically contains from 6 to 10 atoms in the ring portion including one or more heteroatoms. It is generally a 5- or 6-membered ring, containing at least one heteroatom selected from O, S, N, P, Se and Si. It may contain, for example, 1, 2 or 3 heteroatoms.
  • heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, quinolyl and isoquinolyl.
  • a heteroaryl group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically it carries 0, 1, 2 or 3 substituents.
  • Ci -20 alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated.
  • alkylene includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Typically it is Ci-I 0 alkylene, for instance C 1-6 alkylene.
  • Ci -4 alkylene for example methylene, ethylene, i-propylene, n-propylene, t-butylene, s-butylene or n- butylene. It may also be pentylene, hexylene, heptylene, octylene and the various branched chain isomers thereof.
  • An alkylene group may be unsubstituted or substituted, for instance, as specified above for alkyl.
  • a substituted alkylene group carries 1, 2 or 3 substituents, for instance 1 or 2.
  • the prefixes denote the number of carbon atoms, or range of number of carbon atoms.
  • the term "Ci- 4 alkylene,” as used herein, pertains to an alkylene group having from 1 to 4 carbon atoms.
  • groups of alkylene groups include C 1-4 alkylene ("lower alkylene"), Ci -7 alkylene, Ci-I 0 alkylene and Ci -20 alkylene.
  • linear saturated Ci -7 alkylene groups include, but are not limited to,
  • n is an integer from 1 to 7, for example, -CH 2 - (methylene), -CH 2 CH 2 - (ethylene), -CH 2 CH 2 CH 2 - (propylene), and -CH 2 CH 2 CH 2 CH 2 - (butylene).
  • branched saturated Ci -7 alkylene groups include, but are not limited to, -CH(CH 3 )-, -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH 2 CH 3 )-, -CH(CH 2 CH 3 )CH 2 -, and -CH 2 CH(CH 2 CH 3 )CH 2 -.
  • alicyclic saturated C 1-7 alkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex-l,4-ylene).
  • alicyclic partially unsaturated C 1-7 alkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), cyclohexenylene (e.g., 2-cyclohexen-l,4-ylene; 3-cyclohexen-l,2-ylene; 2,5-cyclohexadien-l,4-ylene).
  • cyclopentenylene e.g., 4-cyclopenten-l,3-ylene
  • cyclohexenylene e.g., 2-cyclohexen-l,4-ylene; 3-cyclohexen-l,2-ylene; 2,5-cyclohexadien-l,4-ylene.
  • Ci -20 alkyl groups as defined herein are either uninterrupted or interrupted by one or more heteroatoms or heterogroups, such as S, O or N(R") wherein R" is H, C 1-6 alkyl or aryl (typically phenyl), or by one or more arylene (typically phenylene) groups.
  • the phrase "optionally interrupted” as used herein thus refers to a Ci -20 alkyl group or an alkylene group, as defined above, which is uninterrupted or which is interrupted between adjacent carbon atoms by a heteroatom such as oxygen or sulfur, by a heterogroup such as N(R") wherein R" is H, aryl or Ci-C 6 alkyl, or by an arylene group.
  • a C 1-20 alkyl group such as n-butyl may be interrupted by the heterogroup N(R") as follows: -CH 2 N(R")CH 2 CH 2 CH 3 , -CH 2 CH 2 N(R")CH 2 CH 3 , or -CH 2 CH 2 CH 2 N(R")CH 3 .
  • an alkylene group such as n-butylene may be interrupted by the heterogroup N(R") as follows: -CH 2 N(R")CH 2 CH 2 CH 2 -, -CH 2 CH 2 N(R")CH 2 CH 2 -, or -CH 2 CH 2 CH 2 N(R")CH 2 -.
  • an interrupted group for instance an interrupted C 1-20 alkylene or C 1-20 alkyl group, is interrupted by 1, 2 or 3 heteroatoms or heterogroups or by 1, 2 or 3 arylene (typically phenylene) groups. More typically, an interrupted group, for instance an interrupted C 1-20 alkylene or Ci -20 alkyl group, is interrupted by 1 or 2 heteroatoms or heterogroups or by 1 or 2 arylene (typically phenylene) groups.
  • a Ci -20 alkyl group such as n-butyl may be interrupted by 2 heterogroups N(R") as follows: -CH 2 N(R")CH 2 N(R")CH 2 CH 3 .
  • An arylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, which moiety has from 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has from 5 to 7 or from 5 to 6 ring atoms.
  • An arylene group may be unsubstituted or substituted, for instance, as specified above for aryl.
  • the prefixes e.g., Cs -20 , C 6-20 , C 5-H , C 5-7 , C 5-6 , etc.
  • the term "C 5-6 arylene,” as used herein, pertains to an arylene group having 5 or 6 ring atoms.
  • groups of arylene groups include C 5-20 arylene, C 6-20 arylene, C 5-14 arylene, C 6-14 arylene, C 6-10 arylene, C 5-12 arylene, C 5-10 arylene, C 5-7 arylene, C 5-6 arylene, C 5 arylene, and C 6 arylene.
  • the ring atoms may be all carbon atoms, as in "carboarylene groups” (e.g., C 6-20 carboarylene, C 6-J4 carboarylene or C 6-I0 carboarylene).
  • C 6-20 arylene groups which do not have ring heteroatoms include, but are not limited to, those derived from the compounds discussed above in regard to aryl groups, e.g. phenylene, and also include those derived from aryl groups which are bonded together, e.g. phenylene-phenylene (diphenylene) and phenylene-phenylene-phenylene (triphenylene).
  • the ring atoms may include one or more heteroatoms, as in
  • heteroarylene groups e.g., C 5-I0 heteroarylene.
  • C 5-10 heteroarylene groups include, but are not limited to, those derived from the compounds discussed above in regard to heteroaryl groups.
  • R is an acyloxy substituent, for example, substituted or unsubstituted C 1-20 alkyl group, a substituted or unsubstituted C 3-20 heterocyclyl group, or a substituted or unsubstituted aryl group, typically a Ci -6 alkyl group.
  • phosphonic acid salt represents a group which is a salt of a phosphonic acid group.
  • X + may be an alkali metal cation.
  • X + may be Na + or K + , for example.
  • phosphate ester represents a group of one of the formulae:
  • each R is independently a phosphate ester substituent, for example, -H, substituted or unsubstituted Ci -20 alkyl, substituted or unsubstituted C 3-20 heterocyclyl, C 3-20 heterocyclyl substituted with a further C 3-20 heterocyclyl, substituted or unsubstituted Ci -20 alkylene-C 3-20 heterocyclyl, substituted or unsubstituted C 3-25 cycloalkyl, substituted or unsubstituted C 1-20 alkylene-C 3- 2 5 cycloalkyl, aryl, substituted or unsubstituted Ci -20 alkylene-aryl.
  • amino represents a group of formula -NH 2 .
  • C 1 - Cio alkylamino represents a group of formula -NHR' wherein R' is a Ci-I 0 alkyl group, preferably a Ci -6 alkyl group, as defined previously.
  • di(Ci-io)alkylamino represents a group of formula -NR 'R" wherein R' and R" are the same or different and represent Ci -io alkyl groups, preferably Ci -6 alkyl groups, as defined previously.
  • arylamino represents a group of formula -NHR' wherein R' is an aryl group, preferably a phenyl group, as defined previously.
  • diarylamino represents a group of formula -NR'R" wherein R' and R" are the same or different and represent aryl groups, preferably phenyl groups, as defined previously.
  • arylalkylamino represents a group of formula -NR'R" wherein R' is a C 1- I 0 alkyl group, preferably a Ci -6 alkyl group, and R" is an aryl group, preferably a phenyl group.
  • R 1 and R 2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
  • a C 1-I0 alkylthio group is a said C ⁇ o alkyl group, preferably a Ci -6 alkyl group, attached to a thio group.
  • An arylthio group is an aryl group, preferably a phenyl group, attached to a thio group.
  • a C 1-20 alkoxy group is a said substituted or unsubstituted Ci -20 alkyl group attached to an oxygen atom.
  • a C 1-6 alkoxy group is a said substituted or unsubstituted Ci -6 alkyl group attached to an oxygen atom.
  • a C 1-4 alkoxy group is a substituted or unsubstituted C 1-4 alkyl group attached to an oxygen atom. Said C 1-20 , C 1-6 and C 1-4 alkyl groups are optionally interrupted as defined herein.
  • C 1-4 alkoxy groups include, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).
  • Ci -20 alkoxy groups are -O(Adamantyl), -O-CH 2 -Adamantyl and -0-CH 2 -CH 2 - Adamantyl.
  • An aryloxy group is a substituted or unsubstituted aryl group, as defined herein, attached to an oxygen atom.
  • An example of an aryloxy group is -OPh (phenoxy).
  • a reference to carboxylic acid or carboxyl group also includes the anionic (carboxylate) form (-COO " ), a salt or solvate thereof, as well as conventional protected forms.
  • a reference to an amino group includes the protonated form (-N + HR 1 R 2 ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group.
  • a reference to a hydroxyl group also includes the anionic form (- O " ), a salt or solvate thereof, as well as conventional protected forms.
  • Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; C-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L- forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti- forms; synclinal- and anticlinal-forms; ⁇ - and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers” (or "isomeric forms").
  • a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH 2 OH.
  • a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl.
  • a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C 1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
  • C 1-7 alkyl includes n-propyl and iso-propyl
  • butyl includes n-, iso-, sec-, and tert-butyl
  • methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl
  • keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
  • keto enol enolate (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
  • H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 O and 18 O; and the like.
  • a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
  • Examples of pharmaceutically acceptable salts of the compounds for use in accordance with the present invention include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid; and organic acids such as methanesulfonic acid, benzenesulphonic acid, formic acid, acetic acid, trifiuoroacetic acid, propionic acid, butyric acid, isobutyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, ethanesulfonic acid, aspartic acid, benzoic acid and glutamic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid
  • organic acids such as methanesulfonic acid, benzenesulphonic
  • the salt is a hydrochloride, an acetate, a propionate, a benzoate, a butyrate or an isobutyrate.
  • pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19.
  • a prodrug of a compound which elevates intracellular calcium, an agent which reduces sphingosine storage or an inhibitor of sphingo lipid biosynthesis is a compound which, when metabolised (e.g., in vivo), yields the desired active compound.
  • the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.
  • some prodrugs are 0-acylated (acyloxy) derivatives of the active compound, i.e. physiologically acceptable metabolically labile acylated derivatives.
  • R p may be a C 1 - J o alkyl group, an aryl group or a C 3-2O cycloalkyl group.
  • R p is a C 1-10 alkyl group including, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.
  • Such derivatives may be formed by acylation, for example, of any of the hydroxyl groups (-OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.
  • the free hydroxyl groups on an iminosugar inhibitor of sphingo lipid biosynthesis may be acylated with up to four, typically exactly four, O-acyl groups.
  • the O-acyl groups are enzymatically removed in vivo to provide the non-O-acylated (i.e. hydroxyl-containing) active inhibitor of sphingolipid biosynthesis.
  • Some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester).
  • the compound which elevates intracellular calcium, agent which reduces sphingosine storage or inhibitor of sphingolipid biosynthesis, for use in accordance with the invention can be used in the free form or the salt form.
  • the compound may also be used in prodrug form.
  • the prodrug can itself be used in the free form or the salt form.
  • compound which elevates intracellular calcium means any compound that causes an increase in the concentration of calcium within the cell, typically within the cytosol and/or the lysosomes of the cell, after administration to a patient.
  • the compound which elevates intracellular calcium is a compound which elevates cytosolic calcium or a compound which elevates lysosomal calcium.
  • compound which elevates cytosolic calcium means any compound that causes an increase in the concentration of calcium in the cytosol after administration to a patient.
  • compound which elevates lysosomal calcium means any compound that causes an increase in the concentration of calcium in the lysosomes after administration to a patient.
  • Compounds which elevate intracellular calcium are either known or readily identifiable, without undue experimentation, using known procedures. Such compounds can be identified by measuring the effects of target compounds on intracellular calcium levels (for example, their effect on cytosolic and lysosomal calcium concentrations) and by identifying compounds which elevate the level of intracellular calcium.
  • SERCA inhibitors are known compounds. J. Med. Chem. 2005, 48, 7005-7011 describes thapsigargin and various analogs thereof as potent SERCA inhibitors, and compares the respective activities of the compounds. Eur. J. Biochem, 268, 6318-6327 (2001) details the SERCA inhibitory activity of curcumin. Cyclopiazonic acid is also a known SERCA inhibitor. SERCA inhibitors may be readily identified, without undue experimentation, using known procedures.
  • J. Med. Chem. 2005, 48, 7005-7011 describes how SERCA inhibitors may be designed (for instance, using the structure of a known SERCA inhibitor as a starting point) and then tested for SERCA inhibitory activity.
  • a standard assay for the measurement of SERCA inhibitory activity is described in J. Med. Chem. 2005, 48, p 7010.
  • Eur. J. Biochem, 268, 6318-6327 (2001) also describes how Ca 2+ ATPase activity can be determined, in order to identify SERCA inhibitors, using a phosphate liberation assay described in Longland et al., Cell Calcium 24, 27-34 (1998).
  • the compound which elevates intracellular calcium is typically a SERCA inhibitor.
  • the SERCA inhibitor is selected from curcumin, cyclopiazonic acid and a compound of formula (A):
  • R Z! is selected from hydrogen, hydroxyl, carboxyl, amino, thiol, halo, substituted or unsubstituted Ci -10 alkyl, substituted or unsubstituted Ci -10 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C 1-10 alkylamino, di(Ci-i 0 )alkylamino, amido, acylamido, -0-C 3-25 cycloalkyl and -0-C 3-20 heterocyclyl, wherein said C 1-I0 alkyl is optionally interrupted by N(R'), O, S or arylene; R Z2 is selected from hydrogen, substituted or unsubstituted Ci-io alkyl and acyl, wherein said Ci -I0 alkyl is optionally interrupted by N(R'), O, S or arylene; and either X Z1 is CH and X Z2 is CHR' wherein X Z1 is CH
  • R' is H, Ci -6 alkyl or aryl; and pharmaceutically acceptable salts thereof.
  • R Z1 is selected from H and ester. More typically, R Z1 is selected from H and -OC(O)(CH 2 ) 6 CH 3 .
  • R Z2 is selected from C 1-10 alkyl and acyl. More typically, R 22 is selected from ethyl and acetyl (i.e. -C(O)CH 3 ).
  • X Z1 is CH and X Z2 is C(H)Me.
  • X Z1 is C and X Z2 is C(H)Me.
  • R Z1 is -OC(O)(CH 2 ) 6 CH 3
  • R Z2 is -C(O)CH 3
  • X Z1 is C
  • X Z2 is C(H)Me (i.e. the compounds is thapsigargin).
  • R Z1 is H
  • R Z2 is -C(O)CH 3
  • X Z1 is C
  • X Z2 is C(H)Me (i.e. the compounds is nortrilobolide).
  • R Z1 is H
  • R 22 is ethyl
  • X Z1 is CH
  • X 22 is C(H)Me.
  • R Z1 is H
  • R Z2 is -C(O)CH 3
  • X Z1 is CH
  • X Z2 is C(H)Me.
  • the SERCA inhibitor is selected from thapsigargin, curcumin and cyclopiazonic acid.
  • the chemical structure of curcumin is as follows:
  • thapsigargin The structure of thapsigargin is as follows:
  • the compound which elevates intracellular calcium is other than curcumin.
  • the compound which elevates intracellular calcium is other than curcumin when the disease which has an NPC like cellular phenotype is tuberculosis.
  • Compounds which elevate intracellular calcium, and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention also include modulators of the ryanodine receptor (the sarcoplasmic reticulum Ca 2+ channel, RyR).
  • Ryanodine receptor modulators (RyR modulators) are known compounds, and it is known that these compounds can interact with RyR to stimulate Ca 2+ release and cause an increase in intracellular calcium. Zucci and Ronca-Testoni, Pharmacological Reviews (1997), Vol. 49, No. 1, 1-51 describes many such RyR modulators that stimulate Ca 2+ release and thereby elevate intracellular calcium. Such RyR modulators can be readily identified, without undue experimentation, using known procedures. Pharmacological Reviews (1997), Vol. 49, No. 1, 1-51 describes how RyR modulators may be identified, using Ca 2+ release studies, single channel studies, [ 3 H]ryanodine binding studies or indirect studies.
  • the compound which elevates intracellular calcium is typically a Ryanodine receptor modulator (RyR modulator).
  • RoR modulator a Ryanodine receptor modulator
  • RyR modulators which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention, include, but are not limited to, the following compounds (many of which are described in Pharmacological Reviews (1997), Vol. 49, No. 1, 1-51):
  • R X1 is a substituted or unsubstituted pyrrole ring
  • R X2 , R X3 , R X4 , R X5 , R X6 , R X7 and R x8 which are the same or different, are independently selected from hydrogen, halo, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C 1- I 0 alkyl, aryl, substituted or unsubstituted C 3-25 cycloalkyl, substituted or unsubstituted C 3-20 heterocyclyl, substituted or unsubstituted C 1-10 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, Ci -1O alkylamino, di(Ci.io)alkylamino, amido, acylamido, -0-C 3-25 cycloalkyl and -0-C 3-20 heterocyclyl, wherein said C 1- Io alkyl is optionally interrupted by N(R'), O, S or ary
  • R X ⁇ 9 y is H.
  • R is an unsubstituted pyrrole ring.
  • R and R are both either hydrogen or substituted or unsubstituted C 1-10 alkyl. More typically, R ⁇ and R X3 are both hydrogen.
  • R X4 , R X5 , R x6 , R X7 and R x8 are independently selected from hydroxyl, substituted or unsubstituted C 1-10 alkoxy and acyloxy. More typically, each of R X4 , R X5 , R X6 ,
  • R and R is a hydroxyl group.
  • the ryanoid compound is selected from ryanodine, 9,21-didehydroryanodine, guanidinopropionyl and /7-alanil-ryanodine.
  • R MI , R M2 , R M3 , R M4 and R M5 which are the same or different, are independently selected from hydrogen and substituted or unsubstituted C 1-10 alkyl, provided that at least one of R M1 , R M2 , R M3 , R M4 and R M5 is substituted or unsubstituted Ci -10 alkyl, wherein said Ci -10 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C 1-6 alkyl or aryl; and pharmaceutically acceptable salts thereof.
  • R M1 , R M2 , R M3 , R M4 and R M5 which are the same or different, are independently selected from hydrogen and methyl, provided that at least one of R M1 , R M2 , R M3 , R M4 and R M5 is methyl.
  • R M1 , R M2 , R M3 , R M4 and R M5 are substituted or unsubstituted Ci -10 alkyl (more typically methyl) groups and the remainder are hydrogens.
  • the compound of formula (C) is a methylxanthine.
  • the methyxanthine is selected from any one of the following compounds:
  • E 1 , E 2 , E 3 and E 4 which are the same or different, are independently selected from C(R 05 ) and N provided that no more than two of Ei, E 2 , E 3 and E 4 are N;
  • R 01 is selected from hydrogen, substituted or unsubstituted Ci -1O alkyl, aryl, substituted or unsubstituted C 3-25 cycloalkyl, and substituted or unsubstituted C 3-20 heterocyclyl, wherein said C 1-I o alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci -6 alkyl or aryl; and R° 2 , R , R 04 and each R 05 , which are the same or different, are independently selected from hydrogen, halo, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Ci -10 alkyl, aryl, substituted or unsubstituted C 3-25 cycloalkyl, substituted or unsubstituted C 3-20 heterocyclyl, substituted or unsubstituted C 1 - I o alkoxy, substituted or unsubstituted aryloxy, acy
  • R 01 is selected from hydrogen and Ci -6 alkyl. More typically, R 01 is selected from hydrogen and methyl.
  • R 02 , R° 3 , R° 4 and each R 05 which are the same or different, are independently selected from hydrogen and halo groups. More typically, R° 2 , R 03 , R 04 and each R 05 , which are the same or different, are independently selected from H, Br and Cl.
  • the compound of formula (D) is a carbazole or a carboline compound.
  • the compound of formula (D) is 9-methyl-7-bromoeudistomin D (MBED), which has the following structure:
  • R N1 is a substituted or unsubstituted Ci 4-2O alkyl group, wherein said Ci 4-20 alkyl is optionally interrupted by N(R'), O, S or arylene; and pharmaceutically acceptable salts thereof.
  • R N1 is an unsubstituted, uninterrupted C] 4-20 alkyl group.
  • R N1 is -(CH 2 ) 14 CH 3 (i.e. the compound is palmitoyl carnitine).
  • Adenine nucleotides including, but not limited to the following adenine nucleotides: adenosine 5'-(jff,y-methylene)triphosphate (AMP-PCP), cyclic AMP (cAMP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP).
  • Anthraquinones including, but not limited to the following compounds: Doxorubicin, mitoxantrone, daunorubicin, rubidazone and doxorubicinol • Digoxin, digitoxin and ouabain
  • RyR-modulating peptides including, but not limited to: imperatoxin-a, myotoxin-a, and ryanotoxin • Sulfhydryl reagents, including, but not limited to: N-ethylmaleimide, thimerosal, 5,5'- dithiobis-(2-nitrobenzoic acid), 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, N- succinimidyl 3 -(2-pyridyldithio)propionate • Mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP), tetrasodium-mesotetra- (4-sulfonatophenyl)-porphine
  • TMPyP tetrasodium-mesotetra- (4-sulfonatophenyl)-porphine
  • Disulfonic stilbene derivatives including, but not limited to: 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetoamido-4'-isothiocyanostilbene- 2,2 '-disulfonic acid
  • IP3 receptor modulators are known compounds, and it is known that these compounds can interact with the IP3 receptor to mobilise Ca 2+ and cause an increase in intracellular calcium.
  • IP3 receptor modulators that stimulate Ca 2+ release and thereby elevate intracellular calcium, include inositol 1 ,4,5-trisphosphate (IP3) (Cell Calcium (2002) 32 (5-6), 343-354) and 2- APB (2- aminoethoxydiphenyl borate).
  • IP3 receptor modulators can be readily identified, without undue experimentation, using known assay methods.
  • the compound which elevates intracellular calcium is typically an IP3 receptor modulator.
  • the IP3 receptor modulator is inositol 1,4,5-trisphosphate (IP3) or 2-aminoethoxydiphenyl borate, which have the following structures respectively:
  • NAADP receptor modulators are known compounds and can be readily identified, without undue experimentation, using known assay methods. It is known that these compounds can interact with the NAADP receptor to mobilise Ca 2+ and cause an increase in intracellular calcium (Cell Calcium (2002) 32 (5-6), 343-354).
  • NAADP receptor modulators that stimulate Ca 2+ release and thereby elevate intracellular calcium, include nicotinic acid adenine dinucleotide phosphate (NAADP) (Cell Calcium (2002) 32 (5-6), 343- 354) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS).
  • NAADP nicotinic acid adenine dinucleotide phosphate
  • PPADS pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid
  • the compound which elevates intracellular calcium is typically a NAADP receptor modulator.
  • the NAADP receptor modulator is selected from nicotinic acid adenine dinucleotide phosphate (NAADP) and a compound of the following formula (F):
  • n is 0 or 1 ;
  • R Y1 is a substituted or unsubstituted aryl or heteroaryl group; and pharmaceutically acceptable salts thereof.
  • R Y1 is a substituted or unsubstituted phenyl group. More typically, R Y1 is unsubstituted phenyl or phenyl which is substituted with one or two groups selected from carboxyl, -SO 3 H and -CH 2 PO 3 H 2 . Even more typically, R ⁇ l is selected from any one of the following groups:
  • the compound of formula (F) is pyridoxalphosphate-6-azophenyl-2',4'- disulphonic acid (PPADS), which has the following structure:
  • the SERCA, EP3, RyR and NAADP receptors are classified as intracellular calcium channels, and can induce intracellular calcium elevation directly upon binding to a ligand.
  • Plasma membrane channels function in one of two ways they either release calcium directly following ligand binding or once bound to a ligand, activate the generation of an intracellular second messenger (such as NAADP or cADPr) which subsequently binds to the intracellular calcium channel, the binding triggering calcium release and an elevation in the intracellular calcium concentration.
  • plasma membrane channels include, P2X receptors (direct calcium release) and EDG receptors or Glutamate receptors (mGluR) (activate generation of an intracellular second messenger).
  • compounds which elevate intracellular calcium, and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention also include modulators of the EDG receptor, P2X receptor and Glutamate receptor (mGluR).
  • modulators of the EDG receptor, P2X receptor and Glutamate receptor (mGluR) are known compounds and/or can be readily identified, without undue experimentation, using known assay methods.
  • the compound which elevates intracellular calcium is typically an EDG receptor modulator, a P2X receptor modulator or a Glutamate receptor (mGluR) modulator.
  • P2X receptor modulators include, but are not limited to, compounds of formula (E) as defined above.
  • An example of a P2X receptor modulator is PPADS.
  • EDG receptor modulators include, but are not limited to sphingosine-1 -phosphate (SIP) and lyso-phosphatidyl-choline (LPC).
  • mGluR modulators include, but are not limited to glutamate.
  • the compound which elevates intracellular calcium is a SERCA inhibitor, a Ryanodine receptor modulator, an IP3 receptor modulator, a NAADP receptor modulator, an EDG receptor modulator, a P2X receptor modulator or a Glutamate receptor modulator.
  • the compound which elevates intracellular calcium is l ⁇ ,25- dihydroxyvitamin D 3 or any one of the SERCA inhibitors, Ryanodine receptor modulators, IP3 receptor modulators, NAADP receptor modulators, EDG receptor modulators, P2X receptor modulators or Glutamate receptor modulators defined above, or a pharmaceutically acceptable salt of any of those compounds.
  • the compound which elevates intracellular calcium is a compound selected from: l ⁇ ,25-dihydroxyvitamin D 3 ; curcumin, thapsigargin, cyclopiazonic acid, Cyclic ADP-
  • the compound which elevates intracellular calcium is thapsigargin, curcumin or l ⁇ ,25-dihydroxyvitamin D 3 (l ⁇ ,25(OH) 2 VD 3 ).
  • Diseases which have a NPC like cellular phenotype can be treated with a compound which elevates intracellular calcium in accordance with the present invention.
  • NPC disease is a complex lipid storage disorder that results from inactivation of the NPCl or NPC2 proteins (Vanier MT, Millat G.; Clin Genet 2003; 64: 269-81). This results in a poorly understood series of events that ultimately leads to neurodegeneration and premature death. Defining the early step(s) in the pathogenic cascade has the potential to lead to the identification of novel clinical intervention points, that in the future may be targeted to maximise therapeutic benefit for patients.
  • gangliosides GM2 and GMl have been shown modulate the function of calcium transporting ATPases (Duan et al. Arch Biochem Biophys 2006; 454: 155-9; Zhang et al., Arch Biochem Biophys 2005; 444: 1-6; Zhao et al. Arch Biochem Biophys 2004; 427: 204-12).
  • NPCl null cells have normal ER and mitochondrial calcium levels, but significantly lower calcium in Le/Lys.
  • the first biochemical change detected is a rapid elevation in sphingosine, which is the only lipid stored in NPC storage capable of inducing the Le/Lys calcium defect.
  • sphingosine storage is the primary event in NPC disease pathology, leading to subsequent depletion of Le/Lys calcium, endocytic transport abnormalities followed by GSL, sphingomyelin and cholesterol storage. It was also found that the therapeutic targeting of the calcium defect in NPC is of therapeutic benefit in the NPCl mouse.
  • the present inventors have determined for the first time the chronology of events following inactivation of NPCl using pharmacological agents.
  • the first detectable change the increase in sphingosine
  • SlP sphingosine- 1 phosphate
  • the elevation in sphingosine occured very rapidly, (during the first 30-60min following drug treatment) and then plateaued, suggestive of induction of a compensative change within the cell.
  • the decrease in SlP levels was only transient, as at 4h post-treatment SlP levels increased back to control levels.
  • lysosome is the primary source of sphingosine for SlP formation in healthy cells, but that the cell can compensate for loss of availability of this source of sphingosine, possibly by up- regulating the activity of neutral ceramidase.
  • Sphingosine generated from ceramide degradation has an amine group that is readily protonated.
  • sphingosine pKa of 8.9
  • NPCl should be considered as a candidate to be involved in the salvage pathway of sphingolipid biosynthesis, through potentially facilitating (either directly or indirectly) sphingosine efflux out of lysosomes.
  • the consequence is the rapid accumulation of sphingosine in the lysosome and a reduction in efflux of sphingosine out of the lysosome negatively impacting SlP generation.
  • NPCl pathogenic cascade Following sphingosine storage, the next event in NPCl pathogenic cascade is the development of abnormally low Le/Lys calcium levels. This is likely caused by sphingosine storage, as this was the only NPCl storage lipid capable of exogenously inducing this phenotype in healthy cells. This effect was rapid, suggesting a direct effect of sphingosine itself rather than a downstream metabolite.
  • the present inventors calculated that sphingosine storage levels ( ⁇ 0.75 ⁇ M in U18666A treated RAW cells and ⁇ 1.5 ⁇ M in NPCl mouse brain) approximate the exogenous concentrations ( ⁇ l ⁇ M) required to decrease Le/Lys calcium and subsequently induce an NPCl phenotype in normal cells.
  • Le/Lys calcium release is defective as for the store to be reduced by -70% would require constant calcium efflux, which would be sufficient to promote vesicular fusion and release maintaining function in the endocytic pathway. It is more likely that sphingosine storage inhibits calcium entry into the Le/Lys, so there is insufficient calcium for release to maintain normal fusion and trafficking in the endocytic pathway.
  • sphingosine is a known inhibitor of calcium ATPases and Na + /Ca 2+ exchangers and may inhibit calcium entry by such a mechanism (Futerman AH, Hannun YA; EMBO Rep 2004; 5: 777-82). This mechanism, however, does not require protons as the present inventors have shown that NPC Le/Lys maintains normal pH, in agreement with a previous report (Bach et al.; Clin Chim Acta 1999; 280: 173-9).
  • NPC by elevating cytosolic calcium via different mechanisms were evaluated: thapsigargin (Canova, N.K. et al.; Cell Biol Toxicol 2007; 23: 337-54), activated vitamin D3 (van de Graaf et al.; MoI Biol 2004; 89-90: 303-8) and curcumin (Cell Calcium 2002; 31 : 45-52).
  • thapsigargin Canova, N.K. et al.; Cell Biol Toxicol 2007; 23: 337-54
  • activated vitamin D3 van de Graaf et al.; MoI Biol 2004; 89-90: 303-8
  • curcumin Curcumin
  • curcumin was tested in the NPCl mouse disease model and it resulted in increased life expectancy and slowed the rate of disease progression.
  • agents that can compensate for the underlying calcium homeostatic defect in NPC disease by elevating intracellular (typically cytosolic and/or lysosomal) calcium, by any mechanism, could be used to treat NPC disease and any other disease which has a NPC like cellular phenotype.
  • sphingosine storage has been identified as the earliest measurable event in NPC disease suggesting that NPCl may either directly or indirectly be involved in sphingosine transport out of the lysosome.
  • NPC Niemann-Pick disease type C
  • Nemann-Pick disease type C like cellular phenotype and 'TSIPC like cellular phenotype as used herein are equivalent and mean a cellular phenotype which includes: (a) abnormal cholesterol metabolism and trafficking; (b) abnormal sphingolipid storage and trafficking; (c) defective endocytosis; and (d) defective Le/Lys calcium.
  • the abnormal sphingolipid storage involves the majority of sphingolipids in the cell being present at abnormally elevated levels.
  • (c) comprises defective endocytosis of substantially all biomolecules in the endocytic pathway, including substantially all lipids and biomolecules other than lipids, for instance proteins.
  • (d) comprises a reduction in calcium content within the lumen of acidic endosomes (such as late endosomes or lysosomes).
  • the diseases which have a Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention include (a) "primary" Niemann-Pick disease type C (NPC), and (b) diseases which have a secondary Niemann-Pick disease type C like cellular phenotype.
  • NPC Primary Niemann-Pick disease type C involves a mutation in either the NPCl or NPC2 gene.
  • the disease which has a NPC like cellular phenotype is typically Niemann-Pick disease type C (i.e. "primary" Niemann-Pick disease type C), for instance Niemann-Pick disease type Cl or Niemann-Pick disease type C2. More typically, the disease which has a NPC like cellular phenotype is Niemann-Pick disease type Cl .
  • the term "disease which has a NPC like cellular phenotype” also includes diseases which have a secondary Niemann-Pick disease type C (NPC) like cellular phenotype.
  • NPC Niemann-Pick disease type C
  • disease which has a secondary Niemann-Pick disease type C like cellular phenotype and “disease which has a secondary NPC like cellular phenotype” as used herein are equivalent and generally refer to any disease which does not involve a mutation in either the NPCl gene or the NPC2 gene and which involves:
  • the disease which has a secondary Niemann-Pick disease type C (NPC) like cellular phenotype is a disease which does not involve a mutation in either the NPCl gene or the NPC2 gene but which incurs the accumulation of a biomolecule, the accumulation of which in turn induces a Niemann-Pick disease type C like cellular phenotype.
  • the biomolecule is a class II amphiphile.
  • a class II amphiphile is a detergent-like molecule which is, typically, capable of altering the stability of the lysosome.
  • the class II amphiphile is 7-DHC.
  • the class II amphiphile may be U18666A, 7- ketocholesterol, progesterone or imipramine, all of which are steroids or steroid analogues known to induce free cholesterol storage and abnormal lipid endocytosis.
  • the biomolecule is a class II amphiphile which is a precursor or analogue of cholesterol.
  • the biomolecule may, for instance, be 7-DHC.
  • SLOS Smith-Lemli-Opitz Syndrome
  • the present inventors have observed that 7-DHC accumulates in Smith-Lemli-Opitz Syndrome (SLOS) cells to cause abnormal sphingolipid storage and transport in the LE/Lys system (a Niemann-Pick disease type C like cellular phenotype).
  • SLOS Smith-Lemli-Opitz Syndrome
  • Huntington's disease - (Truchina, E., et al., Hum. MoI. Genet., 2006, 15:3578-91); Cystic Fibrosis - (Gentzsch, M., et al., J. Cell. Sci., 2007, 120:447-455); Pelizaeus-Merzbacher disease - (Simons, M., et al., J. Cell. Biol., 2002, 157:327- 36); and Mucolipidosis II - (Inui, K., et al., Biochem. Int., 1989, 18:1129-35).
  • the disease which has a Niemann-Pick disease type C like cellular phenotype is other than cystic fibrosis.
  • the disease which has a Niemann- Pick disease type C like cellular phenotype is other than Tangier disease.
  • the disease which has a Niemann- Pick disease type C like cellular phenotype is other than Huntington's disease.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype is other than Tangier disease and other than Huntington's disease and other than cystic fibrosis.
  • the disease which has a secondary Niemann-Pick disease type C like cellular phenotype may be a disorder which alters the activity of an enzyme involved in cholesterol synthesis. Typically, therefore, the disease which has a secondary Niemann-Pick disease type
  • C like cellular phenotype is selected from Smith-Lemli-Opitz Syndrome, Tangier disease, Huntington's disease, Cystic Fibrosis, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis, for instance SLOS. More typically, the disease which has a secondary Niemann-Pick disease type C like cellular phenotype is selected from Smith-Lemli-Opitz Syndrome, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis, for instance SLOS.
  • the disease which has a secondary Niemann-Pick disease type C like cellular phenotype is selected from Smith-Lemli-Opitz Syndrome, Pelizaeus- Merzbacher disease and Mucolipidosis II (Icell).
  • the disease which has a secondary Niemann-Pick disease type C like cellular phenotype is Smith-Lemli-Opitz Syndrome (SLOS).
  • SLOS Smith-Lemli-Opitz Syndrome
  • the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type C, Smith-Lemli-Opitz Syndrome, Tangier disease, Huntington's disease, Cystic Fibrosis, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis.
  • the Niemann-Pick type C disease is Niemann- Pick type Cl disease or Niemann-Pick type C2 disease. More typically, the Niemann-Pick type C disease is Niemann-Pick type Cl disease.
  • the disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis is SLOS.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type C and Smith-Lemli-Opitz Syndrome. More typically, the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type Cl, Niemann-Pick disease type C2 and Smith- Lemli-Opitz Syndrome.
  • the diseases which have a Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention also include pathogenic infections which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
  • pathogenic infections are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
  • One such pathogenic infection is the infectious disease tuberculosis. Tuberculosis is a global health problem affecting approximately one third of the world's population and resulting in three million deaths each year.
  • a major current concern is the emergence of strains of Mycobacterium tuberculosis (Mtb) that are resistant to antibiotics.
  • Mtb One of the characteristic hallmarks of tuberculosis is the ability of Mtb to successfully survive within cells of the innate immune system including macrophages and monocytes. Multiple mechanisms are involved in the intracellular survival of Mtb, including defective acidification of the phagosome and inhibition of phosphatidylinositol-dependent trafficking pathways, via secretion of inositol-like lipids by the mycobacteria. Bacilli interact with cell surface complement receptors and are ingested into phagosomes that mature but do not fuse with lysosomes.
  • Binding to the complement receptor is normally followed by an intracellular cytosolic elevation in which calcium can stimulate phago-lysosome fusion.
  • this elevation is substantially reduced; the present inventors have shown that the ability of lysosomes to fuse with late endosomes relies upon calcium release, specifically from the late endosomal/lysosomal compartment itself.
  • insufficient calcium is released from acidic stores there is a complete block in late endosome - lysosome fusion.
  • a severe human disease results from calcium deficiency in acidic the acidic compartment, the lysosomal storage disease termed Niemann-Pick disease type C (NPC).
  • NPC Niemann-Pick disease type C
  • NPC is unusual as it is caused by mutations in two genes, NPCl or NPC2, that function as part of the same cellular pathway. However, the precise mechanistic link between these two genes remains unknown and the functional roles of these proteins remains enigmatic. NPCl encodes a multimembrane spanning protein of the limiting membrane of the late endosome/lysosome where as NPC2 is a soluble cholesterol binding protein of the lysosome. The inventors have discovered that when NPCl is inactivated sphingosine is the first lipid to be stored, suggesting that NPCl plays a role in the transport of sphingosine from the lysosome, where it is normally generated as part of sphingolipid catabolism.
  • Elevated sphingosine in turn causes a defect in calcium entry into acidic stores resulting in greatly reduced calcium release from this compartment. This then prevents LE/Lys fusion (calcium dependent process) and causes the secondary accumulation of lipids (cholesterol, sphingomyelin and glycosphingolipids) that are cargos in transit through the late endocytic pathway.
  • lipids cholesterol, sphingomyelin and glycosphingolipids
  • Other secondary consequences of inhibiting NPCl function include defective endocytosis and failure to clear autophagic vacuoles.
  • the present inventors have therefore investigated whether the NPC1/NPC2 cellular pathway is targeted by pathogenic mycobacteria to promote their survival in late endosomes and found this to be the case (see Fig. 11 and Example 11).
  • the diseases which have a Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention also include pathogenic infections which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
  • the invention provides a compound which elevates intracellular calcium for use in the treatment of a pathogenic infection capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
  • Such pathogenic infections include infection by any pathogen that that secretes a molecule (for instance a lipid or a secondary amine) that can inhibit NPCl function or any pathogen that prevents NPCl from getting to the compartment in which it normally functions (for instance by inhibition of late endosome acidification).
  • a pathogen that secretes a molecule for instance a lipid or a secondary amine
  • any pathogen that prevents NPCl from getting to the compartment in which it normally functions for instance by inhibition of late endosome acidification.
  • intracellular cholesterol storage would also be observed in combination with either of these phenotypes.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infection caused by any one of the following types of bacteria, all of which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype: Mycobacteria, Salmonella, Brucella, Coxiella, and Anaplasma phagocytophilum.
  • the pathogenic infection treated in accordance with the present invention is an infection caused by Mycobacteria (a mycobacterial infection).
  • Such infections include the infectious disease tuberculosis.
  • the invention provides a compound which elevates intracellular calcium for use in the treatment of tuberculosis.
  • the compound which elevates intracellular calcium is other than curcumin. Accordingly, the invention in one embodiment provides a compound which elevates intracellular calcium, which compound is other than curcumin, for use in the treatment of tuberculosis.
  • the mycobacterium which most commonly causes tuberculosis in humans is
  • Mycobacterium tuberculosis Although these species are less common in humans.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infection caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannetti and/or Mycobacterium microtti.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infection caused by Salmonella, Brucella or Coxiella.
  • the infection may be one caused by Salmonella enterica, Brucella abortus or Coxiella burnetti.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is a pathogenic infection caused by Anaplasma phagocytophilum.
  • Anaplasma phagocytophilum is a bacterium that lives inside white blood cells, and causes the disease human granulocytic anaplasmosis (HGA).
  • the invention provides a compound which elevates intracellular calcium for use in the treatment of human granulocytic anaplasmosis.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infectious disease, which infectious disease has has a NPC like cellular phenotype which is treated in accordance with the present invention.
  • the invention in one embodiment therefore provides a compound which elevates intracellular calcium for use in the treatment of an infectious disease, which infectious disease is capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
  • the infectious disease is tuberculosis.
  • the infectious disease is human granulocytic anaplasmosis.
  • the compound which elevates intracellular calcium may be administered, in order to treat or prevent said disease which has a NPC like cellular phenotype, as a dietary or food supplement.
  • the compound which which elevates intracellular calcium is for administration as a dietary supplement.
  • the invention further provides a dietary supplement comprising a compound which elevates intracellular calcium, which dietary supplement is for use in the treatment of a disease which has a NPC like cellular phenotype.
  • NPC Niemann-Pick disease type C
  • NPCl Niemann-Pick disease type Cl
  • NPC2 Niemann-Pick disease type C2
  • the invention provides a compound which reduces sphingosine storage for use in the treatment of Niemann-Pick disease type C.
  • Sphingosine storage can be reduced by disrupting sphingolipid biosynthesis.
  • Sphingolipid biosynthesis can be disrupted by the use of inhibitors of enzymes, such as transferases and synthases, that act upstream of glucosylceramide synthase or galactosylceramide synthase.
  • inhibitors are termed "inhibitors of ceramide biosynthesis”.
  • Inhibitors of ceramide biosynthesis are capable of inhibiting the synthesis of a sphingolipid and thereby reduce sphingosine storage.
  • Enzymes which act upstream of glucosylceramide synthase include serine palmitoyltransferase and dihydroceramide synthase.
  • Inhibitors of serine palmitoyltransferase include L-Cycloserine and Myriocin.
  • Inhibitors of dihydroceramide synthase include Fumonisin and Safingol (L-threo- dihydrosphingosine).
  • Sphingosine storage can also be reduced by inhibiting the degradation of ceramide, for instance by using a sphingomyelinase inhibitor (e.g. scyphostatin, manumycin A, W-7 and imipramine) or a ceramidase inhibitor (e.g. D-MAPP).
  • a sphingomyelinase inhibitor e.g. scyphostatin, manumycin A, W-7 and imipramine
  • a ceramidase inhibitor e.g. D-MAPP
  • compound which reduces sphingosine storage means any compound which reduces sphingosine storage by (a) inhibiting ceramide biosynthesis and/or (b) inhibiting ceramide degradation.
  • the compound which reduces sphingosine storage is an inhibitor of ceramide biosynthesis.
  • the skilled person can readily identify inhibitors of ceramide biosynthesis without undue experimentation, using known procedures (see, for instance, J. Biol. Chem., Vol. 268, No. 36, 1993, 27299-27306).
  • the inhibitor of ceramide biosynthesis is an inhibitor of serine palmitoyltransferase (for instance, L- Cycloserine or Myriocin) or an inhibitor of dihydroceramide synthase (for instance, Fumonisin or S Aggol) .
  • the inhibitor of ceramide biosynthesis is a compound of the following formula (DC):
  • R Ka is H, COOH or an unsubstituted or substituted ester
  • R 003 is an unsubstituted or substituted C 1-6 alkyl
  • R Kc and R ⁇ which are the same or different, are each independently selected from H, unsubstituted or substituted C 1-6 alkyl and unsubstituted or substituted phenyl;
  • R Ke and R ⁇ f which are the same or different, are each independently selected from H, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl; either (a) one of R Kg and R 001 is H and the other is OR ⁇ , wherein R Kr is selected from H, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl, or (b) R Kg and R 001 together form an oxo group;
  • R Kl is H, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted C 1-6 alkoxy and unsubstituted or substituted phenyl;
  • R Kj is H, unsubstituted or substituted C 1-6 alkyl or a group of the following formula
  • R 1 *" and R 1 * 0 which are the same or different, are each independently selected from OH, unsubstituted or substituted C 1-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C 1-6 alkylamino and unsubstituted or substituted di(C 1-6 )alkylamino;
  • R Ixk is H, unsubstituted or substituted C 1-6 alkyl or a group of the following formula (XI):
  • R Kp and R 0 ⁇ which are the same or different, are each independently selected from OH, unsubstituted or substituted C 1-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C 1-6 alkylamino and unsubstituted or substituted di(C 1-6 )alkylamino; and
  • R Km is selected from H and unsubstituted or substituted Ci -2O alkyl, which C 1-20 alkyl is optionally interrupted by N(R'), O, S or phenylene, wherein R' is H, Ci -6 alkyl or phenyl; or a pharmaceutically acceptable salt thereof.
  • r is O and q is 1.
  • R m> is unsubstituted Ci -6 alkyl. More typically, R ⁇ 5 is methyl.
  • R Ka is typically H.
  • R Kc and R Kd are independently selected from H and unsubstituted Ci -6 alkyl. More typically, however, R Kc and R Kd are both H.
  • R Ke and R ⁇ f are independently selected from H and unsubstituted Ci -6 alkyl. More typically, R Ke and R 0 ⁇ are both H. Usually, one of R Kg and R 001 is H and the other is OR 00" , wherein R 1 * is selected from H and unsubstituted C 1-6 alkyl. More typically, however, one of R Kg and R Kh is H and the other is OH. R Kl is typically unsubstituted C 1-6 alkyl, more typically methyl. Typically, R ⁇ j is a group of formula (X). Ususally, R 0 * is a group of formula (XI).
  • R 1 *" R Ko , R Kp and R Kq which are the same or different, are independently selected from H and unsubstituted Ci -6 alkyl. More typically, each of R , I E Xn R Ko , R Kp and R Ixq is H.
  • R Km is typically selected from unsubstituted or substituted C 1- I 0 alkyl. More typically, R Km is an unsubstituted or substituted C 1-6 alkyl.
  • R 1 * 1 " may be, for instance, -CH(CH 3 )(C 4 H 9 ).
  • R is C 1- 6 alkyl substituted with a hydroxyl group. More typically, R 0 * is CH 2 OH. Furthermore, R 1 * 3 is typically COOH or an unsubstituted ester. More typically, R ⁇ is COOH.
  • R Kc and R Kd are independently selected from H and unsubstituted C 1-6 alkyl. More typically, however, R Kc and R Kd are both H.
  • R Ke and R ⁇ f are independently selected from H and unsubstituted C 1-6 alkyl. More typically, R Ke and R ⁇ f are both H.
  • R Kg and R 001 together form an oxo group.
  • R Kl is typically H.
  • R ⁇ j and R 00 ' which may be the same or different, are independently selected from H and unsubstituted C 1-6 alkyl. More typically, R ⁇ j and R 0 ⁇ are both H.
  • R DCm is typically, in this embodiment, selected from unsubstituted or substituted Ci -6 alkyl. More typically, R Km is an unsubstituted Ci -6 alkyl. R Km may be, for instance, methyl.
  • Table 1 shows examples of compounds of formula (IX) which maybe employed in the present invention as compounds which reduce sphingosine storage. Such compounds are inhibitors of ceramide biosynthesis. More specifically, compound 1 (Myriocin) is a serine palmitoyltransferase inhibitor and compound 2 (Fumonisin) is a dihydroceramide synthase inhibitor.
  • Myriocin is a serine palmitoyltransferase inhibitor
  • compound 2 Flumonisin
  • the inhibitor of ceramide biosynthesis is a compound of the following formula (XII):
  • R Xa is H, substituted or unsubstituted Ci -2O alkyl, substituted or unsubstituted C) -20 alkylene-aryl, substituted or unsubstituted Ci -2O alkylene-C 3-20 heteroaryl, substituted or unsubstituted Ci -20 alkylene-C 3-25 cycloalkyl, substituted or unsubstituted Ci -20 alkylene-C 3 .
  • Ci -20 alkyl and Ci -2 Q alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, C] -6 alkyl or aryl; and R 501 and R Xc , which are the same or different, are independently selected from H, unsubstituted or substituted Ci -I0 alkyl and unsubstituted or substituted aryl; or a pharmaceutically acceptable salt thereof.
  • R Xa is H, substituted or unsubstituted Cj -1O alkyl or substituted or unsubstituted phenyl . More typically, R Xa is H, unsubstituted C 1-6 alkyl or unsubstituted phenyl . Even more typically, R Xa is H.
  • R 5 * and R Xc which are the same or different, are independently selected from H, unsubstituted Ci -6 alkyl and unsubstituted phenyl. More typically, R and R are both H.
  • Table 2 shows an example of a compound of formula (XII) which maybe employed in the present invention as a compound which reduces sphingosine storage.
  • the compound (compound 3) is an inhibitor of ceramide biosynthesis. More specifically, compound 3 (L- Cycloserine) is a serine palmitoyltransferase inhibitor.
  • the inhibitor of ceramide biosynthesis is Safingol (L-threo- dihydrosphingosine).
  • Safingol is an inhibitor of dihydroceramide synthase (Meyer, S.G.E. et al., Biochimica et Biophysica Acta 1643 (2003) 1-4).
  • the structure of Safingol is as follows:
  • the compound which reduces sphingosine storage is an inhibitor of ceramide degradation.
  • the skilled person can readily identify inhibitors of ceramide degradation without undue experimentation, using known procedures (see for instance J. Med. Chem. 2008, 51, 219-237 and Bioorg. Med. Chem., 9 (2001) 2901-2904, which describe the identification of sphingomyelinase inhibitor compounds, and Bioorg. Med. Chem., 16 (2008) 1032-1045 which describes the identification of ceramidase inhibitors).
  • the inhibitor of ceramide degradation is a sphingomyelinase inhibitor (for instance an acid sphingomyelinase inhibitor, such as W-7 or imipramine, or a neutral sphingomyelinase inhibitor, such as scyphostatin or manumycin A) or a ceramidase inhibitor (for instance an acid ceramidase inhibitor, such as D-MAPP).
  • a sphingomyelinase inhibitor for instance an acid sphingomyelinase inhibitor, such as W-7 or imipramine, or a neutral sphingomyelinase inhibitor, such as scyphostatin or manumycin A
  • a ceramidase inhibitor for instance an acid ceramidase inhibitor, such as D-MAPP
  • the inhibitor of ceramide degradation is a ceramidase inhibitor of formula (G):
  • R P1 is selected from hydrogen and unsubstituted or substituted Ci -6 alkyl
  • R p2 and R P3 which are the same or different, are independently selected from hydrogen, hydroxyl and unsubstituted or substituted C 1-6 alkyl;
  • R P4 is hydrogen, -NO 2 , -NH 2 or -N(H)C(O)(CH 2 ) n CH 2 Y pl , wherein Y P1 is hydrogen, aryl or heteroaryl, and wherein n is 0 or an integer of 1 to 10;
  • R P5 is selected from unsubstituted or substituted methyl, unsubstituted or substituted ethyl, aryl and heteroaryl;
  • Y P2 is -CH 2 - or -C(O)-;
  • Y P3 is -CH 2 - or -N(H)-;
  • E P1 is N or N + H, provided that E P1 is N when Y P2 is -C(O)-; and m is 0 or an integer of 1 to 14; or a pharmaceutically acceptable salt thereof.
  • R P1 is methyl or hydrogen. More typically, R P1 is hydrogen.
  • R P2 and R P3 are independently selected from hydrogen and hydroxyl. More typically, R P2 is selected from hydrogen and hydroxyl and R P3 is hydroxyl.
  • R P5 is ethyl or heteroaryl.
  • R P5 is heteroaryl, it is typically a cationic heteroaryl group and, more typically, pyridinium, as follows:
  • R is hydrogen or -NO 2 .
  • Y P1 is typically H or a cationic heteroaryl group.
  • the cationic heteroaryl group may be pyridinium.
  • E P1 is N + H
  • Y P2 is -CH 2 - and Y P3 is -CH 2 -.
  • E pl is N
  • Y P2 is -C(O)-
  • Y P3 is either -CH 2 - or -N(H)-.
  • n is an integer of 5 to 9. More typically, m is 5 or 9.
  • E pl is N + H
  • a suitable counter-anion will be present, for instance a halide, typically Br “ or Cl " .
  • a cationic aryl group for instance pyridinium
  • a suitable counter-anion will be present.
  • the counter anion may be a halide, for instance Br " or Cl " .
  • Table 3 shows examples of compounds of formula (G) which may be employed in the present invention as compounds which reduce sphingosine storage. Such compounds are ceramidase inhibitors and are described in Bioorg. Med. Chem., 16 (2008) 1032-1045.
  • the inhibitor of ceramide degradation is a neutral sphingomyelinase inhibitor selected from manumycin A, scyphostatin or a compound of formula (H):
  • R Q1 is C 1-6 alkyl, -R Q2 -aryl or -R Q2 -0H;
  • R ⁇ 2 is unsubstituted or substituted C 1-6 alkylene
  • R ⁇ 3 is unsubstituted or substituted C 1-20 alkyl, wherein said C 1-20 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C 1-6 alkyl or aryl; or a pharmaceutically acceptable salt thereof.
  • R Q1 is methyl, -R Q2 -phenyl or -R Q2 -0H.
  • R Q2 is methylene.
  • R Q3 is butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl. More typically, R Q3 is n-nonyl.
  • Table 4 shows examples of compounds of formula (H) which may be employed in the present invention as compounds which reduce sphingosine storage. Such compounds are neutral sphingomyelinase inhibitors and are described in Bioorg. Med. Chem., 9 (2001) 2901-2904. Table 4
  • the inhibitor of ceramide degradation is an acid sphingomyelinase inhibitor selected from W-5, W-7, dibucaine, mianserin, perhexiline, tamoxifen, trihexyphenidyl, amlodipine, bepridil, astemizole, suloctidil, AY9944, benztropine, camylofin, cloperastine, cocaine, clomiphene, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, paroxetine, sertraline, terfenadine and a compound of formula (J):
  • R l and R G3 which are the same or different, are independently selected from hydrogen, halo, cyano, -CF 3 , -S(O) 2 NMe 2 , -SMe and C 1-6 alkyl;
  • X 01 is C and Y 61 is CH and X 01 and Y 01 are linked by a double bond, or X G1 is selected from N and CH, Y 01 is CH 2 and X G1 and Y 01 are linked by a single bond;
  • R G2 is a group -Y° 2 -R G4 , wherein Y° 2 is substituted or unsubstituted C M alkyl and R G4 is selected from amino, C 1-10 alkylamino, di(C 1- i 0 )alkylamino and unsubstituted or substituted C 3-20 heterocyclyl; provided that X 01 , Y G1 and R G2 may together form the group:
  • R G1 and R G3 are hydrogen and the other is selected from hydrogen, halo, cyano, -CF 3 , -S(O) 2 NMe 2 , -SMe and C 1-6 alkyl. More typically, both R GI and R G3 are hydrogen.
  • the halo group is a chloro group.
  • Z G1 is either -S- or -CH 2 -CH 2 -.
  • X G1 is C and Y 01 is CH and X G1 and Y 01 are linked by a double bond, or X 01 is N and Y 01 is CH 2 and X G1 and Y 01 are linked by a single bond;
  • Y 02 is substituted or unsubstituted ethylene. More typically, Y 02 is selected from -CH 2 -CH 2 and -CH(CH 3 )-CH 2 -; Typically, R G4 is selected from -NMe 2 , -NHMe and the following heterocyclyl groups:
  • the compound which reduces sphingosine storage is an inhibitor of ceramide biosynthesis or an inhibitor of ceramide degradation. More typically, the compound which reduces sphingosine storage is an inhibitor of serine palmitoyltransferase, an inhibitor of dihydroceramide synthase, a sphingomyelinase inhibitor, for instance a neutral sphingomyelinase inhibitor or an acid sphingomyelinase inhibitor, or a ceramidase inhibitor, for instance an acid ceramidase inhibitor.
  • the compound which reduces sphingosine storage is any one of the inhibitors of ceramide biosynthesis and inhibitors of ceramide degradation defined above, or a pharmaceutically acceptable salt of any of those compounds.
  • the compound which reduces sphingosine storage is: Myriocin (ISP-I), Fumonisin, L-Cycloserine, L-t ⁇ reo-dihydrosphingosine (Safingol), D-e-MAPP, LCL16, LCL284, LCL120, B13, LCL15, LCL204, LCL85, W-5, W-7, triflupromazine, terfenadine, suloctidil, sertraline, promethazine, paroxetine, nortriptyline, maprotiline, norfluoxetine, fluoxetine, fendiline, protriptyline, chlorprothixene, promazine, pimethixene, drofenine, doxepine, cyproheptadine, cyclobenzaprine, clomiphene, cloperastine, camylof ⁇ n, bepridil, benztropine, astemizole, aml
  • the compound which reduces sphingosine storage is myriocin (ISP-I).
  • the compound which reduces sphingosine storage may be administered, in order to treat or prevent Niemann-Pick disease type C, as a dietary or food supplement.
  • the compound which reduces sphingosine storage is for administration as a dietary supplement.
  • the invention further provides a dietary supplement comprising a compound which reduces sphingosine storage, which dietary supplement is for use in the treatment of Niemann- Pick disease type C.
  • a compound which elevates intracellular calcium can advantageously be used in combination with an inhibitor of sphingo lipid biosynthesis in order to treat a disease which has a Niemann- Pick disease type C like cellular phenotype.
  • inhibitor of sphingolipid biosynthesis means a compound that is capable of inhibiting the synthesis or expression of a sphingolipid.
  • the sphingolipid is ceramide or a compound which uses ceramide as a backbone, for instance a glycosphingolipid (GSL). More typically, the sphingolipid is a ganglioside. Alternatively, the sphingolipid is a neutral GSL.
  • the inhibitor of sphingolipid biosynthesis may be a compound which reduces sphingosine storage, as defined above, for instance an inhibitor of ceramide biosynthesis or an inhibitor of ceramide degradation.
  • the inhibitor of sphingolipid biosynthesis may be an inhibitor of an enzyme which acts downstream of glucosylceramide synthase or galactosyl ceramide synthase.
  • the inhibitor of sphingolipid biosynthesis could be an inhibitor of a glycosyltransferase or a sulfotransferase, for instance an inhibitor of a glucosyltransferase, sialyltransferase, galactosyltransferasae, ceramide galactosyltransferase, fucosyltransferase or N-acetylhexosaminetransferase, or an inhibitor of glucosylceramide synthase - such inhibitor compounds are further defined below.
  • the compound which elevates intracellular calcium is for use in said treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype by coadministration with an inhibitor of sphingolipid biosynthesis.
  • the invention provides a product which comprises (a) a compound which elevates intracellular calcium and (b) an inhibitor of sphingolipid biosynthesis, for simultaneous, separate or sequential use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype.
  • the invention also provides a compound which elevates intracellular calcium for use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype by coadministration with an inhibitor of sphingolipid biosynthesis.
  • the invention also provides an inhibitor of sphingolipid biosynthesis for use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype by coadministration with a compound which elevates intracellular calcium.
  • the invention also provides a method of treating a disease which has a Niemann- Pick disease type C like cellular phenotype which method comprises administering to a patient in need of such treatment an effective amount of a compound which elevates intracellular calcium and an effective amount of an inhibitor of sphingolipid biosynthesis.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype is a pathogenic infection which is capable of blocking lysosome- phagosome fusion via induction of a NPC like cellular phenotype.
  • the disease which has a Niemann-Pick disease type C like cellular phenotype is tuberculosis.
  • the inhibitor of sphingolipid biosynthesis miglustat (NB-DNJ) was found to promote the normalisation of NPC cellular phenotypes from cells treated with BCG- derived lipids: see Example 13 and Figure 13 hereinbelow.
  • inhibitors of sphingolipid biosynthesis can be identified by incubating and or growing cells in culture in the presence of the putative inhibitor together with an assay for the effect of sphingolipid biosynthesis.
  • assays include the analysis of fluorescently-labelled glycosphingolipid carbohydrate headgroups by HPLC, thin-layer chromatography (TLC) of sphingolipids and analysis of sphingolipids using mass spectrometry (Neville DC, Anal. Biochem. 2004 Aug 15;331(2):275-82; Mellor HR Biochem. J. 2004 Aug l;381(Pt 3):861-6; Hayashi Y.
  • the inhibitor of sphingolipid biosynthesis may be an inhibitor of a glycosyltransferase or a sulfotransferase.
  • the inhibitor of sphingolipid biosynthesis may be an inhibitor of glucosyltransferase, sialyltransferase, galactosyltransferasae, ceramide galactosyltransferase, fucosyltransferase, or N-acetylhexosaminetransferase.
  • the inhibitor of sphingolipid biosynthes is an inhibitor of glucosylceramide synthase.
  • the inhibitor of sphingolipid biosynthesis is an inhibitor of ceramide biosynthesis.
  • the inhibitor may be an inhibitor of serine palmitoyltransferase or an inhibitor of dihydroceramide synthase. Many of these types of inhibitor compounds are described in WO 2008/012555.
  • the inhibitor of sphingolipid biosynthesis maybe an inhibitor of ceramide degradation, for instance a sphingomyelinase inhibitor (e.g. a neutral sphingomyelinase inhibitor or an acid sphingomyelinase inhibitor) or a ceramidase inhibitor (e.g. an acid ceramidase inhibitor).
  • the inhibitor of sphingolipid biosynthesis is a compound of of one of the following formulae (I), (H), (m), (IV), (V), (IX) and (XII):
  • X is O, S or NR 5 ;
  • R 5 is hydrogen, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkylene-aryl, substituted or unsubstituted C 1-20 alkylene-C 3-20 heteroaryl, substituted or unsubstituted Ci -20 alkylene-C 3-25 cycloalkyl, substituted or unsubstituted Ci -20 alkylene- C 3-20 heterocyclyl, substituted or unsubstituted Ci -20 alkylene-O-C 3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C 3-20 heteroaryl, substituted or unsubstituted C 3-25 cycloalkyl or substituted or unsubstituted C 3-20 heterocyclyl, or R 5 forms, together with R 1 , R 1 ', R 4 or R 14 , a substituted or unsubstituted C] -6 alkylene group, wherein said C 1-20 al
  • Y is O, S or CR 6 R 16 ;
  • R 1 , R 1 ', R 4 and R 14 which may be the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted Ci -20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, Ci -I0 alkylamino, di(Ci-io)alkylamino, amido, acylamido, -0-C 3-25 cycloalkyl and -0-C 3-20 heterocyclyl, provided that one of R 1 , R 1 ' , R 4 and R 14 may form, together with R 5 , a substituted or unsubstituted Ci -6 alkylene group, wherein said Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene;
  • R 2 , R 12 , R 3 , R 13 , R 6 and R 16 which may be the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Cj -20 alkyl, substituted or unsubstituted Ci -20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C M0 alkylamino, di(Ci-io)alkylamino, amido, acylamido -0-C 3-25 cycloalkyl and -O-C 3-2 o heterocyclyl, wherein said Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene; R 21 is selected from oxo, -L 30 -R 23 , -L 30 -C(O)N(H)-R 24 and a group of the following formula (VI):
  • L 30 is substituted or unsubstituted Ci -20 alkylene which is optionally interrupted by N(R'), O, S or arylene;
  • R 23 is carboxyl, hydroxyl, ester, phosphonate ester, phosphate ester, phosphoric acid and phosphonic acid;
  • R 24 is Ci -20 alkyl which is unsubstituted or substituted with one or more groups selected from carboxyl, hydroxyl, ester, phosphonate ester, phosphate ester, phosphoric acid and phosphonic acid, wherein said Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene;
  • R 30 is Ci -2O alkyl which is unsubstituted or substituted with one or more groups selected from carboxyl, hydroxyl, ester, amino, phosphonate ester, phosphate ester, phosphoric acid and phosphonic acid, wherein said Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene; and
  • R 22 is hydroxyl, oxo, acyloxy, phosphoric acid or -OC(O)-alk-C(O)OH, wherein alk is substituted or unsubstituted Ci -20 alkylene which is optionally interrupted by N(R'), O, S or arylene;
  • Base is selected from a group of any one of the following formulae (a), (b), (c), (d), (e), (f) and (g):
  • y is 0 or 1 ;
  • R 31 is OH;
  • R 32 is H or OH; or, provided that y is 0, R 31 and R 32 together form -O-C(R 33 )(R 34 )-O-, wherein R 33 and R 34 are independently selected from H and methyl;
  • A is substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkylene-aryl, substituted or unsubstituted C 1-2O alkylene-C 3-20 heteroaryl, substituted or unsubstituted Ci -20 alkylene-C 3-25 cycloalkyl or substituted or unsubstituted Ci -20 alkylene- C 3-20 heterocyclyl, wherein said Ci -20 alkyl and Ci -20 alkylene are optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci -6 alkyl or aryl, or A is a group of any one of the following formulae (g) to (k):
  • L 70 , L 701 and L 702 are independently selected from -0-, -C(R 35 )(R 36 )- and -NH-, wherein R 35 and R 36 are independently selected from H, OH and CH 3 ;
  • R 70 , R 71 and R 701 are selected from OH, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkoxy, substituted or unsubstituted C 1-10 alkylamino and -L 7I -(X 2 ) m -L 72 -R 72 ; wherein m is O or 1; X 2 is O, S, -C(R 45 )(R 46 )- or -O-C(R 45 )(R 46 )-, wherein R 45 and R 46 are independently selected from H, OH, phosphonic acid or a phosphonic acid salt; L 71 and L 72 are independently selected from a single bond and substituted or unsubstituted C 1-20 alkylene, which C 1-20 alkylene is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci -6 alkyl or aryl; and R 72 is C 3-25 cycloal
  • L J is substituted or unsubstituted Ci -20 alkylene
  • X ⁇ is N or C(R K6 ), wherein R K6 is H, COOH or ester;
  • Z ⁇ is O or CH(R 1 "); p is O or 1 ;
  • R K1 , R 1 ", R 10 , R K4 and R 1 " which are the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C 1-I o alkylamino, di(Ci -1 o)alkylamino, amido, acylamido, -0-C 3-25 cycloalkyl and -0-C 3-20 heterocyclyl, wherein said C 1-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
  • R 1 ⁇ and R m which are the same or different, are independently selected from H, substituted or unsubstituted C 1-6 alkyl or substitute
  • R m is H or substituted or unsubstituted C 1-20 alkyl, which C 1-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
  • R Ws is H or substituted or unsubstituted Ci -20 alkyl, which Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene;
  • R We is H, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Ci -20 alkyl, substituted or unsubstituted Ci -20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C M0 alkylamino, di(Ci-i 0 )alkylamino, amido, acylamido, -0-C 3-25 cycloalkyl, -O- C 3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C 3-20 heteroaryl, substituted or unsubstituted C 3-25 cycloalkyl or substituted or unsubstituted C 3-20 heterocyclyl, which Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene;
  • L w is substituted or unsubstituted Ci -20 alkylene which Ci -20 alkylene is optionally interrupted by N(R'), O, S or arylene;
  • R 91 and R 92 which are the same or different, are independently selected from H, substituted or unsubstituted Ci -20 alkyl, substituted or unsubstituted aryl and -L 91 -R 95 , wherein L 91 is substituted or unsubstituted Ci -20 alkylene, wherein said Ci -20 alkyl and said Cj -20 alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, Ci -6 alkyl or aryl, and wherein R 95 is substituted or unsubstituted aryl, amino, Ci -I0 alkylamino or di(Ci-io)alkylamino; R 93 is -L 92 -R 96 , wherein L 92 is a single bond or substituted or unsubstituted Ci -20 alkylene, which Ci -20 alkylene is optionally interrupted by N(R'), O, S or arylene, and wherein R
  • R 94 is H or substituted or unsubstituted Ci -20 alkyl, which Ci -20 alkyl is optionally interrupted by N(R'), O, S or arylene; q is O or 1 ; r is O or 1 ;
  • R Ka is H, COOH or an unsubstituted or substituted ester;
  • R 005 is an unsubstituted or substituted Ci -6 alkyl;
  • R Kc and R Kd which are the same or different, are each independently selected from H, unsubstituted or substituted C 1-6 alkyl and unsubstituted or substituted phenyl;
  • R Ke and R Kf which are the same or different, are each independently selected from H, unsubstituted or substituted Ci -6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl; either (a) one of R Kg and R 0 * is H and the other is OR 1 *, wherein R 1 * is selected from H, unsubstituted or substituted Ci -6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl, or (b) R Kg and R 001 together form an oxo group;
  • R Kl is H, unsubstituted or substituted C 1-6 alkyl, unsubstituted or substituted Ci -6 alkoxy and unsubstituted or substituted phenyl;
  • R Kj is H, unsubstituted or substituted C 1-6 alkyl or a group of the following formula (X):
  • R 1501 and R Ko which are the same or different, are each independently selected from OH, unsubstituted or substituted Ci -6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted Ci -6 alkylamino and unsubstituted or substituted di(C 1-6 )alkylamino;
  • R 1 * 1 * is H, unsubstituted or substituted C 1-6 alkyl or a group of the following formula (XI):
  • R Kp and R Kq which are the same or different, are each independently selected from OH, unsubstituted or substituted Ci -6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C 1-6 alkylamino and unsubstituted or substituted di(C 1-6 )alkylamino;
  • R Km is selected from H and unsubstituted or substituted Ci -20 alkyl, which Ci -20 alkyl is optionally interrupted by N(R'), O, S or phenylene, wherein R' is H, C 1-6 alkyl or phenyl;
  • R Xa is H, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkylene-aryl, substituted or unsubstituted C 1-2O alkylene-C 3- 2 0 heteroaryl, substituted or unsubstituted Ci -20 alkylene-C 3-25 cycloalkyl, substituted or unsubstituted Ci -20 alkylene-C 3- 20 heterocyclyl, substituted or unsubstituted C 1-20 alkylene-O-C 3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C 3-20 heteroaryl, substituted or unsubstituted C 3-25 cycloalkyl or substituted or unsubstituted C 3-20 heterocyclyl wherein said Ci -2O alkyl and C 1-20 alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, C 1-6 alky
  • R 503 and R Xc which are the same or different, are independently selected from H, unsubstituted or substituted C 1-10 alkyl and unsubstituted or substituted aryl; or a pharmaceutically acceptable salt thereof.
  • the inhibitor of sphingolipid biosynthesis may be a compound of any one of formulae (G), (H) and (J) defined above, or a pharmaceutically acceptable salt thereof.
  • the inhibitor of sphingolipid biosynthesis is typically one of the following compounds: N-butyldeoxynojirimycin; N-nonyldeoxynojirimycin; N-butyldeoxygalactonojirimycin; N- 5-adamantane-l-yl-methoxypentyl-deoxynojirimycin; alpha-homogalactonojirimycin; nojirimycin; deoxynojirimycin; N7-oxadecyl-deoxynojirimycin; deoxygalactonojirimycin; N-butyl-deoxygalactonoj irimycin; N-nonyl-deoxygalactonoj irimycin; N-nonyl- ⁇ deoxygalactonojirimycin; N7-oxanonyl-6deoxy-DGJ; alpha-homoallonoj irimycin; beta-1- C-butyl-deoxygalactonoj i
  • the inhibitor of sphingo lipid biosynthesis may alternatively be any one of the compounds of formula (DI) described in R. Wang et al., Biooorg. & Med. Chem., Vol. 5, No. 4, pp 661-672, 1997; X. Wang et al., Medicinal Research Reviews, Vol. 23, No. 1, 32- 47, 2003; Schafer et al., J. Org. Chem. 2000, 65, 24-29; and Qiao et al., J. Am. Chem. Soc, 1996, 118, 7653-7662, and in WO 2008/012555.
  • DI formula
  • the inhibitor of sphingolipid biosynthesis may alternatively be any one of the following compounds: D-e-MAPP, LCL16, LCL284, LCL120, B13, LCL15, LCL204, LCL85, W-5, W-7, trifiupromazine, terfenadine, suloctidil, sertraline, promethazine, paroxetine, nortriptyline, maprotiline, norfluoxetine, fluoxetine, fendiline, protriptyline, chlorprothixene, promazine, pimethixene, drofenine, doxepine, cyproheptadine, cyclobenzaprine, clomiphene, cloperastine, camylofin, bepridil, benztropine, astemizole, amlodipine, trimipramine, trihexylphenidyl, trifluoroperazin, thioridazin, thioproperazine,
  • R is CH 3 or CH 2 C 6 Hs; or a pharmaceutically acceptable salt thereof.
  • the inhibitor of sphingolipid biosynthesis employed is N- butyldeoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ). More typically, the inhibitor of sphingolipid biosynthesis is NB-D ⁇ J.
  • NB-DGJ is the galactose analogue of NB-D ⁇ J.
  • NB-DGJ inhibits GSL biosynthesis comparably to NB-D ⁇ J but lacks certain side effect activities associated with NB-D ⁇ J. There has been extensive use of NB-DGJ in mouse models of GSL storage diseases and it is very well tolerated.
  • the inhibitor of sphingolipid biosynthesis employed in combination with the compound which elevates intracellular calcium is NB-DGJ.
  • the compound which elevates intracellular calcium is curcumin.
  • a compound which elevates intracellular calcium, a compound which reduces sphingosine storage or an inhibitor of sphingolipid biosynthesis, for use in accordance with the present invention can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously.
  • the compound may therefore be given by injection or infusion.
  • the compound which elevates intracellular calcium may be presented for administration in a liposome.
  • the compound may be encapsulated or entrapped in the liposome and then administered to the patient to be treated.
  • Active ingredients encapsulated by liposomes may reduce toxicity, increase efficacy, or both.
  • liposomes are thought to interact with cells by stable absorption, endocytosis, lipid transfer, and fusion (R.B. Egerdie et al., 1989, J. Urol. 142:390).
  • the dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a compound is administered alone to adult humans is 0.0001 to 50 mg/kg, most commonly in the range of 0.001 to 10 mg/kg, body weight, for instance 0.01 to 1 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg/kg body weight, preferably from 0.0001 to 0.1 mg/kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule.
  • a dose to treat human patients may range from about 0.1 mg to about 1000 mg of a compound for use in accordance with the invention, more typically from about 10 mg to about 1000 mg of a compound for use in accordance with the invention.
  • a typical dose may be about 100 mg to about 300 mg of the compound.
  • a dose may be administered once a day (QID), twice per day (BE)), or more frequently, depending on the pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism, and excretion of the particular compound.
  • toxicity factors may influence the dosage and administration regimen.
  • the pill, capsule, or tablet When administered orally, the pill, capsule, or tablet may be ingested daily or less frequently for a specified period of time. The regimen may be repeated for a number of cycles of therapy.
  • a compound is formulated for use as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier or diluent.
  • the compositions are typically prepared following conventional methods and are administered in a pharmaceutically suitable form.
  • the compound may be administered in any conventional form, for instance as follows: A) Orally, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is present as such, or mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides for example polyoxyethylene sorbitan monooleate.
  • Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
  • compositions for use in accordance with the invention may also be in the form of oil-in- water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occuring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan mono- oleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsion may also contain sweetening and flavouring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose.
  • a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.
  • Such formulations may also contain a demulcent, a preservative and flavouring and coloring agents;
  • sterile injectable aqueous or oleaginous suspensions This suspension may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic paternally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • hi addition fatty acids such as oleic acid find use in the preparation of injectables;
  • a suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and poly-ethylene glycols
  • Example 1 Abnormally low lysosomal Ca 2+ levels are caused by sphingosine storage in NPCl cells
  • Lysosomal calcium levels are decreased following only Ih treatment ( Figure 2, graph B) which correlates with elevations in sphingosine levels at 30 min and Ih post Ul 8666a treatment ( Figure 2, graph C). This suggests that sphingosine storage is the primary event in NPCl pathology and leads to the lysosomal calcium defect which ultimately results in downstream lipid storage.
  • NPCl null CHO cells with l ⁇ M thapsigargin for Ih leads to a correction in defective transport of BODIP Y-LacCer from the endocytic system to the Golgi ( Figure 3.1, A, panels I to IV). This is associated with a gradual reduction in GSL levels back to wild- type levels after 24h treatment with thapsigargin ( Figure 3.1, graph B). Cholesterol localization (filipin, Fig. 3.2, A, panels I to IV) and cholesterol levels (amplex red cholesterol oxidase assay, Fig. 3.2, graph B) also return to normal at Ih post thapsigargin treatment.
  • NPCl knock-out mice were fed a diet of pelleted mouse chow with or without curcumin amounting to a dosage of 150mg/kg/day from weaning at 3 weeks of age until death.
  • Curcumin fed mice had superficial improvements in coat condition and also improved gait compared to ataxic untreated NPC mice ( Figure 5, A, panels I to IV). Curcumin fed NPCl mice also exhibited improved weight gain and a delay in subsequent weight loss accompanied by an increase in life expectancy ( Figure 5, B). Additional functional benefits were seen in motor function as measured by the open field test ( Figure 5, C), curcumin fed NPCl mice remained functional for an additional 3 weeks ( ⁇ 33% improvement) compared to untreated NPC mice.
  • Example 6 Altered endocytosis in diseases with associated NPC-like phenotypes can also be corrected following curcumin treatment
  • graphs corrects the defective transport of BODIPY- LacCer that is observed under cholesterol null growth conditions (LPDS, Figure 6 micrographs) illustrating that increasing intracellular calcium is a potential therapy for any disorder where the function of the NPCl protein is inhibited.
  • Example 7 Curcumin reduces NPCl-null CHO cell lysosomal storage
  • Treatment with 30 ⁇ M curcumin (an inhibitor of the sarco/endoplasmic reticulum calcium ATPase) for the indicated time leads to a reduction in lysotracker staining indicative of a reduction in lysosomal storage.
  • Images are representative of NPCl-null CHO cell lysotracker staining (top) and 24h curcumin treated NPCl-null CHO cells (bottom): a reduction in fluorescence is clearly visible.
  • Example 8 increased life expectancy of NPCl mice fed curcumin and further benefit upon combination of curcumin with NB-DNJ
  • Untreated NPCl knock-out mice loose weight rapidly between 7-11 weeks age and are all dead by week 11.
  • NPCl knock-out mice fed a diet of mouse chow supplemented with 150mg/kg/day curcumin have a similar decrease in body weight compared to untreated NPCl mice but survive until 13.5 weeks of age (23% increase).
  • Combination treatment see diamond- shaped data points in Figure 8) of curcumin and NB-DNJ (NB-DNJ alone leads to a 1 week increase in lifespan of NPCl mice, Zervas et al. Current Biology 2001) leads to a reduction in decline of NPCl knock-out mice body weight and an increase in survival to 16 weeks (45% increase) indicative of synergy between the two drugs (see Figure 8).
  • Example 9 - ISP-I partially corrects the reduction in NPCl-null CHO cell lysosomal calcium levels
  • Wild-type (see upper, black trace in Figure 9), NPCl-null (see middle, light grey trace in Figure 9) and 15OnM ISP-1/myriocin treated NPCl-null (see lower, dark grey trace in Figure 9) CHO cells were labeled with Calcium Green 1-AM and Fura Red- AM to measure intracellular calcium. Lysosomal calcium was released following addition of ionomycin to permeabilise all membranes to calcium (apart from the lysosomal membrane) and then GPN (a substrate of cathepsin C which upon cleavage induces lysosomal osmotic lysis).
  • GPN a substrate of cathepsin C which upon cleavage induces lysosomal osmotic lysis.
  • NPCl -null cells have a characteristic reduction in lysosomal calcium compared to wild-types.
  • 15OnM ISP-I an inhibitor of serine palmitoyltransferase, the first sphingolipid biosynthetic enzyme
  • NPCl lysosomal calcium is elevated almost to normal levels (see Figure 9).
  • Example 10 ISP-I (myriocin) reduces NPCl-null CHO cell lysosomal storage
  • Treatment with 15OnM ISP- 1 /myriocin (an inhibitor of serine palmitoyltransferase the first enzyme in sphingolipid biosynthesis) for the indicated time leads to a reduction in lysotracker staining indicative of a reduction in lysosomal storage (see Figure 10 (A), third and fourth micrographs, and Figure 10 (B), third and fourth bars of the graph).
  • Images (A) are representative microscopy images of lysotracker staining similar to the 96 well plate assay.
  • In (B) a reduction in fluorescence is clearly visible at 3 and 5 days post-treatment.
  • Example 11 Live mycobacteriutn bovis (BCG) and mycotic acid lipids (M.A.) from mycobacterium tuberculosis (Tb) induce an NPCl phenotype in RAW mouse macrophages
  • RAW cells were infected overnight with either live BCG, live Mycobacterium smegmatis (M. smeg) or mycolic acid lipids isolated from Tb, fixed and stained with the indicated probes for sphingomyelin (lysenin), ganglioside GMl and endocytosis (cholera toxin) or cholesterol (filipin). Punctate accumulation of all lipids is indicative of an NPCl storage phenotype and dysfunctional endocytosis, this combination of phenotypes is specific for NPCl. As can be seen from the micrographs in Figure 11, this NPCl phenotype was observed with live BCG and lipids from Tb but not from the non-pathogenic M. smeg. Live mycobacterium bovis (BCG) and mycolic acid lipids (M.A.) from Mycobacterium tuberculosis (Tb) therefore induce an NPCl phenotype in RAW mouse macrophages.
  • BCG live
  • Example 12 Live BCG infection depletes lysosomal calcium which can be overcome using calcium agonists (curcumin) to clear the infection
  • RAW cells grown in the presence of the calcium indicator Rhod-dextran for 24h to label lysosomes, were infected overnight with either live or heat killed (HK) BCG.
  • Figure 12 A shows that infection with live-BCG depletes calcium in the lysosome (reduced Rhod- dextran signal, representative images of each experiment are shown below the graph) whereas infection with dead HK-BCG cannot induce this phenotype.
  • Figure 12B shows that treatment of live BCG infected cells with curcumin for 4h to elevate cytosolic calcium levels leads to clearance of infectious mycbacteria (reduction in GFP-labelled BCG signal) and a reduction in cholesterol storage (filipin stained).
  • Example 13 Depletion of sphingolipids with miglustat (NB-DNJ) reverses the accumulation of sphingomyelin induced by BCG and Tb secreted lipids
  • RAW macrophages were pre-treated with either vehicle (water) or 5OmM MB-DNJ for 24h prior to addition of either BCG or Tb secreted lipids (mycolates) for a further 48h at a concentration of 5mg/ml to induce the NPCl -like phenotype.
  • Cells were subsequently fixed and stained with lysenin (blue, or dark grey shading in Figure 13) for sphingomyelin or Hoechst (green, or light grey shading in Figure 13) for the nuclei.
  • micrographs in Figure 13 show that under normal conditions sphingomyelin can be found as punctate structures on the plasma membrane, but following treatment with MB-DNJ there is an elevation in sphingomyelin levels as a result of increased flux through that biosynthetic pathway. Addition of BCG or Tb mycolates induces intracellular storage of large quantities of sphingomyelin in compartments that surround the nucleus and spread out through the cytosol (lysosomes).
  • Tablets each weighing 0.15 g and containing 25 mg of a compound which elevates intracellular calcium or a compound which reduces sphingosine storage, for use in accordance with the invention, are manufactured as follows:
  • the active compound, lactose and half of the corn starch are mixed. The mixture is then forced through a sieve 0.5 mm mesh size.
  • Corn starch (10 g) is suspended in warm water (90 ml). The resulting paste is used to granulate the powder. The granulate is dried and broken up into small fragments on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium is added, carefully mixed and processed into tablets.
  • the compound which elevates intracellular calcium or the compound which reduces sphingosine storage, for use in accordance with the invention is dissolved in most of the water (35° 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate.
  • the batch is then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.
  • the active compound is dissolved in the glycofurol.
  • the benzyl alcohol is then added and dissolved, and water added to 3 ml.
  • the mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).
  • the compound which elevates intracellular calcium or the compound which reduces sphingosine storage, for use in accordance with the invention is dissolved in a mixture of the glycerol and most of the purified water.
  • An aqueous solution of the sodium benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavour.
  • the volume is made up with purified water and mixed well.

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Abstract

The present invention provides a compound which elevates intracellular calcium for use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype. The invention further provides a compound which reduces sphingosine storage for use in the treatment of Niemann-Pick disease type C.

Description

TREATING NIEMANN-PICK DISEASE TYPE C AND RELATED DISORDERS BY ELEVATING
INTRACELLULAR CALCIUM
FIELD OF THE INVENTION
The present invention relates to the treatment of diseases which have a Niemann- Pick disease type C (NPC) like cellular phenotype including, but not limited to, Niemann- Pick disease type C.
BACKGROUND TO THE INVENTION
Niemann-Pick disease type C (NPC) is an inherited neurodegenerative late endosomal/lysosomal (Le/Lys) lipid storage disorder. It is caused by mutations in the NPCl gene that encodes a transmembrane protein of the Le/Lys. NPCl has been suggested to facilitate endocytic transport and lysosomal lipid efflux. Additionally, a clinically identical disease (also called NPC) can result from mutation of an unrelated gene, NPC2, that encodes a soluble protein of the Le/Lys. Currently, the function of NPCl is unknown whereas NPC2 is a protein that transports cholesterol from inner lysosomal vesicles to the limiting membrane of the lysosome.
An unusual feature of NPC disease is the broad range of lipid species that accumulate, including cholesterol, sphingomyelin, glycosphingolipids (GSLs) and unusually sphingosine. Currently, the mechanism(s) leading to this complex pattern of storage remains unknown. How lipid storage causes the neurodegenerative phenotype is also not fully understood.
Current therapeutic approaches for NPCl are limited. No clinical benefit has been achieved by reducing cholesterol storage (via statins, dietary reduction or knock-out of LDL receptors (Erickson et al., J Inherit Metab Dis 2000, 23: 54-62; Patterson et al., Neurology 1993; 43: 61-4). However, some benefit has been reported in a clinical trial using an inhibitor of GSL biosynthesis: the imino sugar drug, miglustat (Patterson et al., Rev Neurol (separata) 2006; 43: 8). Together these findings suggest that the mechanisms of pathogenesis are complex and cannot be attributed simply to cholesterol or GSL storage alone. There is therefore a need to develop improved treatments for NPC and related disorders which have a Niemann-Pick disease type C (NPC) like cellular phenotype, for instance Smith-Lemli-Opitz Syndrome (SLOS) and Niemann-Pick disease type C2 (NPC2). SUMMARY OF THE INVENTION
The present invention relates to the findings presented herein that NPCl cells have a large reduction in the late endosomal/lysosomal calcium store. In a drug-induced NPC model, the first measurable event in the pathogenic cascade was sphingosine storage that in turn caused calcium depletion in the late endosome/lysosome. This then led to defective endocytic transport/fusion and the secondary storage of cholesterol, sphingomyelin and glycosphingolipids. The unique calcium phenotype, caused by sphingosine storage, represents a novel target for therapeutic intervention for the treatment of diseases having an NPC-like cellular phenotype.
Agents that elevate intracellular calcium were found to restore a normal cellular phenotype in NPC and SLOS cells, and to prolong the survival of and improve the function of the NPCl mouse. Such agents are therefore of potential therapeutic benefit to Niemann- Pick disease type C patients, for instance Niemann-Pick disease type Cl patients, Niemann-Pick disease type C2 patients and to patients of other diseases which have a Niemann-Pick disease type C (NPC) like cellular phenotype, for instance Smith-Lemli- Opitz Syndrome (SLOS).
Accordingly, the present invention provides a compound which elevates intracellular calcium for use in the treatment of a disease which has a Niemann-Pick disease type C (NPC) like cellular phenotype.
The invention also provides a method of treating a disease which has a NPC like cellular phenotype, which method comprises administering to a patient in need of such treatment an effective amount of a compound which elevates intracellular calcium.
The invention also provides a pharmaceutical composition for use in treating a disease which has a NPC like cellular phenotype, comprising a pharmaceutically acceptable carrier or diluent and a compound which elevates intracellular calcium.
The invention also provides the use of a compound which elevates intracellular calcium in the manufacture of a medicament for the treatment of a disease which has a NPC like cellular phenotype. The invention also provides an agent for the treatment of a disease which has a
NPC like cellular phenotype, comprising a compound which elevates intracellular calcium.
It has also now been found that pathogens which are capable of blocking phagosome-lysosome fusion, including M. tuberculosis, target the Niemann-Pick Disease type C (NPC) cellular pathway in order to promote their survival in late endosomes. Infections caused by such pathogens, including the infectious disease tuberculosis, are also therefore "diseases which have a NPC like cellular phenotype" that can be treated in accordance with the present invention. Examples 11 to 13 hereinbelow support that therapies which are effective in treating NPC disease, including administration of agents that elevate intracellular calcium and compounds that inhibit sphingolipid biosynthesis, are effective in promoting clearance of such pathogens from infected cells.
Accordingly, in one embodiment, the disease which has a Niemann-Pick disease type C (NPC) like cellular phenotype is a pathogenic infection, which pathogenic infection is capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype. In one preferred embodiment, the pathogenic infection is tuberculosis. hi another aspect, the invention provides a compound which reduces sphingosine storage for use in the treatment of Niemann-Pick disease type C.
The invention also provides a method of treating Niemann-Pick disease type C, which method comprises administering to a patient in need of such treatment an effective amount of a compound which reduces sphingosine storage.
The invention also provides a pharmaceutical composition for use in treating Niemann-Pick disease type C, comprising a pharmaceutically acceptable carrier or diluent and a compound which reduces sphingosine storage. The invention also provides the use of a compound which reduces sphingosine storage in the manufacture of a medicament for the treatment of Niemann-Pick disease type C.
The invention also provides an agent for the treatment of Niemann-Pick disease type C, comprising a compound which reduces sphingosine storage.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows six graphs, A to F, in which:
A is a graph of ER calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with lOOμM ryanodine in wild type cells (black line) and NPCl cells (grey line).
B is a graph of ER calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with lμM thapsigargin in wild type cells (black line) and NPCl cells (grey line). C is a graph of mitochondrial calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with 2μM CCCP in wild type cells (black line) and NPCl cells (grey line).
D is a graph of Le/Lys calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with 200μM GPN in wild type cells (black line) and NPCl cells (grey line).
E is a graph of Le/Lys calcium release (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes after treatment with 50OnM bafilomycin Al in wild type cells (black line) and NPCl cells (grey line). F is a graph of Le/Lys calcium release (y axis) using 200μM GPN in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of minutes, in normal cells, after 5 min pre-incubation with 1 μM sphinganine (line 1), 0.1 μM sphingosine (line 3), 1 μM sphingosine (line 4) and after no preincubation (control; line 2).
Figure 2 shows: A (upper panel / first row): micrographs of RAW macrophages treated with Ul 8666a over a 24h timecourse, leading to a blockage in transport of BODIPY labelled lactosylceramide (BODIPY-LacCer) from late endosomes (punctate appearance) to the Golgi (perinuclear crescent staining) at 2h post-treatment, persisting up to 24h.
A (lower panel / second row): micrographs of RAW macrophages treated with Ul 8666a over a 24h timecourse, showing that cholesterol levels are normal for the first 4h of treatment but are elevated after 8h and 24h.
B: graph of lysosomal calcium level (measured with GPN) (y axis) in arbitrary fluorescence units (deltaF/FO) versus time (x axis) in units of hours of Ul 8666a treatment.
C: graph of the sphingosine level (y axis) in units of μM sphingosine / mg protein versus Ul 8666a incubation time (x axis) in units of hours.
D: graph of the GSL level (measured by HPLC) (y axis) in units of pmol GSL / μg protein versus Ul 8666a incubation time (x axis) in units of hours.
Figure 3 shows:
1: A, micrographs I to IV: show that treatment of NPCl null CHO cells with lμM thapsigargin for Ih leads to a correction in defective transport of BODIPY-LacCer from the endocytic system to the Golgi
1 : B is a bar chart showing the GSL level (y axis) in units of pmol GSL / μg protein in normal cells (left hand bars) and NPCl cells (right hand bars) after 1 hour (white bars), 4 hours (dark grey bars) and 24 hours (light grey bars) of treatment with lμM thapsigargin and without thapsigargin treatment (control; black bars).
2: A, micrographs I to IV: show that cholesterol localization (filipin) returns to normal at Ih post thapsigargin treatment. 2: B is a bar chart showing cholesterol levels (y axis) in units of μg cholesterol / μg protein in RA25 cells (left hand bars) and CT43 cells (right hand bars) after 1 hour (dark grey bars), 4 hours (light grey bars) and 24 hours (white bars) of treatment with lμM thapsigargin and without thapsigargin treatment (control; black bars).
3: A, micrographs I to FV: show the levels and localization of neutral lipids using nile red; correct localization of neutral lipids to the perinuclear ER is observed, illustrating that the stored cholesterol in NPCl cells can be delivered to the ER for utilization following elevation of cytosolic calcium.
3: B is bar chart showing the levels of cholesterol esters (y axis) in units of μg cholesterol esters / μg protein in RA25 cells (left hand bars) and CT43 cells (right hand bars) after 1 hour (dark grey bars) and 4 hours (white bars) of treatment with lμM thapsigargin and without thapsigargin treatment (control; black bars).
Figure 4 consists of micrographs of BODIP Y-LacCer transport in wild- type (first row / upper panel) and NPCl null glial cells (second row / lower panel) which are untreated (fist column), treated with lμM thapsigargin (second column), 30μM curcumin (third column) and 1OnM lα, 25-dihydroxyvitamin D3 (lα,25(OH)2VD3) (fourth column).
Figure 5 shows:
A, micrographs I to IV: show that curcumin-fed mice (right hand column, micrographs π and rV) had superficial improvements in coat condition and also improved gait compared to ataxic untreated NPC mice (left hand column, micrographs I and IH). B: graph of mouse weight (y axis) in units of grams versus time (x axis) in units of weeks. The data points represented by solid black squares are for NPCl knock-out mice which were fed a diet of pelleted mouse chow without curcumin from weaning at 3 weeks of age until death. The data points represented by hollow black circles are for NPCl knockout mice which were fed a diet of pelleted mouse chow with curcumin amounting to a dosage of 150mg/kg/day from weaning at 3 weeks of age until death.
C: bar chart of mouse exploration activity in units of rearing/activity per minute (y axis) versus age (x axis) in units of weeks. The black bars are for NPCl knock-out mice which were fed a diet of pelleted mouse chow with curcumin amounting to a dosage of 150mg/kg/day from weaning at 3 weeks of age until death. The white bars are for NPCl knock-out mice which were fed a diet of pelleted mouse chow without curcumin from weaning at 3 weeks of age until death. The grey bars are for wild type NPCl mice which were fed a diet of pelleted mouse chow without curcumin from weaning at 3 weeks of age until death.
Figure 6 shows images of BODIPY-LacCer transport in wild-type (first row / upper left panel) and SLOS murine embryonic fibroblast cells (first row / upper right panel & second row both panels) which are untreated (fist column), or grown in lipoprotein deficient serum (LPDS) (second column) with 30μM curcumin (second column / lower panel). Figure 6 also show micrographs of GPN mediated lysosomal calcium release in units of arbitrary fluorescence (right hand side 4 panels) of wild-type (upper row) and SLOS (lower row) fibroblasts grown in foetal calf serum (left hand side column) or LPDS (right hand side column).
Figure 7 shows a graph of lysotracker fluorescence absorption (indicative of lysosomal storage) (y axis) in fluorescence units versus time (x axis) in units of hours for NPCl -null CHO cells either without ("npcl") or with ("4hs", "8hs", "16hs") treatment with 30 μM curcumin for the time indicated. The two micrographs (labelled "CHO" and "curcumin") show NPCl -null CHO cell lysotracker staining ("CHO") and 24-hour 30μM curcumin- treated NPCl -null CHO cell lysotracker staining ("curcumin"). Figure 8 is a graph of mouse weight (y axis) in units of grams versus age (x axis) in units of weeks. The data points represented by triangles are for untreated NPCl knock-out mice. The data points represented by circles are for NPCl knock-out mice fed a diet supplemented with 150 mg/kg/day curcumin. The data points represented by diamonds are for NPCl knock-out mice treated with a combination of curcumin and NB-DNJ. Figure 9 is a graph of fluorescence (indicative of intracellular calcium levels) (y axis) in fluorescence units versus time (x axis) in units of minutes for wild-type (upper, black trace), NPCl -null (middle, light grey trace) and 15OnM myriocin-treated NPCl (lower, dark grey trace) CHO cells labelled with Calcium Green 1-AM and Fura Red- AM. Figure 10 shows: A: Four micrographs, showing lysotracker-stained wild type CHO cells (CHO WT),
NPC-I null CHO cells (CHO NPCl), NPCl cells treated with 15OnM ISP-I (myriocin) for 3 days (NPCl+ISP-1 3 DAYS) and for 5 days (NPCl+ISP-1 5 DAYS). B: A bar chart of fluorescence absorption (indicative of lysosomal storage) (y axis) for various CHO cells (x axis): either wild type (WT), NPCl -null (NPCl), NPCl treated with 15OnM ISP-I (myriocin) for 3 days (NPCl+ISP-1 3 DAYS) or NPCl treated with 15OnM ISP-I (myriocin) for 5 days (NPCl+ISP-1 5 DAYS). Figure 11 consists of micrographs which show that live Mycobacterium bovis (BCG) and mycolic acid lipids (M. A.) from Mycobacterium tuberculosis (Tb) induce an NPCl phenotype in RAW mouse macrophages.
Figure 12 comprises micrographs which show that live BCG infection depletes lysosomal calcium, and that this can be overcome using a calcium agonist (curcumin) to clear the infection. Fig. 2A contains a graph showing the concentration of intralysosomal calcium in μM (y axis) in control RAW cells (left hand bar), cells infected overnight with live BCG (middle bar) and cells infected with heat-killed BCG (right hand bar).
Figure 13 consists of micrographs of RAW macrophages which show that depletion of sphingolipids using an inhibitor of sphingo lipid biosynthesis (miglustat, NB-DNJ) reverses the accumulation of sphingomyelin induced by BCG and Tb secreted lipids.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions apply to the compounds defined herein which elevate intracellular calcium, the agents which reduce sphingosine storage and the inhibitors of sphingolipid biosynthesis:
A Ci-20 alkyl group is an unsubstituted or substituted, straight or branched chain saturated hydrocarbon radical. Typically it is C1-Io alkyl, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl, or Ci-6 alkyl, for example methyl, ethyl, propyl, butyl, pentyl or hexyl, or CM alkyl, for example methyl, ethyl, i-propyl, n-propyl, t- butyl, s-butyl or n-butyl. When an alkyl group is substituted it typically bears one or more substituents selected from substituted or unsubstituted Ci-2O alkyl, substituted or unsubstituted aryl (as defined herein), cyano, amino, CMO alkylamino, di(Ci-i0)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, Ci-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, - SH), Ci-io alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester. Examples of substituted alkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups. The term alkaryl, as used herein, pertains to a Ci-20 alkyl group in which at least one hydrogen atom has been replaced with an aryl group. Examples of such groups include, but are not limited to, benzyl (phenylmethyl, PhCH2-), benzhydryl (Ph2CH-), trityl (triphenylmethyl, Ph3C-), phenethyl (phenylethyl, Ph-CH2CH2-), styryl (Ph-CH=CH-), cinnamyl (Ph-CH=CH-CH2-). Typically a substituted Ci-2O alkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
A C3-25 cycloalkyl group is an unsubstituted or substituted alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound, which moiety has from 3 to 25 carbon atoms (unless otherwise specified), including from 3 to 25 ring atoms. Thus, the term "cycloalkyl" includes the sub-classes cycloalkyenyl and cycloalkynyl. Examples of groups OfC3-25 cycloalkyl groups include C3-20 cycloalkyl, C3-15 cycloalkyl, C3-10 cycloalkyl, C3-7 cycloalkyl. When a C3-25 cycloalkyl group is substituted it typically bears one or more substituents selected from C1-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, Ci-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, -SH), Ci-I0 alkylthio, arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically a substituted C3-25 cycloalkyl group carries 1, 2 or 3 substituents, for instance 1 or 2. Examples OfC3-25 cycloalkyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds, which C3-25 cycloalkyl groups are unsubstituted or substituted as defined above: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7), methylcyclohexane (C7), dimethylcyclohexane (C8), menthane
unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7), methylcyclohexene (C7), dimethylcyclohexene (C8); saturated polycyclic hydrocarbon compounds: thujane (C10), carane (C1O), pinane (Ci0), bomane (C1O), norcarane (C7), noφinane (C7), norbornane (C7), adamantane (C]0), decalin (decahydronaphthalene) (C10);
Figure imgf000010_0001
unsaturated polycyclic hydrocarbon compounds: camphene (C10), limonene (C10), pinene (C10),
Figure imgf000010_0002
polycyclic hydrocarbon compounds having an aromatic ring: indene (C9), indane (e.g., 2,3-dihydro-lH-indene) (C9), tetraline
(1,2,3,4-tetrahydronaphthalene) (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (Ci5), aceanthrene (Cj6), cholanthrene (C20).
A C3-2O heterocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. When a C3-20 heterocyclyl group is substituted it typically bears one or more substituents selected from C1-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, Ci-I0 alkylamino, di(Ci-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, -SH), C1-I0 alkylthio, arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically a substituted C3-20 heterocyclyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
Examples of groups of heterocyclyl groups include C3-2oheterocyclyl, C5-20heterocyclyl, C3-15heterocyclyl, C5-15heterocyclyl, C3-i2heterocyclyl, C5-12heterocyclyl, C3-ioheterocyclyl, C5-i0heterocyclyl, C3-7heterocyclyl, C^heterocyclyl, and Cs-βheterocyclyl.
Examples of (non-aromatic) monocyclic C3-20 heterocyclyl groups include, but are not limited to, those derived from: N1 : aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline
(e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1 : oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7); Si : thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane
(tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1Oi: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), moφholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
NiS1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6); N2O1 : oxadiazine (C6);
O1Si: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1OiSi: oxathiazine (C6).
Examples of substituted (non-aromatic) monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. C3-20 heterocyclyl includes groups derived from heterocyclic compounds of the following structure:
Figure imgf000011_0001
wherein x is 0 or 1 ; z is CHR84; and R80, R81 , R82, R83 and R84, which are the same or different, are independently selected from H, Ci-6 alkyl, OH, acyloxy, SH, Ci-6 alkoxy, aryloxy, amino, Ci-I0 alkylamino, di(Ci.io)alkylamino, amido, acylamido and a group derived from a second group of the following structure:
Figure imgf000012_0001
in which second group x is 0 or 1; z is CHR84; and R80, R81, R82, R83 and R84, which are the same or different, are independently selected from H, C1-6 alkyl, OH, acyloxy, SH, Ci-6 alkoxy, aryloxy, amino, Ci-io alkylamino, di(Ci.io)alkylamino, amido and acylamido. The term "group derived from" in this case means that the group is a monovalent moiety obtained by removing the R80, R81, R82, R83 or R84 atom from a carbon atom of the above compounds. Thus, C3-20 heterocyclyl includes groups of the following structure:
Figure imgf000012_0002
wherein each of the ring carbon atoms is independently unsubstituted or substituted with Ci-6 alkyl, OH, acyloxy, SH, Ci-6 alkoxy, aryloxy, amino, C]-I0 alkylamino, di(Ci-io)alkylamino, amido and acylamido.
C3-20 heterocyclyl also includes groups in which two heterocyclic rings are linked by an oxygen atom. Thus, C3-20 heterocyclyl includes disaccharide groups, in which two monosaccharide heterocyclic rings are linked with an oxygen atom. Accordingly, C3-20 heterocyclyl includes groups of the following formula (m):
Figure imgf000012_0003
wherein each Rm, which is the same or different, is independently selected from Ci-6 alkyl, OH, acyloxy, SH, C1-6 alkoxy, aryloxy, amino, Ci-I0 alkylamino, di(Ci-i0)alkylamino, amido and acylamido. Thus, the following disaccharide group is one example of a substituted C3-20 heterocyclic group:
Figure imgf000013_0001
Examples of C3-20 heterocyclyl groups which are also aryl groups are described below as heteroaryl groups.
An aryl group is a substituted or unsubstituted, monocyclic or bicyclic aromatic group which typically contains from 6 to 14 carbon atoms, preferably from 6 to 10 carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl and indanyl groups. An aryl group is unsubstituted or substituted. When an aryl group as defined above is substituted it typically bears one or more substituents selected from Ci-C6 alkyl which is unsubstituted (to form an aralkyl group), aryl which is unsubstituted, cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfhydryl (i.e. thiol, -SH), Ci-10 alkylthio, arylthio, sulfonic acid, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically it carries 0, 1, 2 or 3 substituents. A substituted aryl group may be substituted in two positions with a single C1-6 alkylene group, or with a bidentate group represented by the formula -X-Ci-6 alkylene, or -X-Ci-6 alkylene-X-, wherein X is selected from O, S and NR, and wherein R is H, aryl or Ci-6 alkyl. Thus a substituted aryl group may be an aryl group fused with a cycloalkyl group or with a heterocyclyl group. The term aralkyl as used herein, pertains to an aryl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with a Ci-6 alkyl group. Examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), and cumenyl (or cumyl, from cumene), and duryl (from durene). The ring atoms of an aryl group may include one or more heteroatoms (as in a heteroaryl group). Such an aryl group (a heteroaryl group) is a substituted or unsubstituted mono- or bicyclic heteroaromatic group which typically contains from 6 to 10 atoms in the ring portion including one or more heteroatoms. It is generally a 5- or 6-membered ring, containing at least one heteroatom selected from O, S, N, P, Se and Si. It may contain, for example, 1, 2 or 3 heteroatoms. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, quinolyl and isoquinolyl. A heteroaryl group may be unsubstituted or substituted, for instance, as specified above for aryl. Typically it carries 0, 1, 2 or 3 substituents.
A Ci-20 alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Typically it is Ci-I0 alkylene, for instance C1-6 alkylene. Typically it is Ci-4 alkylene, for example methylene, ethylene, i-propylene, n-propylene, t-butylene, s-butylene or n- butylene. It may also be pentylene, hexylene, heptylene, octylene and the various branched chain isomers thereof. An alkylene group may be unsubstituted or substituted, for instance, as specified above for alkyl. Typically a substituted alkylene group carries 1, 2 or 3 substituents, for instance 1 or 2. In this context, the prefixes (e.g., Cj-4, C1-7, C1-20, C2-7, C3-7, etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term "Ci-4alkylene," as used herein, pertains to an alkylene group having from 1 to 4 carbon atoms. Examples of groups of alkylene groups include C1-4 alkylene ("lower alkylene"), Ci-7 alkylene, Ci-I0 alkylene and Ci-20 alkylene. Examples of linear saturated Ci-7 alkylene groups include, but are not limited to,
-(CH2)n- where n is an integer from 1 to 7, for example, -CH2- (methylene), -CH2CH2- (ethylene), -CH2CH2CH2- (propylene), and -CH2CH2CH2CH2- (butylene).
Examples of branched saturated Ci-7 alkylene groups include, but are not limited to, -CH(CH3)-, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-.
Examples of linear partially unsaturated Ci-7 alkylene groups include, but is not limited to, -CH=CH- (vinylene), -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH- CH=CH-CH2-CH2-, -CH=CH-CH2-CH=CH-, and -CH=CH-CH2-CH2-CH=CH-.
Examples of branched partially unsaturated Ci-7 alkylene groups include, but is not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, and -CH=CH-CH(CH3)-. Examples of alicyclic saturated C1-7 alkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex-l,4-ylene).
Examples of alicyclic partially unsaturated C1-7 alkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), cyclohexenylene (e.g., 2-cyclohexen-l,4-ylene; 3-cyclohexen-l,2-ylene; 2,5-cyclohexadien-l,4-ylene).
C1-20 alkylene and Ci-20 alkyl groups as defined herein are either uninterrupted or interrupted by one or more heteroatoms or heterogroups, such as S, O or N(R") wherein R" is H, C1-6 alkyl or aryl (typically phenyl), or by one or more arylene (typically phenylene) groups. The phrase "optionally interrupted" as used herein thus refers to a Ci-20 alkyl group or an alkylene group, as defined above, which is uninterrupted or which is interrupted between adjacent carbon atoms by a heteroatom such as oxygen or sulfur, by a heterogroup such as N(R") wherein R" is H, aryl or Ci-C6 alkyl, or by an arylene group. For instance, a C1-20 alkyl group such as n-butyl may be interrupted by the heterogroup N(R") as follows: -CH2N(R")CH2CH2CH3, -CH2CH2N(R")CH2CH3, or -CH2CH2CH2N(R")CH3. Similarly, an alkylene group such as n-butylene may be interrupted by the heterogroup N(R") as follows: -CH2N(R")CH2CH2CH2-, -CH2CH2N(R")CH2CH2-, or -CH2CH2CH2N(R")CH2-. Typically an interrupted group, for instance an interrupted C1-20 alkylene or C1-20 alkyl group, is interrupted by 1, 2 or 3 heteroatoms or heterogroups or by 1, 2 or 3 arylene (typically phenylene) groups. More typically, an interrupted group, for instance an interrupted C1-20 alkylene or Ci-20 alkyl group, is interrupted by 1 or 2 heteroatoms or heterogroups or by 1 or 2 arylene (typically phenylene) groups. For instance, a Ci-20 alkyl group such as n-butyl may be interrupted by 2 heterogroups N(R") as follows: -CH2N(R")CH2N(R")CH2CH3.
An arylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, which moiety has from 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has from 5 to 7 or from 5 to 6 ring atoms. An arylene group may be unsubstituted or substituted, for instance, as specified above for aryl.
In this context, the prefixes (e.g., Cs-20, C6-20, C5-H, C5-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C5-6 arylene," as used herein, pertains to an arylene group having 5 or 6 ring atoms. Examples of groups of arylene groups include C5-20 arylene, C6-20 arylene, C5-14 arylene, C6-14 arylene, C6-10 arylene, C5-12 arylene, C5-10 arylene, C5-7 arylene, C5-6 arylene, C5 arylene, and C6 arylene.
The ring atoms may be all carbon atoms, as in "carboarylene groups" (e.g., C6-20 carboarylene, C6-J4 carboarylene or C6-I0 carboarylene). Examples of C6-20 arylene groups which do not have ring heteroatoms (i.e., C6-20 carboarylene groups) include, but are not limited to, those derived from the compounds discussed above in regard to aryl groups, e.g. phenylene, and also include those derived from aryl groups which are bonded together, e.g. phenylene-phenylene (diphenylene) and phenylene-phenylene-phenylene (triphenylene). Alternatively, the ring atoms may include one or more heteroatoms, as in
"heteroarylene groups" (e.g., C5-I0 heteroarylene).
Examples of C5-10 heteroarylene groups include, but are not limited to, those derived from the compounds discussed above in regard to heteroaryl groups.
As used herein the term oxo represents a group of formula: =O As used herein the term acyl represents a group of formula: -C(=O)R, wherein R is an acyl substituent, for example, a substituted or unsubstituted C1-20 alkyl group, a substituted or unsubstituted C3-20 heterocyclyl group, or a substituted or unsubstituted aryl group. Examples of acyl groups include, but are not limited to, -C(=O)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=O)C(CH3)3 (t-butyryl), and -C(=O)Ph (benzoyl, phenone).
As used herein the term acyloxy (or reverse ester) represents a group of formula: -OC(=O)R, wherein R is an acyloxy substituent, for example, substituted or unsubstituted C1-20 alkyl group, a substituted or unsubstituted C3-20heterocyclyl group, or a substituted or unsubstituted aryl group, typically a Ci-6 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(O)CH3 (acetoxy), -OC(=O)CH2CH3, -OC(=O)C(CH3)3, -OC(=O)Ph, and -OC(=O)CH2Ph.
As used herein the term ester (or carboxylate, carboxylic acid ester or oxycarbonyl) represents a group of formula: -C(=0)0R, wherein R is an ester substituent, for example, a substituted or unsubstituted C1-20 alkyl group, a substituted or unsubstituted C3-20 heterocyclyl group, or a substituted or unsubstituted aryl group (typically a phenyl group). Examples of ester groups include, but are not limited to, -C(=O)OCH3, -C(=O)OCH2CH3, -C(=O)OC(CH3)3, and -C(=O)OPh.
As used herein the term phosphonic acid represents a group of the formula: -P(=O)(OH)2. As would be understood by the skilled person, a phosphonic acid group can exist in protonated and deprotonated forms (i.e. -P(=O)(OH)2, -P(=O)(O')2 and -P(=O)(OH)(O")) all of which are within the scope of the term "phosphonic acid".
As used herein the term phosphonic acid salt represents a group which is a salt of a phosphonic acid group. For example a phosphonic acid salt may be a group of the formula -P(=O)(OH)(O"X+) wherein X is a monovalent cation. X+ may be an alkali metal cation. X+ may be Na+ or K+, for example.
As used herein the term phosphonate ester represents a group of one of the formulae: -P(=O)(OR)2 and -P(=O)(OR)O" wherein each R is independently a phosphonate ester substituent, for example, -H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C3-20 heterocyclyl, C3-2O heterocyclyl substituted with a further C3-20 heterocyclyl, substituted or unsubstituted C1-20 alkylene-C3-2o heterocyclyl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted C1-20 alkylene-C3-25 cycloalkyl, aryl, substituted or unsubstituted C1-20 alkylene-aryl. Examples of phosphonate ester groups include, but are not limited to, -P(=O)(OCH3)2, -P(=O)(OCH2CH3)2, -P(O)(O- t-Bu)2, and -P(O)(OPh)2,
As used herein the term phosphoric acid represents a group of the formula: -OP(=O)(OH)2. As used herein the term phosphate ester represents a group of one of the formulae:
-OP(=O)(OR)2 and -0P(=0)(0R)0" wherein each R is independently a phosphate ester substituent, for example, -H, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted C3-20 heterocyclyl, C3-20 heterocyclyl substituted with a further C3-20 heterocyclyl, substituted or unsubstituted Ci-20 alkylene-C3-20 heterocyclyl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted C1-20 alkylene-C3-25 cycloalkyl, aryl, substituted or unsubstituted Ci-20 alkylene-aryl. Examples of phosphate ester groups include, but are not limited to, -0P(O)(0CH3)2, -0P(O)(0CH2CH3)2, -OP(=O)(O-t-Bu)2, and -OP(O)(OPh)2.
As used herein the term amino represents a group of formula -NH2. The term C1- Cio alkylamino represents a group of formula -NHR' wherein R' is a Ci-I0 alkyl group, preferably a Ci-6 alkyl group, as defined previously. The term di(Ci-io)alkylamino represents a group of formula -NR 'R" wherein R' and R" are the same or different and represent Ci -io alkyl groups, preferably Ci-6 alkyl groups, as defined previously. The term arylamino represents a group of formula -NHR' wherein R' is an aryl group, preferably a phenyl group, as defined previously. The term diarylamino represents a group of formula -NR'R" wherein R' and R" are the same or different and represent aryl groups, preferably phenyl groups, as defined previously. The term arylalkylamino represents a group of formula -NR'R" wherein R' is a C1-I0 alkyl group, preferably a Ci-6 alkyl group, and R" is an aryl group, preferably a phenyl group.
As used herein the term amido represents a group of formula: -C(=O)NR R , wherein R and R are independently amino substituents, as defined for di(C1-10)alkylamino groups. Examples of amido groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -CC=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as amido groups in which R and R , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
As used herein the term acylamido represents a group of formula: -NR1CC=O)R2, wherein R1 is an amide substituent, for example, hydrogen, a Ci-20alkyl group, a C3-20 heterocyclyl group, an aryl group, preferably hydrogen or a C1-20 alkyl group, and R2 is an acyl substituent, for example, a Ci-2O alkyl group, a C3-20 heterocyclyl group, or an aryl group, preferably hydrogen or a C1-20 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=0)CH3 , -NHC(=O)CH2CH3, -NHC(=O)Ph, -NHC(=O)C15H31 and -NHCC=O)C9H19. Thus, a substituted C1-20 alkyl group may comprise an acylamido substituent defined by the formula -NHC(=0)-Ci-2o alkyl, such as -NHCC=O)Ci5H3I or -NHCC=O)C9Hi9. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
Figure imgf000018_0001
succinimidyl maleimidyl ' phthalimidyl A C1-I0 alkylthio group is a said C^o alkyl group, preferably a Ci-6 alkyl group, attached to a thio group. An arylthio group is an aryl group, preferably a phenyl group, attached to a thio group.
A C1-20 alkoxy group is a said substituted or unsubstituted Ci-20 alkyl group attached to an oxygen atom. A C1-6 alkoxy group is a said substituted or unsubstituted Ci-6 alkyl group attached to an oxygen atom. A C1-4 alkoxy group is a substituted or unsubstituted C1-4 alkyl group attached to an oxygen atom. Said C1-20, C1-6 and C1-4 alkyl groups are optionally interrupted as defined herein. Examples of C1-4 alkoxy groups include, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy). Further examples of Ci-20 alkoxy groups are -O(Adamantyl), -O-CH2-Adamantyl and -0-CH2-CH2- Adamantyl. An aryloxy group is a substituted or unsubstituted aryl group, as defined herein, attached to an oxygen atom. An example of an aryloxy group is -OPh (phenoxy).
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid or carboxyl group (-C00H) also includes the anionic (carboxylate) form (-COO"), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N+HR1R2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (- O"), a salt or solvate thereof, as well as conventional protected forms.
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; C-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L- forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti- forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").
Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers," as used herein, are structural (or constitutional) isomers
(i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C1-7alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). The above exclusion does not pertain to tautomeric forms, for example, keto, enol, and enolate forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Figure imgf000020_0001
keto enol enolate
Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting known methods, in a known manner. Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, protected forms and prodrugs thereof.
Examples of pharmaceutically acceptable salts of the compounds for use in accordance with the present invention include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid and phosphoric acid; and organic acids such as methanesulfonic acid, benzenesulphonic acid, formic acid, acetic acid, trifiuoroacetic acid, propionic acid, butyric acid, isobutyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, ethanesulfonic acid, aspartic acid, benzoic acid and glutamic acid. Typically the salt is a hydrochloride, an acetate, a propionate, a benzoate, a butyrate or an isobutyrate. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19.
A prodrug of a compound which elevates intracellular calcium, an agent which reduces sphingosine storage or an inhibitor of sphingo lipid biosynthesis, is a compound which, when metabolised (e.g., in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties. For example, some prodrugs are 0-acylated (acyloxy) derivatives of the active compound, i.e. physiologically acceptable metabolically labile acylated derivatives. During metabolism, the one or more -O-acyl (acyloxy) groups (-O-C(=O)RP) are cleaved to yield the active drug. Rp may be a C1-Jo alkyl group, an aryl group or a C3-2O cycloalkyl group. Typically, Rp is a C1-10 alkyl group including, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. Such derivatives may be formed by acylation, for example, of any of the hydroxyl groups (-OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Thus, the free hydroxyl groups on an iminosugar inhibitor of sphingo lipid biosynthesis (for instance DNJ, DGJ, or an N- alkylated derivative of DNJ or DGJ such as NB-DNJ or NB-DGJ) may be acylated with up to four, typically exactly four, O-acyl groups. The O-acyl groups are enzymatically removed in vivo to provide the non-O-acylated (i.e. hydroxyl-containing) active inhibitor of sphingolipid biosynthesis. Some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.
The compound which elevates intracellular calcium, agent which reduces sphingosine storage or inhibitor of sphingolipid biosynthesis, for use in accordance with the invention can be used in the free form or the salt form. The compound may also be used in prodrug form. The prodrug can itself be used in the free form or the salt form.
The term "compound which elevates intracellular calcium", as used herein, means any compound that causes an increase in the concentration of calcium within the cell, typically within the cytosol and/or the lysosomes of the cell, after administration to a patient. Typically, the compound which elevates intracellular calcium is a compound which elevates cytosolic calcium or a compound which elevates lysosomal calcium. The term "compound which elevates cytosolic calcium", as used herein, means any compound that causes an increase in the concentration of calcium in the cytosol after administration to a patient.
The term "compound which elevates lysosomal calcium", as used herein, means any compound that causes an increase in the concentration of calcium in the lysosomes after administration to a patient.
Compounds which elevate intracellular calcium are either known or readily identifiable, without undue experimentation, using known procedures. Such compounds can be identified by measuring the effects of target compounds on intracellular calcium levels (for example, their effect on cytosolic and lysosomal calcium concentrations) and by identifying compounds which elevate the level of intracellular calcium.
Grynkiewicz et al., J. Biol. Chem., Vol. 260, No. 6, 1985, 3440-3450, describes how the concentration of cytosolic free calcium can be measured using highly fluorescent Ca2+ indicators. Furthermore, Miyawaki et al., Proc. Natl. Acad. Sci., Vol. 96, 1999, 2135- 2140 discusses how cellular Ca2+ can be measured quantitively using fluorescent cameleon indicator proteins (based on green fluorescent protein variants and calmodulin). Thomas et al., Cell Calcium (2000) 28 (4), 213-223, compares various fluorescent Ca2+ indicators and their use in measuring calcium levels. Finally, Christeinsen et al., Journal of Cell Science 115 (3), 599-607 (2002) describes the measurement of the concentration of free calcium in the lysosome, using both ratiometric and time-resolved fluorescence microscopy of probes. These articles cover the main ways of measuring elevations of calcium within cells and can be used on microscopes, FACS, fluorimeters or fluorescent plate readers. Compounds which elevate intracellular calcium, including compounds which elevate cytosolic calcium and compounds which elevate lysosomal calcium, can readily be identified using such methods, without undue burden.
Compounds which elevate intracellular calcium and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention include, but are not limited to, inhibitors of the sarco/endoplasmic reticulum calcium ATPase (SERCA inhibitors). SERCA inhibitors are known compounds. J. Med. Chem. 2005, 48, 7005-7011 describes thapsigargin and various analogs thereof as potent SERCA inhibitors, and compares the respective activities of the compounds. Eur. J. Biochem, 268, 6318-6327 (2001) details the SERCA inhibitory activity of curcumin. Cyclopiazonic acid is also a known SERCA inhibitor. SERCA inhibitors may be readily identified, without undue experimentation, using known procedures. J. Med. Chem. 2005, 48, 7005-7011 describes how SERCA inhibitors may be designed (for instance, using the structure of a known SERCA inhibitor as a starting point) and then tested for SERCA inhibitory activity. A standard assay for the measurement of SERCA inhibitory activity is described in J. Med. Chem. 2005, 48, p 7010. Eur. J. Biochem, 268, 6318-6327 (2001) also describes how Ca2+ ATPase activity can be determined, in order to identify SERCA inhibitors, using a phosphate liberation assay described in Longland et al., Cell Calcium 24, 27-34 (1998).
Accordingly, the compound which elevates intracellular calcium is typically a SERCA inhibitor. Typically, the SERCA inhibitor is selected from curcumin, cyclopiazonic acid and a compound of formula (A):
Figure imgf000023_0001
wherein: RZ! is selected from hydrogen, hydroxyl, carboxyl, amino, thiol, halo, substituted or unsubstituted Ci-10 alkyl, substituted or unsubstituted Ci-10 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C1-10 alkylamino, di(Ci-i0)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said C1-I0 alkyl is optionally interrupted by N(R'), O, S or arylene; RZ2 is selected from hydrogen, substituted or unsubstituted Ci-io alkyl and acyl, wherein said Ci-I0 alkyl is optionally interrupted by N(R'), O, S or arylene; and either XZ1 is CH and XZ2 is CHR' wherein XZ1 and XZ2 are bonded by a C-C single bond, or XZ1 is C and XZ2 is CR' wherein XZ1 and XZ2 are bonded by a C=C double bond; and
R' is H, Ci-6 alkyl or aryl; and pharmaceutically acceptable salts thereof.
Typically, RZ1 is selected from H and ester. More typically, RZ1 is selected from H and -OC(O)(CH2)6CH3. Typically, RZ2 is selected from C1-10 alkyl and acyl. More typically, R22 is selected from ethyl and acetyl (i.e. -C(O)CH3).
Typically, XZ1 is CH and XZ2 is C(H)Me. Alternatively, XZ1 is C and XZ2 is C(H)Me.
In one embodiment, RZ1 is -OC(O)(CH2)6CH3, RZ2 is -C(O)CH3, XZ1 is C and XZ2 is C(H)Me (i.e. the compounds is thapsigargin).
In another embodiment, RZ1 is H, RZ2 is -C(O)CH3, XZ1 is C and XZ2 is C(H)Me (i.e. the compounds is nortrilobolide).
In another embodiment, RZ1 is H, R22 is ethyl, XZ1 is CH and X22 is C(H)Me.
In another embodiment, RZ1 is H, RZ2 is -C(O)CH3, XZ1 is CH and XZ2 is C(H)Me.
More typically, the SERCA inhibitor is selected from thapsigargin, curcumin and cyclopiazonic acid. The chemical structure of curcumin is as follows:
Figure imgf000024_0001
The structure of thapsigargin is as follows:
Figure imgf000024_0002
In one embodiment of the present invention the compound which elevates intracellular calcium is other than curcumin. hi another embodiment, the compound which elevates intracellular calcium is other than curcumin when the disease which has an NPC like cellular phenotype is tuberculosis.
Compounds which elevate intracellular calcium, and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention also include modulators of the ryanodine receptor (the sarcoplasmic reticulum Ca2+ channel, RyR). Ryanodine receptor modulators (RyR modulators) are known compounds, and it is known that these compounds can interact with RyR to stimulate Ca2+ release and cause an increase in intracellular calcium. Zucci and Ronca-Testoni, Pharmacological Reviews (1997), Vol. 49, No. 1, 1-51 describes many such RyR modulators that stimulate Ca2+ release and thereby elevate intracellular calcium. Such RyR modulators can be readily identified, without undue experimentation, using known procedures. Pharmacological Reviews (1997), Vol. 49, No. 1, 1-51 describes how RyR modulators may be identified, using Ca2+ release studies, single channel studies, [3H]ryanodine binding studies or indirect studies.
Accordingly, the compound which elevates intracellular calcium is typically a Ryanodine receptor modulator (RyR modulator).
RyR modulators, which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention, include, but are not limited to, the following compounds (many of which are described in Pharmacological Reviews (1997), Vol. 49, No. 1, 1-51):
• Cyclic ADP-Ribose (cADPr) (Cell Calcium (2002) 32 (5-6) 343-354)
• Ryanoid compounds, including but not limited to compounds of the following formula (B):
Figure imgf000025_0001
wherein:
RX1 is a substituted or unsubstituted pyrrole ring;
RX2, RX3, RX4, RX5, RX6, RX7 and Rx8 which are the same or different, are independently selected from hydrogen, halo, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C1-I0 alkyl, aryl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted C3-20 heterocyclyl, substituted or unsubstituted C1-10 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, Ci-1O alkylamino, di(Ci.io)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said C1-Io alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci-6 alkyl or aryl; and either ZX1 is CH and Z^ is CH2RX9 wherein ZX1 and Z^ are bonded by a C-C single bond, or ZX1 is C and 7p is CHR' wherein ZX1 and 7p are bonded by a C=C double bond; and R >XΛ9yis H, C1-6 alkyl or aryl; and pharmaceutically acceptable salts thereof.
Typically, R XΛ9y is H.
Typically, R is an unsubstituted pyrrole ring.
Typically, R and R are both either hydrogen or substituted or unsubstituted C1-10 alkyl. More typically, R^ and RX3 are both hydrogen.
Typically, RX4, RX5, Rx6, RX7 and Rx8 are independently selected from hydroxyl, substituted or unsubstituted C1-10 alkoxy and acyloxy. More typically, each of RX4, RX5, RX6,
R and R is a hydroxyl group.
Typically, the ryanoid compound is selected from ryanodine, 9,21-didehydroryanodine, guanidinopropionyl and /7-alanil-ryanodine.
The structures of ryanodine and 9,21-didehydroryanodine are as follows:
Figure imgf000026_0001
ryanodine 9,21 -didehydroryanodine
Compounds of the following formula (C):
Figure imgf000026_0002
wherein:
RMI, RM2, RM3, RM4 and RM5, which are the same or different, are independently selected from hydrogen and substituted or unsubstituted C1-10 alkyl, provided that at least one of RM1, RM2, RM3, RM4 and RM5 is substituted or unsubstituted Ci-10 alkyl, wherein said Ci-10 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C1-6 alkyl or aryl; and pharmaceutically acceptable salts thereof. Typically, RM1, RM2, RM3, RM4 and RM5, which are the same or different, are independently selected from hydrogen and methyl, provided that at least one of RM1, RM2, RM3, RM4 and RM5 is methyl.
Typically, one, two or three of RM1, RM2, RM3, RM4 and RM5 are substituted or unsubstituted Ci-10 alkyl (more typically methyl) groups and the remainder are hydrogens.
Typically, the compound of formula (C) is a methylxanthine. Typically, the methyxanthine is selected from any one of the following compounds:
Figure imgf000027_0001
Figure imgf000027_0002
1 ,3-dimethylxanthine
Figure imgf000027_0003
Compounds of the following formula (D):
Figure imgf000027_0004
wherein:
E1, E2, E3 and E4, which are the same or different, are independently selected from C(R05) and N provided that no more than two of Ei, E2, E3 and E4 are N;
R01 is selected from hydrogen, substituted or unsubstituted Ci-1O alkyl, aryl, substituted or unsubstituted C3-25 cycloalkyl, and substituted or unsubstituted C3-20 heterocyclyl, wherein said C1-Io alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci-6 alkyl or aryl; and R°2, R , R04 and each R05, which are the same or different, are independently selected from hydrogen, halo, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Ci-10 alkyl, aryl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted C3-20 heterocyclyl, substituted or unsubstituted C1-Io alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, Ci-I0 alkylamino, di(C1-1o)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-2o heterocyclyl, wherein said Ci-I0 alkyl is optionally interrupted by N(R'), O, S or arylene; and pharmaceutically acceptable salts thereof.
Typically, R01 is selected from hydrogen and Ci-6 alkyl. More typically, R01 is selected from hydrogen and methyl.
Typically, R02, R°3, R°4 and each R05, which are the same or different, are independently selected from hydrogen and halo groups. More typically, R°2, R03, R04 and each R05, which are the same or different, are independently selected from H, Br and Cl.
Typically, the compound of formula (D) is a carbazole or a carboline compound. Typically, the compound of formula (D) is 9-methyl-7-bromoeudistomin D (MBED), which has the following structure:
Figure imgf000028_0001
• Carnitine compounds of the following formula (E):
Figure imgf000028_0002
wherein:
RN1 is a substituted or unsubstituted Ci4-2O alkyl group, wherein said Ci4-20 alkyl is optionally interrupted by N(R'), O, S or arylene; and pharmaceutically acceptable salts thereof.
Typically, RN1 is an unsubstituted, uninterrupted C]4-20 alkyl group. In one embodiment, RN1 is -(CH2)14CH3 (i.e. the compound is palmitoyl carnitine). • Adenine nucleotides, including, but not limited to the following adenine nucleotides: adenosine 5'-(jff,y-methylene)triphosphate (AMP-PCP), cyclic AMP (cAMP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP).
• Adenosine • L-thyroxine
• Sulmazole, isomazole (the enantiomer of sulmazole), and sulmazole analogs lacking the methylsulfinyl oxygen of sulmazole
• Anthraquinones, including, but not limited to the following compounds: Doxorubicin, mitoxantrone, daunorubicin, rubidazone and doxorubicinol • Digoxin, digitoxin and ouabain
• Milrinone (l,6-dihydro-2-methyl-6-oxo-(3,4-bipyridine)-5-carbonitrile) and 1,1'- diheptyl-4,4'-bipyridinium bromide
• Suramin (s>rø-bis(m-aminobenzoyl-m-amino-/?-methylbenzoyl- 1 -naphthyl-amino- 4,6,8-trisulfonate)carbamide) • Halogenated hydrocarbons, phenols and biphenyls, including, but not limited to, the following compounds: halothane (2-bromo-2-chloro-l,l,l-trifluoroethane); enflurane (2-chloro- 1 -(difluoromethoxy)- 1 , 1 ,2-trifluoroethane); isoflurane (2-chloro-2- (difluoromethoxy)- 1,1,1 -trifluoroethane); 4-chloro-m-cresol; δ-hexachloro- cyclohexane; chlorinated phenol; chlorocresol; a C1-20 linear, branched or cyclic alkane which is substituted with at least one halo group (typically with at least one chloro, bromo or fluoro group) and which is otherwise unsubstituted or substituted; phenol which is substituted with a Ci-1O alkyl group and with at least one halo group (typically chloro); and biphenyl which is substituted with at least two halo groups, typically with at least two chloro groups. • 4-(C2-C9 alkyl)phenol
• FK-506 (tacrolimus), rapamycin, quinolidomicin Al, bastadin 5, bastadin 7, bastadin 19
• RyR-modulating peptides, including, but not limited to: imperatoxin-a, myotoxin-a, and ryanotoxin • Sulfhydryl reagents, including, but not limited to: N-ethylmaleimide, thimerosal, 5,5'- dithiobis-(2-nitrobenzoic acid), 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, N- succinimidyl 3 -(2-pyridyldithio)propionate • Mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP), tetrasodium-mesotetra- (4-sulfonatophenyl)-porphine
• Disulfonic stilbene derivatives including, but not limited to: 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetoamido-4'-isothiocyanostilbene- 2,2 '-disulfonic acid
• Acetic anhydride, maleic anhydride, diethylpyrocarbonate, quercetin, miconazole, clotrimazole, ketonazole, 1-heptanol, 1-octanol, DPI 201-106, cyclosporine A, TMB-8, 2,3-butanedione monoxime, methylenedioxyndenes
Compounds which elevate intracellular calcium, and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention also include modulators of the inositol 1,4,5-trisphosphate (EP3) receptor. IP3 receptor modulators are known compounds, and it is known that these compounds can interact with the IP3 receptor to mobilise Ca2+ and cause an increase in intracellular calcium. Such IP3 receptor modulators, that stimulate Ca2+ release and thereby elevate intracellular calcium, include inositol 1 ,4,5-trisphosphate (IP3) (Cell Calcium (2002) 32 (5-6), 343-354) and 2- APB (2- aminoethoxydiphenyl borate). Such IP3 receptor modulators can be readily identified, without undue experimentation, using known assay methods.
Accordingly, the compound which elevates intracellular calcium is typically an IP3 receptor modulator. Typically, the IP3 receptor modulator is inositol 1,4,5-trisphosphate (IP3) or 2-aminoethoxydiphenyl borate, which have the following structures respectively:
Figure imgf000030_0001
Compounds which elevate intracellular calcium, and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention also include modulators of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) receptor. NAADP receptor modulators are known compounds and can be readily identified, without undue experimentation, using known assay methods. It is known that these compounds can interact with the NAADP receptor to mobilise Ca2+ and cause an increase in intracellular calcium (Cell Calcium (2002) 32 (5-6), 343-354). Such NAADP receptor modulators, that stimulate Ca2+ release and thereby elevate intracellular calcium, include nicotinic acid adenine dinucleotide phosphate (NAADP) (Cell Calcium (2002) 32 (5-6), 343- 354) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS).
Accordingly, the compound which elevates intracellular calcium is typically a NAADP receptor modulator. Typically, the NAADP receptor modulator is selected from nicotinic acid adenine dinucleotide phosphate (NAADP) and a compound of the following formula (F):
Figure imgf000031_0001
wherein n is 0 or 1 ; and
RY1 is a substituted or unsubstituted aryl or heteroaryl group; and pharmaceutically acceptable salts thereof.
Typically, RY1 is a substituted or unsubstituted phenyl group. More typically, RY1 is unsubstituted phenyl or phenyl which is substituted with one or two groups selected from carboxyl, -SO3H and -CH2PO3H2. Even more typically, Rγl is selected from any one of the following groups:
Figure imgf000031_0002
Figure imgf000031_0003
Typically, the compound of formula (F) is pyridoxalphosphate-6-azophenyl-2',4'- disulphonic acid (PPADS), which has the following structure:
Figure imgf000032_0001
The SERCA, EP3, RyR and NAADP receptors are classified as intracellular calcium channels, and can induce intracellular calcium elevation directly upon binding to a ligand. Plasma membrane channels, on the other hand, function in one of two ways they either release calcium directly following ligand binding or once bound to a ligand, activate the generation of an intracellular second messenger (such as NAADP or cADPr) which subsequently binds to the intracellular calcium channel, the binding triggering calcium release and an elevation in the intracellular calcium concentration. Such plasma membrane channels include, P2X receptors (direct calcium release) and EDG receptors or Glutamate receptors (mGluR) (activate generation of an intracellular second messenger).
Accordingly, compounds which elevate intracellular calcium, and which can be used to treat diseases that have an NPC like cellular phenotype in accordance with the present invention also include modulators of the EDG receptor, P2X receptor and Glutamate receptor (mGluR). Such modulators are known compounds and/or can be readily identified, without undue experimentation, using known assay methods.
Accordingly, the compound which elevates intracellular calcium is typically an EDG receptor modulator, a P2X receptor modulator or a Glutamate receptor (mGluR) modulator.
P2X receptor modulators include, but are not limited to, compounds of formula (E) as defined above. An example of a P2X receptor modulator is PPADS. EDG receptor modulators include, but are not limited to sphingosine-1 -phosphate (SIP) and lyso-phosphatidyl-choline (LPC). mGluR modulators include, but are not limited to glutamate. In one embodiment, the compound which elevates intracellular calcium is a SERCA inhibitor, a Ryanodine receptor modulator, an IP3 receptor modulator, a NAADP receptor modulator, an EDG receptor modulator, a P2X receptor modulator or a Glutamate receptor modulator.
Typically, the compound which elevates intracellular calcium is lα,25- dihydroxyvitamin D3 or any one of the SERCA inhibitors, Ryanodine receptor modulators, IP3 receptor modulators, NAADP receptor modulators, EDG receptor modulators, P2X receptor modulators or Glutamate receptor modulators defined above, or a pharmaceutically acceptable salt of any of those compounds.
Typically, the compound which elevates intracellular calcium is a compound selected from: lα,25-dihydroxyvitamin D3; curcumin, thapsigargin, cyclopiazonic acid, Cyclic ADP-
Ribose, ryanodine, 9,21-didehydroryanodine, guanidinopropionyl,/?-alanil-ryanodine, caffeine, 3,7-dimethylxanthine, 1,7-dimethylxanthine, 1,3-dimethylxanthine, 3,9-dimethylxanthine, 9- methyl-7-bromoeudistomin D, palmitoyl carnitine, adenosine 5'-(/9,)'-methylene)triphosphate, cyclic AMP, adenosine diphosphate, adenosine monophosphate, adenosine, L-thyroxine, sulmazole, isomazole, doxorubicin, mitoxantrone, daunorubicin, rubidazone, doxorubicinol, digoxin, digitoxin, ouabain, l,6-dihydro-2-methyl-6-oxo-(3,4-bipyridine)-5-carbonitrile, 1,1'- diheptyl-4,4'-bipyridinium bromide, 5ym-bis(m-aminobenzoyl-w-amino-p-methylbenzoyl-l- naphthyl-amino-4,6,8-trisulfonate)carbamide, 2-bromo-2-chloro- 1,1,1 -trifluoroethane, 2- chloro- 1 -(difluoromethoxy)- 1 , 1 ,2-trifluoroethane, 2-chloro-2-(difluoromethoxy)- 1,1,1- trifluoroethane), 4-chloro-w-cresol, δ-hexachloro-cyclohexane, chlorocresol, 4-(C2-Cg alkyl)phenol, tacrolimus, rapamycin, quinolidomicin Al, bastadin 5, bastadin 7, bastadin 19, imperatoxin-a, myotoxin-a, ryanotoxin, N-ethylmaleimide, thimerosal, 5,5'-dithiobis-(2- nitrobenzoic acid), 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, N-succinimidyl 3-(2- pyridyldithio)propionate, mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP), tetrasodium-mesotetra-(4-sulfonatophenyl)-porphine, 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid, 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, acetic anhydride, maleic anhydride, diethylpyrocarbonate, quercetin, miconazole, clotrimazole, ketonazole, 1- heptanol, 1-octanol, DPI 201-106, cyclosporine A, TMB-8, 2,3-butanedione monoxime, inositol 1,4,5-trisphosphate, 2-aminoethoxydiphenyl borate, nicotinic acid adenine dinucleotide phosphate, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, sphingosine- 1 -phosphate, lyso-phosphatidyl-choline, and glutamate; or a pharmaceutically acceptable salt thereof.
More typically, the compound which elevates intracellular calcium is thapsigargin, curcumin or lα,25-dihydroxyvitamin D3 (lα,25(OH)2VD3). Diseases which have a NPC like cellular phenotype can be treated with a compound which elevates intracellular calcium in accordance with the present invention.
NPC disease is a complex lipid storage disorder that results from inactivation of the NPCl or NPC2 proteins (Vanier MT, Millat G.; Clin Genet 2003; 64: 269-81). This results in a poorly understood series of events that ultimately leads to neurodegeneration and premature death. Defining the early step(s) in the pathogenic cascade has the potential to lead to the identification of novel clinical intervention points, that in the future may be targeted to maximise therapeutic benefit for patients. The relative pathological significance of the various lipids that accumulate in NPC remains controversial (Sturley SL et al., Biochim Biophys Acta 2004, 1685: 83-7; Vanier MT, Millat G.; Clin Genet 2003; 64: 269-81; Xie C et al., Am J Physiol 1999; 276: E336-44; Zervas M. et al. Curr Biol 2001; 11: 1283-7).
An emerging factor in the pathogenesis of the primary lysosomal sphingolipid storage disorders (e.g. Gaucher, Sandhoff and GMl gangliosidosis) is abnormal endoplasmic reticulum (ER) calcium homeostasis (Futerman AH., Med Sci (Paris) 2005; 21: 16-9). In Gaucher disease, the accumulation of glucosylceramide (GlcCer) leads to enhanced calcium release from the ryanodine receptor (RyR) leading to increased susceptibility to neurotoxic (Futerman AH., Med Sci (Paris) 2005; 21: 16-9). Furthermore, gangliosides GM2 and GMl have been shown modulate the function of calcium transporting ATPases (Duan et al. Arch Biochem Biophys 2006; 454: 155-9; Zhang et al., Arch Biochem Biophys 2005; 444: 1-6; Zhao et al. Arch Biochem Biophys 2004; 427: 204-12). In both Sandhoff disease (Pelled et al.; J Biol Chem 2003; 278: 29496-501) and GMl gangliosidosis (Tessitore et al.; MoI Cell 2004; 15: 753-66), lysosomal ganglioside storage leads to depletion of the ER calcium store via inhibition of the sarco/endoplasmic reticulum Ca2+-ATPaSe (SERCA). The depletion of the ER calcium store in GMl gangliosidosis leads to initiation of the unfolded protein response (UPR), which contributes to neuropathology (Tessitore et al.; MoI Cell 2004; 15: 753-66). hi this study, it was found that, unlike the primary sphingolipidoses, NPCl null cells have normal ER and mitochondrial calcium levels, but significantly lower calcium in Le/Lys. Following inactivation of NPCl the first biochemical change detected is a rapid elevation in sphingosine, which is the only lipid stored in NPC storage capable of inducing the Le/Lys calcium defect. This indicates that sphingosine storage is the primary event in NPC disease pathology, leading to subsequent depletion of Le/Lys calcium, endocytic transport abnormalities followed by GSL, sphingomyelin and cholesterol storage. It was also found that the therapeutic targeting of the calcium defect in NPC is of therapeutic benefit in the NPCl mouse.
The present inventors have determined for the first time the chronology of events following inactivation of NPCl using pharmacological agents. The first detectable change, the increase in sphingosine, was accompanied by a concomitant decrease in sphingosine- 1 phosphate (SlP). The elevation in sphingosine occured very rapidly, (during the first 30-60min following drug treatment) and then plateaued, suggestive of induction of a compensative change within the cell. Interestingly, the decrease in SlP levels was only transient, as at 4h post-treatment SlP levels increased back to control levels. These data suggest that the lysosome is the primary source of sphingosine for SlP formation in healthy cells, but that the cell can compensate for loss of availability of this source of sphingosine, possibly by up- regulating the activity of neutral ceramidase.
Sphingosine generated from ceramide degradation has an amine group that is readily protonated. In the protonated state in the lysosome, (Kagedal K. et al. Biochem J 2001; 359: 335-43) sphingosine (pKa of 8.9) would be incapable of crossing the lysosomal membrane unless facilitated by a sphingosine transporter, the nature of which is unknown. On the basis of this report, NPCl should be considered as a candidate to be involved in the salvage pathway of sphingolipid biosynthesis, through potentially facilitating (either directly or indirectly) sphingosine efflux out of lysosomes. When inactivated, the consequence is the rapid accumulation of sphingosine in the lysosome and a reduction in efflux of sphingosine out of the lysosome negatively impacting SlP generation.
Following sphingosine storage, the next event in NPCl pathogenic cascade is the development of abnormally low Le/Lys calcium levels. This is likely caused by sphingosine storage, as this was the only NPCl storage lipid capable of exogenously inducing this phenotype in healthy cells. This effect was rapid, suggesting a direct effect of sphingosine itself rather than a downstream metabolite. The present inventors calculated that sphingosine storage levels (~0.75μM in U18666A treated RAW cells and ~1.5μM in NPCl mouse brain) approximate the exogenous concentrations (~lμM) required to decrease Le/Lys calcium and subsequently induce an NPCl phenotype in normal cells. Without wishing to be bound by theory, it is unlikely that Le/Lys calcium release is defective as for the store to be reduced by -70% would require constant calcium efflux, which would be sufficient to promote vesicular fusion and release maintaining function in the endocytic pathway. It is more likely that sphingosine storage inhibits calcium entry into the Le/Lys, so there is insufficient calcium for release to maintain normal fusion and trafficking in the endocytic pathway. Whilst the mechanisms of Le/Lys calcium entry are unknown, sphingosine is a known inhibitor of calcium ATPases and Na+/Ca2+ exchangers and may inhibit calcium entry by such a mechanism (Futerman AH, Hannun YA; EMBO Rep 2004; 5: 777-82). This mechanism, however, does not require protons as the present inventors have shown that NPC Le/Lys maintains normal pH, in agreement with a previous report (Bach et al.; Clin Chim Acta 1999; 280: 173-9).
In this study, normal ER and mitochondrial calcium homeostasis was observed in NPC cells. These data suggest that in contrast to the primary glycosphingolipidoses (Med Sci (Paris) 2005; 21 : 16-9), stored GSLs in NPC cells cannot reach the ER and are instead trapped in the Le/Lys system (further illustrating that a transport defect occurs prior to lipid storage). These findings of normal ER calcium homeostasis in NPCl cells are in agreement with a previous report where KCl induced depolarization, leading to calcium release from the ER, was identical between control and NPCl null primary neurons. The following compounds, which compensate for the low Le/Lys calcium levels in
NPC by elevating cytosolic calcium via different mechanisms, were evaluated: thapsigargin (Canova, N.K. et al.; Cell Biol Toxicol 2007; 23: 337-54), activated vitamin D3 (van de Graaf et al.; MoI Biol 2004; 89-90: 303-8) and curcumin (Cell Calcium 2002; 31 : 45-52). These drugs permit normalisation of the fusion and trafficking machinery in NPC cells by increasing levels of calcium in the cytosol that can be utilised by the calcium- dependent proteins involved in the endocytic pathway, hi all cases, these pharmacological agents or natural products corrected the NPC phenotype at the cellular level. One of these agents, curcumin, was tested in the NPCl mouse disease model and it resulted in increased life expectancy and slowed the rate of disease progression. Thus, agents that can compensate for the underlying calcium homeostatic defect in NPC disease by elevating intracellular (typically cytosolic and/or lysosomal) calcium, by any mechanism, could be used to treat NPC disease and any other disease which has a NPC like cellular phenotype. hi summary, sphingosine storage has been identified as the earliest measurable event in NPC disease suggesting that NPCl may either directly or indirectly be involved in sphingosine transport out of the lysosome. When defective sphingosine builds up this in turn induces a novel calcium homeostatic defect characterised by low Ca+* levels in acidic stores. Emptying of acidic store Ca+"1" is an early step following induction of NPCl disease in model systems suggesting that defective calcium homeostasis plays an early role in pathogenesis. Both sphingosine storage and emptying of acidic store calcium occur before storage of cholesterol and GSLs. The low calcium status of the LE/Lys in NPC does not permit sufficient calcium release from LE/Lys to correct vesicular fusion events, which are calcium dependent. This may therefore explain why there is such a profound block in LE to Lys transport in NPC disease. These data therefore reveal a new mechanism underlying pathogenesis in NPC disease and identify a novel clinical intervention strategy (increasing intracellular calcium) for treating Niemann-Pick disease type C or any disease which involves NPCl dysfunction or has a Niemann-Pick disease type C (NPC) like cellular phenotype.
Diseases which have a Niemann-Pick disease type C (NPC) like cellular phenotype
The terms "Niemann-Pick disease type C like cellular phenotype" and 'TSIPC like cellular phenotype" as used herein are equivalent and mean a cellular phenotype which includes: (a) abnormal cholesterol metabolism and trafficking; (b) abnormal sphingolipid storage and trafficking; (c) defective endocytosis; and (d) defective Le/Lys calcium.
Typically, the abnormal sphingolipid storage (b) involves the majority of sphingolipids in the cell being present at abnormally elevated levels. Typically, (c) comprises defective endocytosis of substantially all biomolecules in the endocytic pathway, including substantially all lipids and biomolecules other than lipids, for instance proteins. Typically (d) comprises a reduction in calcium content within the lumen of acidic endosomes (such as late endosomes or lysosomes).
The diseases which have a Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention include (a) "primary" Niemann-Pick disease type C (NPC), and (b) diseases which have a secondary Niemann-Pick disease type C like cellular phenotype.
Primary Niemann-Pick disease type C (NPC) involves a mutation in either the NPCl or NPC2 gene. The disease which has a NPC like cellular phenotype is typically Niemann-Pick disease type C (i.e. "primary" Niemann-Pick disease type C), for instance Niemann-Pick disease type Cl or Niemann-Pick disease type C2. More typically, the disease which has a NPC like cellular phenotype is Niemann-Pick disease type Cl .
The term "disease which has a NPC like cellular phenotype" also includes diseases which have a secondary Niemann-Pick disease type C (NPC) like cellular phenotype.
The, terms "disease which has a secondary Niemann-Pick disease type C like cellular phenotype" and "disease which has a secondary NPC like cellular phenotype" as used herein are equivalent and generally refer to any disease which does not involve a mutation in either the NPCl gene or the NPC2 gene and which involves:
- a mutation in a gene other than NPC 1 or NPC2; an alteration in the function of a protein other than NPCl protein or NPC2 protein; or
- a process that causes mislocalisation of NPCl protein or NPC2 protein, wherein said mutation of the gene other than NPCl or NPC2, said alteration in the function of the protein other than NPCl protein or NPC2 protein, or said mislocalisation of NPCl protein or NPC2 protein, results in a Niemann-Pick disease type C like cellular phenotype.
Typically, the disease which has a secondary Niemann-Pick disease type C (NPC) like cellular phenotype is a disease which does not involve a mutation in either the NPCl gene or the NPC2 gene but which incurs the accumulation of a biomolecule, the accumulation of which in turn induces a Niemann-Pick disease type C like cellular phenotype. Usually, the biomolecule is a class II amphiphile. A class II amphiphile is a detergent-like molecule which is, typically, capable of altering the stability of the lysosome. Typically, the class II amphiphile is 7-DHC. Alternatively, the class II amphiphile may be U18666A, 7- ketocholesterol, progesterone or imipramine, all of which are steroids or steroid analogues known to induce free cholesterol storage and abnormal lipid endocytosis. More typically, the biomolecule is a class II amphiphile which is a precursor or analogue of cholesterol. Thus the biomolecule may, for instance, be 7-DHC. The present inventors have observed that 7-DHC accumulates in Smith-Lemli-Opitz Syndrome (SLOS) cells to cause abnormal sphingolipid storage and transport in the LE/Lys system (a Niemann-Pick disease type C like cellular phenotype).
Diseases which have a secondary Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention include, but are not limited to, the following conditions:
Smith-Lemli-Opitz Syndrome (SLOS); Tangier disease - (Neufeld, E.B., et al., J. Biol. Chem., 2004, 279:15571-8);
Huntington's disease - (Truchina, E., et al., Hum. MoI. Genet., 2006, 15:3578-91); Cystic Fibrosis - (Gentzsch, M., et al., J. Cell. Sci., 2007, 120:447-455); Pelizaeus-Merzbacher disease - (Simons, M., et al., J. Cell. Biol., 2002, 157:327- 36); and Mucolipidosis II - (Inui, K., et al., Biochem. Int., 1989, 18:1129-35). hi one embodiment of the present invention, the disease which has a Niemann-Pick disease type C like cellular phenotype is other than cystic fibrosis.
In another embodiment of the present invention, the disease which has a Niemann- Pick disease type C like cellular phenotype is other than Tangier disease.
In another embodiment of the present invention, the disease which has a Niemann- Pick disease type C like cellular phenotype is other than Huntington's disease.
In one embodiment, therefore, the disease which has a Niemann-Pick disease type C like cellular phenotype is other than Tangier disease and other than Huntington's disease and other than cystic fibrosis.
The disease which has a secondary Niemann-Pick disease type C like cellular phenotype may be a disorder which alters the activity of an enzyme involved in cholesterol synthesis. Typically, therefore, the disease which has a secondary Niemann-Pick disease type
C like cellular phenotype is selected from Smith-Lemli-Opitz Syndrome, Tangier disease, Huntington's disease, Cystic Fibrosis, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis, for instance SLOS. More typically, the disease which has a secondary Niemann-Pick disease type C like cellular phenotype is selected from Smith-Lemli-Opitz Syndrome, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis, for instance SLOS.
In another embodiment, the disease which has a secondary Niemann-Pick disease type C like cellular phenotype is selected from Smith-Lemli-Opitz Syndrome, Pelizaeus- Merzbacher disease and Mucolipidosis II (Icell).
More typically, the disease which has a secondary Niemann-Pick disease type C like cellular phenotype is Smith-Lemli-Opitz Syndrome (SLOS).
In one embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type C, Smith-Lemli-Opitz Syndrome, Tangier disease, Huntington's disease, Cystic Fibrosis, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis. Typically, the Niemann-Pick type C disease is Niemann- Pick type Cl disease or Niemann-Pick type C2 disease. More typically, the Niemann-Pick type C disease is Niemann-Pick type Cl disease. Typically, the disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis is SLOS.
Typically, the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type C and Smith-Lemli-Opitz Syndrome. More typically, the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type Cl, Niemann-Pick disease type C2 and Smith- Lemli-Opitz Syndrome.
The diseases which have a Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention also include pathogenic infections which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype. One such pathogenic infection is the infectious disease tuberculosis. Tuberculosis is a global health problem affecting approximately one third of the world's population and resulting in three million deaths each year. A major current concern is the emergence of strains of Mycobacterium tuberculosis (Mtb) that are resistant to antibiotics. One of the characteristic hallmarks of tuberculosis is the ability of Mtb to successfully survive within cells of the innate immune system including macrophages and monocytes. Multiple mechanisms are involved in the intracellular survival of Mtb, including defective acidification of the phagosome and inhibition of phosphatidylinositol-dependent trafficking pathways, via secretion of inositol-like lipids by the mycobacteria. Bacilli interact with cell surface complement receptors and are ingested into phagosomes that mature but do not fuse with lysosomes.
Binding to the complement receptor is normally followed by an intracellular cytosolic elevation in which calcium can stimulate phago-lysosome fusion. However, it has been found that in M. tuberculosis-infected macrophages this elevation is substantially reduced; the present inventors have shown that the ability of lysosomes to fuse with late endosomes relies upon calcium release, specifically from the late endosomal/lysosomal compartment itself. When insufficient calcium is released from acidic stores there is a complete block in late endosome - lysosome fusion. A severe human disease results from calcium deficiency in acidic the acidic compartment, the lysosomal storage disease termed Niemann-Pick disease type C (NPC). NPC is unusual as it is caused by mutations in two genes, NPCl or NPC2, that function as part of the same cellular pathway. However, the precise mechanistic link between these two genes remains unknown and the functional roles of these proteins remains enigmatic. NPCl encodes a multimembrane spanning protein of the limiting membrane of the late endosome/lysosome where as NPC2 is a soluble cholesterol binding protein of the lysosome. The inventors have discovered that when NPCl is inactivated sphingosine is the first lipid to be stored, suggesting that NPCl plays a role in the transport of sphingosine from the lysosome, where it is normally generated as part of sphingolipid catabolism.
Elevated sphingosine in turn causes a defect in calcium entry into acidic stores resulting in greatly reduced calcium release from this compartment. This then prevents LE/Lys fusion (calcium dependent process) and causes the secondary accumulation of lipids (cholesterol, sphingomyelin and glycosphingolipids) that are cargos in transit through the late endocytic pathway. Other secondary consequences of inhibiting NPCl function include defective endocytosis and failure to clear autophagic vacuoles. The present inventors have therefore investigated whether the NPC1/NPC2 cellular pathway is targeted by pathogenic mycobacteria to promote their survival in late endosomes and found this to be the case (see Fig. 11 and Example 11). They have additionally found that therapies that are effective in NPC disease and in correcting NPC cellular phenotypes are also therefore effective in promoting clearance of pathogenic mycobateria from infected cells, hi particular, an agent that elevates cytosolic calcium (curcumin) was tested (see Example 12 and Fig. 12), as was an inhibitor of sphingolipid biosynthesis (miglustat; see Example 13 and Fig. 13). Both of these agents were found to promote the normalisation of NPC cellular phenotypes from cells treated with BCG-derived lipids (that inhibit NPC1/NPC2 function). These classes of agents therefore have therapeutic potential for treating any pathogenic infection which is capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
Accordingly, the diseases which have a Niemann-Pick disease type C like cellular phenotype which can be treated in accordance with the present invention also include pathogenic infections which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
In one embodiment, therefore, the invention provides a compound which elevates intracellular calcium for use in the treatment of a pathogenic infection capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
Such pathogenic infections include infection by any pathogen that that secretes a molecule (for instance a lipid or a secondary amine) that can inhibit NPCl function or any pathogen that prevents NPCl from getting to the compartment in which it normally functions (for instance by inhibition of late endosome acidification). Typically, intracellular cholesterol storage would also be observed in combination with either of these phenotypes. In one embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infection caused by any one of the following types of bacteria, all of which are capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype: Mycobacteria, Salmonella, Brucella, Coxiella, and Anaplasma phagocytophilum.
More typically, the pathogenic infection treated in accordance with the present invention is an infection caused by Mycobacteria (a mycobacterial infection). Such infections include the infectious disease tuberculosis.
Accordingly, in one embodiment, the invention provides a compound which elevates intracellular calcium for use in the treatment of tuberculosis.
In one embodiment, the compound which elevates intracellular calcium is other than curcumin. Accordingly, the invention in one embodiment provides a compound which elevates intracellular calcium, which compound is other than curcumin, for use in the treatment of tuberculosis. The mycobacterium which most commonly causes tuberculosis in humans is
Mycobacterium tuberculosis. However, other mycobacteria, such as Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannetti, and Mycobacterium microtti, also cause tuberculosis (although these species are less common in humans).
Thus, in one embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infection caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannetti and/or Mycobacterium microtti. hi another embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infection caused by Salmonella, Brucella or Coxiella. Thus, for instance, the infection may be one caused by Salmonella enterica, Brucella abortus or Coxiella burnetti.
In another embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is a pathogenic infection caused by Anaplasma phagocytophilum. Anaplasma phagocytophilum is a bacterium that lives inside white blood cells, and causes the disease human granulocytic anaplasmosis (HGA).
Accordingly, in another embodiment, the invention provides a compound which elevates intracellular calcium for use in the treatment of human granulocytic anaplasmosis. In one embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype which is treated in accordance with the present invention is an infectious disease, which infectious disease has has a NPC like cellular phenotype which is treated in accordance with the present invention. The invention in one embodiment therefore provides a compound which elevates intracellular calcium for use in the treatment of an infectious disease, which infectious disease is capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype. Typically, the infectious disease is tuberculosis. In another embodiment, the infectious disease is human granulocytic anaplasmosis.
The compound which elevates intracellular calcium may be administered, in order to treat or prevent said disease which has a NPC like cellular phenotype, as a dietary or food supplement.
Accordingly, in one embodiment, the compound which which elevates intracellular calcium is for administration as a dietary supplement.
The invention further provides a dietary supplement comprising a compound which elevates intracellular calcium, which dietary supplement is for use in the treatment of a disease which has a NPC like cellular phenotype.
The finding that sphingosine storage is the primary measurable event in NPC disease pathology suggests that Niemann-Pick disease type C (NPC), and in particular Niemann-Pick disease type Cl (NPCl) and Niemann-Pick disease type C2 (NPC2), can be treated with an agent which reduces sphingosine storage. This is supported by the data presented herein in Example 10 and Figure 10, which shows that ISP-I (also known as myriocin), a compound which reduces sphingosine storage, reduces lysosomal storage in NPCl -null CHO cells and could therefore be used to treat NPC.
Accordingly, the invention provides a compound which reduces sphingosine storage for use in the treatment of Niemann-Pick disease type C.
Sphingosine storage can be reduced by disrupting sphingolipid biosynthesis. Sphingolipid biosynthesis can be disrupted by the use of inhibitors of enzymes, such as transferases and synthases, that act upstream of glucosylceramide synthase or galactosylceramide synthase. Such inhibitors are termed "inhibitors of ceramide biosynthesis". Inhibitors of ceramide biosynthesis are capable of inhibiting the synthesis of a sphingolipid and thereby reduce sphingosine storage. Enzymes which act upstream of glucosylceramide synthase include serine palmitoyltransferase and dihydroceramide synthase. Inhibitors of serine palmitoyltransferase include L-Cycloserine and Myriocin. Inhibitors of dihydroceramide synthase include Fumonisin and Safingol (L-threo- dihydrosphingosine).
Sphingosine storage can also be reduced by inhibiting the degradation of ceramide, for instance by using a sphingomyelinase inhibitor (e.g. scyphostatin, manumycin A, W-7 and imipramine) or a ceramidase inhibitor (e.g. D-MAPP).
The term "compound which reduces sphingosine storage", as used herein, means any compound which reduces sphingosine storage by (a) inhibiting ceramide biosynthesis and/or (b) inhibiting ceramide degradation. hi one embodiment, the compound which reduces sphingosine storage is an inhibitor of ceramide biosynthesis. The skilled person can readily identify inhibitors of ceramide biosynthesis without undue experimentation, using known procedures (see, for instance, J. Biol. Chem., Vol. 268, No. 36, 1993, 27299-27306). Typically, the inhibitor of ceramide biosynthesis is an inhibitor of serine palmitoyltransferase (for instance, L- Cycloserine or Myriocin) or an inhibitor of dihydroceramide synthase (for instance, Fumonisin or S afingol) .
Typically, the inhibitor of ceramide biosynthesis is a compound of the following formula (DC):
Figure imgf000044_0001
in which: q is 0 or 1 ; r is 0 or 1 ;
RKa is H, COOH or an unsubstituted or substituted ester;
R003 is an unsubstituted or substituted C1-6 alkyl;
RKc and Rκ , which are the same or different, are each independently selected from H, unsubstituted or substituted C1-6 alkyl and unsubstituted or substituted phenyl;
RKe and Rκf, which are the same or different, are each independently selected from H, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl; either (a) one of RKg and R001 is H and the other is OR^, wherein RKr is selected from H, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl, or (b) RKg and R001 together form an oxo group;
RKl is H, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted C1-6 alkoxy and unsubstituted or substituted phenyl; RKj is H, unsubstituted or substituted C1-6 alkyl or a group of the following formula
(X):
Figure imgf000045_0001
in which R1*" and R1*0, which are the same or different, are each independently selected from OH, unsubstituted or substituted C1-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C1-6 alkylamino and unsubstituted or substituted di(C1-6)alkylamino;
RIxk is H, unsubstituted or substituted C1-6 alkyl or a group of the following formula (XI):
Figure imgf000045_0002
in which RKp and R0^, which are the same or different, are each independently selected from OH, unsubstituted or substituted C1-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C1-6 alkylamino and unsubstituted or substituted di(C1-6)alkylamino; and
RKm is selected from H and unsubstituted or substituted Ci-2O alkyl, which C1-20 alkyl is optionally interrupted by N(R'), O, S or phenylene, wherein R' is H, Ci-6 alkyl or phenyl; or a pharmaceutically acceptable salt thereof. hi one embodiment, r is O and q is 1. Typically, in that embodiment, Rm> is unsubstituted Ci-6 alkyl. More typically, R^5 is methyl. Furthermore, RKa is typically H. Usually RKc and RKd are independently selected from H and unsubstituted Ci-6 alkyl. More typically, however, RKc and RKd are both H. Typically, RKe and Rκf are independently selected from H and unsubstituted Ci-6 alkyl. More typically, RKe and R0^ are both H. Usually, one of RKg and R001 is H and the other is OR00", wherein R1* is selected from H and unsubstituted C1-6 alkyl. More typically, however, one of RKg and RKh is H and the other is OH. RKl is typically unsubstituted C1-6 alkyl, more typically methyl. Typically, Rκj is a group of formula (X). Ususally, R0* is a group of formula (XI). Typically, R1*", RKo, RKp and RKq, which are the same or different, are independently selected from H and unsubstituted Ci-6 alkyl. More typically, each of R , IEXn RKo, RKp and RIxq is H. RKm is typically selected from unsubstituted or substituted C1-I0 alkyl. More typically, RKm is an unsubstituted or substituted C1-6 alkyl. R1*1" may be, for instance, -CH(CH3)(C4H9).
In another embodiment, r is 1 and q is 0. Typically, in this embodiment, R is C1- 6 alkyl substituted with a hydroxyl group. More typically, R0* is CH2OH. Furthermore, R1*3 is typically COOH or an unsubstituted ester. More typically, R^ is COOH. Usually RKc and RKd are independently selected from H and unsubstituted C1-6 alkyl. More typically, however, RKc and RKd are both H. Typically, RKe and Rκf are independently selected from H and unsubstituted C1-6 alkyl. More typically, RKe and Rκf are both H. Usually, in this embodiment, RKg and R001 together form an oxo group. RKl is typically H. Typically, in this embodiment, Rκj and R00', which may be the same or different, are independently selected from H and unsubstituted C1-6 alkyl. More typically, Rκj and R0^ are both H. RDCm is typically, in this embodiment, selected from unsubstituted or substituted Ci-6 alkyl. More typically, RKm is an unsubstituted Ci-6 alkyl. RKm may be, for instance, methyl.
Table 1 shows examples of compounds of formula (IX) which maybe employed in the present invention as compounds which reduce sphingosine storage. Such compounds are inhibitors of ceramide biosynthesis. More specifically, compound 1 (Myriocin) is a serine palmitoyltransferase inhibitor and compound 2 (Fumonisin) is a dihydroceramide synthase inhibitor.
Table 1
Figure imgf000046_0001
Figure imgf000047_0001
Typically, the inhibitor of ceramide biosynthesis is a compound of the following formula (XII):
Figure imgf000047_0002
in which:
RXa is H, substituted or unsubstituted Ci-2O alkyl, substituted or unsubstituted C)-20 alkylene-aryl, substituted or unsubstituted Ci-2O alkylene-C3-20 heteroaryl, substituted or unsubstituted Ci-20 alkylene-C3-25 cycloalkyl, substituted or unsubstituted Ci-20 alkylene-C3. 20 heterocyclyl, substituted or unsubstituted Ci-20 alkylene-O-C3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C3-20 heteroaryl, substituted or unsubstituted C3-25 cycloalkyl or substituted or unsubstituted C3-20 heterocyclyl wherein said Ci-20 alkyl and Ci-2Q alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, C]-6 alkyl or aryl; and R501 and RXc, which are the same or different, are independently selected from H, unsubstituted or substituted Ci-I0 alkyl and unsubstituted or substituted aryl; or a pharmaceutically acceptable salt thereof.
Typically, RXa is H, substituted or unsubstituted Cj-1O alkyl or substituted or unsubstituted phenyl . More typically, RXa is H, unsubstituted C1-6 alkyl or unsubstituted phenyl . Even more typically, RXa is H.
Typically, R5* and RXc, which are the same or different, are independently selected from H, unsubstituted Ci-6 alkyl and unsubstituted phenyl. More typically, R and R are both H.
Table 2 shows an example of a compound of formula (XII) which maybe employed in the present invention as a compound which reduces sphingosine storage. The compound (compound 3) is an inhibitor of ceramide biosynthesis. More specifically, compound 3 (L- Cycloserine) is a serine palmitoyltransferase inhibitor.
Table 2
Figure imgf000048_0002
Typically, the inhibitor of ceramide biosynthesis is Safingol (L-threo- dihydrosphingosine). Safingol is an inhibitor of dihydroceramide synthase (Meyer, S.G.E. et al., Biochimica et Biophysica Acta 1643 (2003) 1-4). The structure of Safingol is as follows:
Figure imgf000048_0001
In one embodiment, the compound which reduces sphingosine storage is an inhibitor of ceramide degradation. The skilled person can readily identify inhibitors of ceramide degradation without undue experimentation, using known procedures (see for instance J. Med. Chem. 2008, 51, 219-237 and Bioorg. Med. Chem., 9 (2001) 2901-2904, which describe the identification of sphingomyelinase inhibitor compounds, and Bioorg. Med. Chem., 16 (2008) 1032-1045 which describes the identification of ceramidase inhibitors). Typically, the inhibitor of ceramide degradation is a sphingomyelinase inhibitor (for instance an acid sphingomyelinase inhibitor, such as W-7 or imipramine, or a neutral sphingomyelinase inhibitor, such as scyphostatin or manumycin A) or a ceramidase inhibitor (for instance an acid ceramidase inhibitor, such as D-MAPP).
Typically, the inhibitor of ceramide degradation is a ceramidase inhibitor of formula (G):
Figure imgf000049_0001
wherein:
RP1 is selected from hydrogen and unsubstituted or substituted Ci-6 alkyl; Rp2 and RP3, which are the same or different, are independently selected from hydrogen, hydroxyl and unsubstituted or substituted C1-6 alkyl;
RP4 is hydrogen, -NO2, -NH2 or -N(H)C(O)(CH2)nCH2Ypl, wherein YP1 is hydrogen, aryl or heteroaryl, and wherein n is 0 or an integer of 1 to 10;
RP5 is selected from unsubstituted or substituted methyl, unsubstituted or substituted ethyl, aryl and heteroaryl; YP2 is -CH2- or -C(O)-; YP3 is -CH2- or -N(H)-;
EP1 is N or N+H, provided that EP1 is N when YP2 is -C(O)-; and m is 0 or an integer of 1 to 14; or a pharmaceutically acceptable salt thereof.
Typically, RP1 is methyl or hydrogen. More typically, RP1 is hydrogen. Typically, RP2 and RP3 are independently selected from hydrogen and hydroxyl. More typically, RP2 is selected from hydrogen and hydroxyl and RP3 is hydroxyl.
Typically, RP5 is ethyl or heteroaryl. When RP5 is heteroaryl, it is typically a cationic heteroaryl group and, more typically, pyridinium, as follows:
Figure imgf000049_0002
Typically, R is hydrogen or -NO2. However, when R is -N(H)C(O)(CH2)nCH2Ypl, YP1 is typically H or a cationic heteroaryl group. The cationic heteroaryl group may be pyridinium.
Typically, EP1 is N+H, YP2 is -CH2- and YP3 is -CH2-. Alternatively, typically Epl is N, YP2 is -C(O)- and YP3 is either -CH2- or -N(H)-.
Typically, m is an integer of 5 to 9. More typically, m is 5 or 9.
It is understood that when Epl is N+H, a suitable counter-anion will be present, for instance a halide, typically Br" or Cl". Similarly, when a cationic aryl group, for instance pyridinium, is present as a substituent, a suitable counter-anion will be present. Again, the counter anion may be a halide, for instance Br" or Cl".
Table 3 shows examples of compounds of formula (G) which may be employed in the present invention as compounds which reduce sphingosine storage. Such compounds are ceramidase inhibitors and are described in Bioorg. Med. Chem., 16 (2008) 1032-1045.
Table 3
Figure imgf000050_0001
Figure imgf000051_0001
Typically, the inhibitor of ceramide degradation is a neutral sphingomyelinase inhibitor selected from manumycin A, scyphostatin or a compound of formula (H):
Figure imgf000052_0001
wherein:
RQ1 is C1-6 alkyl, -RQ2-aryl or -RQ2-0H;
R^2 is unsubstituted or substituted C1-6 alkylene; and
R^3 is unsubstituted or substituted C1-20 alkyl, wherein said C1-20 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C1-6 alkyl or aryl; or a pharmaceutically acceptable salt thereof.
Typically, RQ1 is methyl, -RQ2-phenyl or -RQ2-0H. Typically, RQ2 is methylene. Typically, RQ3 is butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl. More typically, RQ3 is n-nonyl.
The chemical structure of manumycin A is as follows:
Figure imgf000052_0002
The chemical structure of scyphostatin is as follows:
Figure imgf000052_0003
Table 4 shows examples of compounds of formula (H) which may be employed in the present invention as compounds which reduce sphingosine storage. Such compounds are neutral sphingomyelinase inhibitors and are described in Bioorg. Med. Chem., 9 (2001) 2901-2904. Table 4
Figure imgf000053_0002
Typically, the inhibitor of ceramide degradation is an acid sphingomyelinase inhibitor selected from W-5, W-7, dibucaine, mianserin, perhexiline, tamoxifen, trihexyphenidyl, amlodipine, bepridil, astemizole, suloctidil, AY9944, benztropine, camylofin, cloperastine, cocaine, clomiphene, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, paroxetine, sertraline, terfenadine and a compound of formula (J):
Figure imgf000053_0001
wherein:
R l and RG3, which are the same or different, are independently selected from hydrogen, halo, cyano, -CF3, -S(O)2NMe2, -SMe and C1-6 alkyl;
ZG1 is selected from -S-, -CH2-CH2-, -CH=CH- and -CH2-O-;
X01 is C and Y61 is CH and X01 and Y01 are linked by a double bond, or XG1 is selected from N and CH, Y01 is CH2 and XG1 and Y01 are linked by a single bond; RG2 is a group -Y°2-RG4, wherein Y°2 is substituted or unsubstituted CM alkyl and RG4 is selected from amino, C1-10 alkylamino, di(C1-i0)alkylamino and unsubstituted or substituted C3-20 heterocyclyl; provided that X01, YG1 and RG2 may together form the group:
Figure imgf000054_0001
or a pharmaceutically acceptable salt thereof.
Typically, one of RG1 and RG3 is hydrogen and the other is selected from hydrogen, halo, cyano, -CF3, -S(O)2NMe2, -SMe and C1-6 alkyl. More typically, both RGI and RG3 are hydrogen. Typically, the halo group is a chloro group. Typically, ZG1 is either -S- or -CH2-CH2-.
Typically, XG1 is C and Y01 is CH and XG1 and Y01 are linked by a double bond, or X01 is N and Y01 is CH2 and XG1 and Y01 are linked by a single bond;
Typically, Y02 is substituted or unsubstituted ethylene. More typically, Y02 is selected from -CH2-CH2 and -CH(CH3)-CH2-; Typically, RG4 is selected from -NMe2, -NHMe and the following heterocyclyl groups:
Figure imgf000054_0002
The structures of various acid sphingomyelinase inhibitors of formula (J), and of the acid sphingomyelinase inhibitors W-5, W-7, dibucaine, mianserin, perhexiline, tamoxifen, trihexylphenidyl, amlodipine, bepridil, astemizole, suloctidil, AY9944, benztropine, camylofin, cloperastine, cocaine, clomiphene, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, paroxetine, sertraline and terfenadine, are shown in the follwing Table 5. These inhibitors are described in J. Med. Chem. 2008, 57, 219-237 and may be employed in the present invention as compounds which reduce sphingosine storage. Table 5
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
In one embodiment, the compound which reduces sphingosine storage is an inhibitor of ceramide biosynthesis or an inhibitor of ceramide degradation. More typically, the compound which reduces sphingosine storage is an inhibitor of serine palmitoyltransferase, an inhibitor of dihydroceramide synthase, a sphingomyelinase inhibitor, for instance a neutral sphingomyelinase inhibitor or an acid sphingomyelinase inhibitor, or a ceramidase inhibitor, for instance an acid ceramidase inhibitor.
Typically, the compound which reduces sphingosine storage is any one of the inhibitors of ceramide biosynthesis and inhibitors of ceramide degradation defined above, or a pharmaceutically acceptable salt of any of those compounds.
More typically, the compound which reduces sphingosine storage is: Myriocin (ISP-I), Fumonisin, L-Cycloserine, L-tΛreo-dihydrosphingosine (Safingol), D-e-MAPP, LCL16, LCL284, LCL120, B13, LCL15, LCL204, LCL85, W-5, W-7, triflupromazine, terfenadine, suloctidil, sertraline, promethazine, paroxetine, nortriptyline, maprotiline, norfluoxetine, fluoxetine, fendiline, protriptyline, chlorprothixene, promazine, pimethixene, drofenine, doxepine, cyproheptadine, cyclobenzaprine, clomiphene, cloperastine, camylofϊn, bepridil, benztropine, astemizole, amlodipine, trimipramine, trihexylphenidyl, trifluoroperazin, thioridazin, thioproperazine, tamoxifen, quinacrine, propericiane, perhexiline, mianserin, prochlorperazine, imipramine, dibucaine, desipramine, clomipramine, alimenazine, amitriptyline, cyamemazine, cocaine, chlorpromazine, AY9944, manumycin A, scyphostatin or a compound of any one of the following formulae:
Figure imgf000060_0001
wherein R is CH3 or CH2C6H5; or a pharmaceutically acceptable salt thereof.
In one embodiment, the compound which reduces sphingosine storage is myriocin (ISP-I).
The compound which reduces sphingosine storage may be administered, in order to treat or prevent Niemann-Pick disease type C, as a dietary or food supplement.
Accordingly, in one embodiment, the compound which reduces sphingosine storage is for administration as a dietary supplement. The invention further provides a dietary supplement comprising a compound which reduces sphingosine storage, which dietary supplement is for use in the treatment of Niemann- Pick disease type C.
It is a further finding of the present invention that a compound which elevates intracellular calcium, as defined herein, can advantageously be used in combination with an inhibitor of sphingo lipid biosynthesis in order to treat a disease which has a Niemann- Pick disease type C like cellular phenotype.
The term "inhibitor of sphingolipid biosynthesis", as used herein, means a compound that is capable of inhibiting the synthesis or expression of a sphingolipid. Typically, the sphingolipid is ceramide or a compound which uses ceramide as a backbone, for instance a glycosphingolipid (GSL). More typically, the sphingolipid is a ganglioside. Alternatively, the sphingolipid is a neutral GSL.
The inhibitor of sphingolipid biosynthesis may be a compound which reduces sphingosine storage, as defined above, for instance an inhibitor of ceramide biosynthesis or an inhibitor of ceramide degradation. Alternativelty, the inhibitor of sphingolipid biosynthesis may be an inhibitor of an enzyme which acts downstream of glucosylceramide synthase or galactosyl ceramide synthase. Thus the inhibitor of sphingolipid biosynthesis could be an inhibitor of a glycosyltransferase or a sulfotransferase, for instance an inhibitor of a glucosyltransferase, sialyltransferase, galactosyltransferasae, ceramide galactosyltransferase, fucosyltransferase or N-acetylhexosaminetransferase, or an inhibitor of glucosylceramide synthase - such inhibitor compounds are further defined below.
Accordingly, in one embodiment of the first aspect of the invention, the compound which elevates intracellular calcium is for use in said treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype by coadministration with an inhibitor of sphingolipid biosynthesis.
Furthermore, in another aspect, the invention provides a product which comprises (a) a compound which elevates intracellular calcium and (b) an inhibitor of sphingolipid biosynthesis, for simultaneous, separate or sequential use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype. The invention also provides a compound which elevates intracellular calcium for use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype by coadministration with an inhibitor of sphingolipid biosynthesis.
The invention also provides an inhibitor of sphingolipid biosynthesis for use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype by coadministration with a compound which elevates intracellular calcium.
The invention also provides a method of treating a disease which has a Niemann- Pick disease type C like cellular phenotype which method comprises administering to a patient in need of such treatment an effective amount of a compound which elevates intracellular calcium and an effective amount of an inhibitor of sphingolipid biosynthesis. hi one embodiment, the disease which has a Niemann-Pick disease type C like cellular phenotype is a pathogenic infection which is capable of blocking lysosome- phagosome fusion via induction of a NPC like cellular phenotype. hi one embodiment, for instance, the disease which has a Niemann-Pick disease type C like cellular phenotype is tuberculosis. The inhibitor of sphingolipid biosynthesis miglustat (NB-DNJ) was found to promote the normalisation of NPC cellular phenotypes from cells treated with BCG- derived lipids: see Example 13 and Figure 13 hereinbelow.
The skilled person can readily identify inhibitors of sphingolipid biosynthesis without undue experimentation, using known procedures. For instance, inhibitors of sphingolipid biosynthesis can be identified by incubating and or growing cells in culture in the presence of the putative inhibitor together with an assay for the effect of sphingolipid biosynthesis. Such assays include the analysis of fluorescently-labelled glycosphingolipid carbohydrate headgroups by HPLC, thin-layer chromatography (TLC) of sphingolipids and analysis of sphingolipids using mass spectrometry (Neville DC, Anal. Biochem. 2004 Aug 15;331(2):275-82; Mellor HR Biochem. J. 2004 Aug l;381(Pt 3):861-6; Hayashi Y. et al., Anal. Biochem. 2005 Oct 15;345(2): 181-6; Sandhoff, R. et al., J. Biol. Chem., vol. 277, no. 23, 20386-20398, 2002; Sandhoff, R. et al., J. Biol. Chem., vol. 280, no. 29, 27310- 27318, 2005; Platt, F.M. et al., J. Biol. Chem., vol. 269, issue 11, 8362-8365, 1994; Platt, F.M. et al., J. Biol. Chem., vol. 269, issue 43, 27108-27114, 1994).
There are several classes of compounds which can affect the metabolism of sphingolipids, and thereby inhibit sphingolipid biosynthesis; these include compounds of formulae (G), (H) and (J), defined above, and compounds of formulae (I), (IT), (HT), (FV), (V), (IX), (XH) defined below. Any of these compounds can be used in combination with a compound which elevates intracellular calcium, as defined above, in order to treat a disease which has a Niemann-Pick disease type C like cellular phenotype.
The inhibitor of sphingolipid biosynthesis may be an inhibitor of a glycosyltransferase or a sulfotransferase. When the inhibitor of sphingolipid biosynthesis is an inhibitor of a glycosyltransferase, it may be an inhibitor of glucosyltransferase, sialyltransferase, galactosyltransferasae, ceramide galactosyltransferase, fucosyltransferase, or N-acetylhexosaminetransferase. Typically, the inhibitor of sphingolipid biosynthes is an inhibitor of glucosylceramide synthase. Alternatively, in one embodiment, the inhibitor of sphingolipid biosynthesis is an inhibitor of ceramide biosynthesis. The inhibitor may be an inhibitor of serine palmitoyltransferase or an inhibitor of dihydroceramide synthase. Many of these types of inhibitor compounds are described in WO 2008/012555. Alternatively, the inhibitor of sphingolipid biosynthesis maybe an inhibitor of ceramide degradation, for instance a sphingomyelinase inhibitor (e.g. a neutral sphingomyelinase inhibitor or an acid sphingomyelinase inhibitor) or a ceramidase inhibitor (e.g. an acid ceramidase inhibitor). Typically, the inhibitor of sphingolipid biosynthesis is a compound of of one of the following formulae (I), (H), (m), (IV), (V), (IX) and (XII):
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000063_0002
Figure imgf000063_0003
wherein:
X is O, S or NR5;
R5 is hydrogen, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C1-20 alkylene-aryl, substituted or unsubstituted C1-20 alkylene-C3-20 heteroaryl, substituted or unsubstituted Ci-20 alkylene-C3-25 cycloalkyl, substituted or unsubstituted Ci-20 alkylene- C3-20 heterocyclyl, substituted or unsubstituted Ci-20 alkylene-O-C3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C3-20 heteroaryl, substituted or unsubstituted C3-25 cycloalkyl or substituted or unsubstituted C3-20 heterocyclyl, or R5 forms, together with R1, R1 ', R4 or R14, a substituted or unsubstituted C]-6 alkylene group, wherein said C1-20 alkyl and C1-20 alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, Ci-6 alkyl or aryl; n is O or 1 ;
Y is O, S or CR6R16; R1, R1 ', R4 and R14, which may be the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted Ci-20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, Ci-I0 alkylamino, di(Ci-io)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, provided that one of R1 , R1 ' , R4 and R14 may form, together with R5, a substituted or unsubstituted Ci-6 alkylene group, wherein said Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
R2, R12, R3, R13, R6 and R16, which may be the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Cj-20 alkyl, substituted or unsubstituted Ci-20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, CM0 alkylamino, di(Ci-io)alkylamino, amido, acylamido -0-C3-25 cycloalkyl and -O-C3-2o heterocyclyl, wherein said Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene; R21 is selected from oxo, -L30-R23, -L30-C(O)N(H)-R24 and a group of the following formula (VI):
Figure imgf000064_0001
L30 is substituted or unsubstituted Ci-20 alkylene which is optionally interrupted by N(R'), O, S or arylene;
R23 is carboxyl, hydroxyl, ester, phosphonate ester, phosphate ester, phosphoric acid and phosphonic acid;
R24 is Ci-20 alkyl which is unsubstituted or substituted with one or more groups selected from carboxyl, hydroxyl, ester, phosphonate ester, phosphate ester, phosphoric acid and phosphonic acid, wherein said Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
R30 is Ci-2O alkyl which is unsubstituted or substituted with one or more groups selected from carboxyl, hydroxyl, ester, amino, phosphonate ester, phosphate ester, phosphoric acid and phosphonic acid, wherein said Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene; and
R22 is hydroxyl, oxo, acyloxy, phosphoric acid or -OC(O)-alk-C(O)OH, wherein alk is substituted or unsubstituted Ci-20 alkylene which is optionally interrupted by N(R'), O, S or arylene;
Base is selected from a group of any one of the following formulae (a), (b), (c), (d), (e), (f) and (g):
Figure imgf000065_0001
(a) (b) (C) (d)
Figure imgf000065_0002
(e) (f) (g) . y is 0 or 1 ;
R31 is OH; R32 is H or OH; or, provided that y is 0, R31 and R32 together form -O-C(R33)(R34)-O-, wherein R33 and R34 are independently selected from H and methyl;
A is substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C1-20 alkylene-aryl, substituted or unsubstituted C1-2O alkylene-C3-20 heteroaryl, substituted or unsubstituted Ci-20 alkylene-C3-25 cycloalkyl or substituted or unsubstituted Ci-20 alkylene- C3-20 heterocyclyl, wherein said Ci-20 alkyl and Ci-20 alkylene are optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci-6 alkyl or aryl, or A is a group of any one of the following formulae (g) to (k):
-
Figure imgf000065_0003
(9) (h) (i)
Figure imgf000066_0001
0) (k)
L70, L701 and L702 are independently selected from -0-, -C(R35)(R36)- and -NH-, wherein R35 and R36 are independently selected from H, OH and CH3;
R70, R71 and R701 are selected from OH, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C1-20 alkoxy, substituted or unsubstituted C1-10 alkylamino and -L7I-(X2)m-L72-R72; wherein m is O or 1; X2 is O, S, -C(R45)(R46)- or -O-C(R45)(R46)-, wherein R45 and R46 are independently selected from H, OH, phosphonic acid or a phosphonic acid salt; L71 and L72 are independently selected from a single bond and substituted or unsubstituted C1-20 alkylene, which C1-20 alkylene is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, Ci-6 alkyl or aryl; and R72 is C3-25 cycloalkyl or C3- 20 heterocyclyl;
LJ is substituted or unsubstituted Ci-20 alkylene;
RJ1, RJ2, RJ3, RJ4, RJ5, RJ6 and RJ7, which are the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted Ci-20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, Ci-I0 alkylamino, di(C1-10)alkylamino, amido, acylamido, -N(H)C(O)CH=CH-RJ8, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene, and wherein RJ8 is substituted or unsubstituted Ci-20 alkyl; L and L , which are the same or different, are independently selected from a single bond and substituted or unsubstituted Ci-20 alkylene;
Xκ is N or C(RK6), wherein RK6 is H, COOH or ester;
Zκ is O or CH(R1"); p is O or 1 ; RK1, R1", R10, RK4 and R1", which are the same or different, are independently selected from hydrogen, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C1-20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C1-Io alkylamino, di(Ci-1o)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said C1-20 alkyl is optionally interrupted by N(R'), O, S or arylene; R1^ and Rm, which are the same or different, are independently selected from H, substituted or unsubstituted C1-6 alkyl or substituted or unsubstituted phenyl;
R1^ is H, substituted or unsubstituted aryl, -CH=CHR^, or substituted or unsubstituted Ci-20 alkyl, which Ci-2O alkyl is optionally interrupted by N(R'), O, S or arylene wherein R' is H, C1-6 alkyl or aryl; R^0 is H, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted phenyl or -C(O)R^8;
Rm is H or substituted or unsubstituted C1-20 alkyl, which C1-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
RWs is H or substituted or unsubstituted Ci-20 alkyl, which Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
RWe is H, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted Ci-20 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, CM0 alkylamino, di(Ci-i0)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl, -O- C3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C3-20 heteroaryl, substituted or unsubstituted C3-25 cycloalkyl or substituted or unsubstituted C3-20 heterocyclyl, which Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene;
Lw is substituted or unsubstituted Ci-20 alkylene which Ci-20 alkylene is optionally interrupted by N(R'), O, S or arylene;
R91 and R92, which are the same or different, are independently selected from H, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted aryl and -L91-R95, wherein L91 is substituted or unsubstituted Ci-20 alkylene, wherein said Ci-20 alkyl and said Cj-20 alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, Ci-6 alkyl or aryl, and wherein R95 is substituted or unsubstituted aryl, amino, Ci-I0 alkylamino or di(Ci-io)alkylamino; R93 is -L92-R96, wherein L92 is a single bond or substituted or unsubstituted Ci-20 alkylene, which Ci-20 alkylene is optionally interrupted by N(R'), O, S or arylene, and wherein R96 is amido or substituted or unsubstituted aryl;
R94 is H or substituted or unsubstituted Ci-20 alkyl, which Ci-20 alkyl is optionally interrupted by N(R'), O, S or arylene; q is O or 1 ; r is O or 1 ;
RKa is H, COOH or an unsubstituted or substituted ester; R005 is an unsubstituted or substituted Ci-6 alkyl;
RKc and RKd, which are the same or different, are each independently selected from H, unsubstituted or substituted C1-6 alkyl and unsubstituted or substituted phenyl;
RKe and RKf, which are the same or different, are each independently selected from H, unsubstituted or substituted Ci-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl; either (a) one of RKg and R0* is H and the other is OR1*, wherein R1* is selected from H, unsubstituted or substituted Ci-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl, or (b) RKg and R001 together form an oxo group;
RKl is H, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted Ci-6 alkoxy and unsubstituted or substituted phenyl;
RKj is H, unsubstituted or substituted C1-6 alkyl or a group of the following formula (X):
Figure imgf000068_0001
in which R1501 and RKo, which are the same or different, are each independently selected from OH, unsubstituted or substituted Ci-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted Ci-6 alkylamino and unsubstituted or substituted di(C1-6)alkylamino;
R1*1* is H, unsubstituted or substituted C1-6 alkyl or a group of the following formula (XI):
Figure imgf000068_0002
in which RKp and RKq, which are the same or different, are each independently selected from OH, unsubstituted or substituted Ci-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C1-6 alkylamino and unsubstituted or substituted di(C1-6)alkylamino; RKm is selected from H and unsubstituted or substituted Ci-20 alkyl, which Ci-20 alkyl is optionally interrupted by N(R'), O, S or phenylene, wherein R' is H, C1-6 alkyl or phenyl;
RXa is H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C1-20 alkylene-aryl, substituted or unsubstituted C1-2O alkylene-C3-20 heteroaryl, substituted or unsubstituted Ci-20 alkylene-C3-25 cycloalkyl, substituted or unsubstituted Ci-20 alkylene-C3- 20 heterocyclyl, substituted or unsubstituted C1-20 alkylene-O-C3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C3-20 heteroaryl, substituted or unsubstituted C3-25 cycloalkyl or substituted or unsubstituted C3-20 heterocyclyl wherein said Ci-2O alkyl and C1-20 alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, C1-6 alkyl or aryl; and
R503 and RXc, which are the same or different, are independently selected from H, unsubstituted or substituted C1-10 alkyl and unsubstituted or substituted aryl; or a pharmaceutically acceptable salt thereof. Such compounds are described and exemplified in WO 2008/012555. Alternatively, the inhibitor of sphingolipid biosynthesis may be a compound of any one of formulae (G), (H) and (J) defined above, or a pharmaceutically acceptable salt thereof.
The inhibitor of sphingolipid biosynthesis is typically one of the following compounds: N-butyldeoxynojirimycin; N-nonyldeoxynojirimycin; N-butyldeoxygalactonojirimycin; N- 5-adamantane-l-yl-methoxypentyl-deoxynojirimycin; alpha-homogalactonojirimycin; nojirimycin; deoxynojirimycin; N7-oxadecyl-deoxynojirimycin; deoxygalactonojirimycin; N-butyl-deoxygalactonoj irimycin; N-nonyl-deoxygalactonoj irimycin; N-nonyl- βdeoxygalactonojirimycin; N7-oxanonyl-6deoxy-DGJ; alpha-homoallonoj irimycin; beta-1- C-butyl-deoxygalactonoj irimycin; l,5-dideoxy-l,5-imino-D-glucitol, l,5-(Butylimino)-l,5- dideoxy-D-glucitol ; 1 , 5 -(Methylimino)- 1 , 5 -dideoxy-D-glucitol ; 1 , 5 -(Hexylimino)- 1,5- dideoxy-D-glucitol; 1 ,5-(Nonylylimino)- 1 ,5-dideoxy-D-glucitol; 1 ,5-(2-Ethylbutylimino)- 1 ,5-dideoxy-D-glucitol; 1 ,5-(2-Methylpentylimino)- 1 ,5-dideoxy-D-glucitol; 1,5- (Benzyloxycarbonylimino)- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-(Phenylacetylimino)- 1,5-dideoxy-D-glucitol, tetraacetate; l,5-(Benzoylimino)-l,5-dideoxy-D-glucitol, tetraacetate;l,5-(Butylimino)-l,5-dideoxy-D-glucitol, tetraacetate; l,5-(Ethyl malonylimino)- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-(Hexylimino)- 1 ,5-dideoxy-D- glucitol, tetraacetate; l,5-(Nonylimino)- 1,5-dideoxy-D-glucitol, tetraacetate; 1,5- (Benzyloxycarbonylimino)- 1 ,5-dideoxy-D-glucitol, tetraisobutyrate; 1 ,5-(Butylimino)- 1,5- dideoxy-D-glucitol, tetrabutyrate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-D-glucitol, tetrapropionate; l,5-(Butylimino)-l,5-dideoxy-D-glucitol, tetrabenzoate; 1,5-Dideoxy-1,5- imino-D-glucitol, tetraisobutyrate; 1 ,5-(Hydrocinnamoyliniino)- 1 ,5-dideoxy-D-glucitol, tetraacetate; l,5-(Methyl malonylimino)-l,5-dideoxy-D-glucitol, tetraacetate; 1,5-
(Butylimino)- 1 ,5-dideoxy-D-glucitol, tetraisobutyrate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-4R,6- O-(phenylmethylene)-D-glucitol, diacetate; 1 ,5-[(Phenoxymethyl)carbonylimino]- 1 ,5- dideoxy-D-glucitol, tetraacetate; l,5-[(Ethylbutyl)imino]-l,5-dideoxy-D-glucitol, tetraacetate; l,5-(Butylimino)-l,5-dideoxy-D-glucitol, 2,3 -diacetate; l,5-(Hexylimino)-l,5- dideoxy-4R,6-O-(phenylmethylene)-D-glucitol, diacetate; l,5-(Hexylimino)-l,5-dideoxy- D-glucitol, 2,3-diacetate; l,5-[(2-Methylpentyl)imino]-l,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-D-glucitol, 6-acetate; 1 ,5-[(3-Nicotinoyl)imino]- 1 ,5- dideoxy-D-glucitol, tetraacetate; 1 ,5-(Cinnamoylimino)- 1 ,5-dideoxy-D-glucitol, tetraacetate; l,5-(Butylimino)-l,5-dideoxy-D-glucitol, 2,3-dibutyrate; l,5-(Butylimino)- l,5-dideoxy-4R,6-O-(phenylmethylene)-D-glucitol, 2,3-dibutyrate; 1,5- (Phenylacetylimino)- 1 ,5-dideoxy-D-glucitol, tetraisobutyrate; 1 ,5-[(4- Chlorophenyl)acetylimino]- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-[(4- Biphenyl)acetylimino]-l,5-dideoxy-D-glucitol, tetraacetate; 1,5- (Benzyloxycarbonylimino)- 1 ,5-dideoxy-D-glucitol, tetrabutyrate; 1 ,5-Dideoxy- 1 ,5-imino- D-glucitol, tetrabutyrate; 3,4,5-piperidinetriol,l-propyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, l-pentyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5- piperidinetriol, l-heptyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- butyl-2- (hydroxymethyl)-, (2S, 3S, 4R, 5S); 3,4,5-piperidinetriol, l-nonyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol,l- (1-ethyl) propyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (3-methyl) butyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (2-phenyl) ethyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (3-phenyl) propyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol,l- (1-ethyl) hexyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (2-ethyl) butyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetrioI,l-[(2R)-(2-methyI-2-phenyl) ethyl]-2-(hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, l-[(2S)-(2-methyl-2- phenyl) ethyl] -2-(hydroxymethyl)-, (2S, 3R, 4R, 5S), β-L-homofuconojirimycin; propyl 2- acetamido-2-deoxy-4-O-(y9-D-galactopyranosyl)-3-O-(2-(N-09-L- homofuconojirimycinyl))ethyl)-α-D-glucopyranoside; D,L-threo-l-phenyl-2- decanoylamino-3-moφholino-l-propanol (PDMP); D,L-threo-l-phenyl-2- hexadecanoylamino-3-moφholino-l-propanol (PPMP); D-threo-l-phenyl-2- palmitoilamino-3-pyrrolidino- 1 -propanol (P4); 4'-hydroxy-D-threo- 1 -phenyl-2- palmitoilamino-3-pyrrolidino-l-propanol (4'-hydroxy-P4); 3',4'-ethylenedioxy-P4 (EtDO- P4); 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine; cytidin-5'-yl sialylethylphosphonate; sialic acid; Soyasaponin I; Fumonisin, Myriocin; L-cycloserine; L-threo- dihydrosphingosine (Safingol);
Figure imgf000071_0001
The inhibitor of sphingo lipid biosynthesis may alternatively be any one of the compounds of formula (DI) described in R. Wang et al., Biooorg. & Med. Chem., Vol. 5, No. 4, pp 661-672, 1997; X. Wang et al., Medicinal Research Reviews, Vol. 23, No. 1, 32- 47, 2003; Schafer et al., J. Org. Chem. 2000, 65, 24-29; and Qiao et al., J. Am. Chem. Soc, 1996, 118, 7653-7662, and in WO 2008/012555.
The inhibitor of sphingolipid biosynthesis may alternatively be any one of the following compounds: D-e-MAPP, LCL16, LCL284, LCL120, B13, LCL15, LCL204, LCL85, W-5, W-7, trifiupromazine, terfenadine, suloctidil, sertraline, promethazine, paroxetine, nortriptyline, maprotiline, norfluoxetine, fluoxetine, fendiline, protriptyline, chlorprothixene, promazine, pimethixene, drofenine, doxepine, cyproheptadine, cyclobenzaprine, clomiphene, cloperastine, camylofin, bepridil, benztropine, astemizole, amlodipine, trimipramine, trihexylphenidyl, trifluoroperazin, thioridazin, thioproperazine, tamoxifen, quinacrine, propericiane, perhexiline, mianserin, prochlorperazine, imipramine, dibucaine, desipramine, clomipramine, alimenazine, amitriptyline, cyamemazine, cocaine, chlorpromazine, AY9944, manumycin A, scyphostatin and a compound of any one of the following formulae: Hig
Figure imgf000072_0001
wherein R is CH3 or CH2C6Hs; or a pharmaceutically acceptable salt thereof.
Typically, the inhibitor of sphingolipid biosynthesis employed is N- butyldeoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ). More typically, the inhibitor of sphingolipid biosynthesis is NB-DΝJ. NB-DGJ is the galactose analogue of NB-DΝJ. NB-DGJ inhibits GSL biosynthesis comparably to NB-DΝJ but lacks certain side effect activities associated with NB-DΝJ. There has been extensive use of NB-DGJ in mouse models of GSL storage diseases and it is very well tolerated.
Thus, in one embodiment, the inhibitor of sphingolipid biosynthesis employed in combination with the compound which elevates intracellular calcium is NB-DGJ. Typically, in this embodiment, the compound which elevates intracellular calcium is curcumin.
A compound which elevates intracellular calcium, a compound which reduces sphingosine storage or an inhibitor of sphingolipid biosynthesis, for use in accordance with the present invention, can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously. The compound may therefore be given by injection or infusion.
The compound which elevates intracellular calcium, the compound which reduces sphingosine storage or the inhibitor of sphingolipid biosynthesis, may be presented for administration in a liposome. Thus, the compound may be encapsulated or entrapped in the liposome and then administered to the patient to be treated. Active ingredients encapsulated by liposomes may reduce toxicity, increase efficacy, or both. Notably, liposomes are thought to interact with cells by stable absorption, endocytosis, lipid transfer, and fusion (R.B. Egerdie et al., 1989, J. Urol. 142:390).
The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a compound is administered alone to adult humans is 0.0001 to 50 mg/kg, most commonly in the range of 0.001 to 10 mg/kg, body weight, for instance 0.01 to 1 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg/kg body weight, preferably from 0.0001 to 0.1 mg/kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule. Typically a dose to treat human patients may range from about 0.1 mg to about 1000 mg of a compound for use in accordance with the invention, more typically from about 10 mg to about 1000 mg of a compound for use in accordance with the invention. A typical dose may be about 100 mg to about 300 mg of the compound. A dose may be administered once a day (QID), twice per day (BE)), or more frequently, depending on the pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism, and excretion of the particular compound. In addition, toxicity factors may influence the dosage and administration regimen. When administered orally, the pill, capsule, or tablet may be ingested daily or less frequently for a specified period of time. The regimen may be repeated for a number of cycles of therapy.
A compound is formulated for use as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier or diluent. The compositions are typically prepared following conventional methods and are administered in a pharmaceutically suitable form. The compound may be administered in any conventional form, for instance as follows: A) Orally, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is present as such, or mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides for example polyoxyethylene sorbitan monooleate. The said aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, such as sucrose or saccharin.
Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
Pharmaceutical compositions for use in accordance with the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occuring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan mono- oleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.
Such formulations may also contain a demulcent, a preservative and flavouring and coloring agents;
B) Parenterally, either subcutaneously, or intravenously, or intramuscularly, or intrasternally, or by infusion techniques, in the form of sterile injectable aqueous or oleaginous suspensions. This suspension may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic paternally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
Among the acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. hi addition fatty acids such as oleic acid find use in the preparation of injectables;
C) By inhalation, in the form of aerosols or solutions for nebulizers; D) Rectally, in the form of suppositories prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and poly-ethylene glycols; E) Topically, in the form of creams, ointments, jellies, collyriums, solutions or suspensions.
F) Vaginally, in the form of 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. The present invention is further illustrated in the Examples which follow:
EXAMPLES
Example 1 - Abnormally low lysosomal Ca2+ levels are caused by sphingosine storage in NPCl cells
Human B lymphoblasts, loaded with 3mM Fura-2AM at room temperature for 45 minutes, were treated with either lOOμM ryanodine (Figure 1, graph A) or lμM thapsigargin (Figure 1 , graph B) to initiate ER calcium release. To release the mitochondrial calcium pool, B lymphoblasts were treated with 2μM CCCP, a mitochondrial uncoupler (Figure 1, graph C). No differences were observed between normal and NPCl cells in calcium release from these compartments. Calcium release from the late endosomal/lysosomal organelles was induced using either 200μM GPN (a substrate of cathepsin C which results in osmotic lysis of the organelle when cleaved) (Figure 1, graph D) or 50OnM bafilomycin Al (an inhibitor of the H+ pumping vacuolar- ATPase) (Figure 1, graph E). It was observed that NPCl cells had -65% reduction in the late endosomal/lysosomal calcium pool compared to normal cells (Figure 1, graphs D and E). Potential candidates that would cause this phenotype were screened by screening the effects of all the NPCl storage lipids individually on the late endosomal/lysosomal calcium pool of normal cells. Cells were pre-incubated with each individual lipid for 5 minutes prior to addition of GPN. Of all the lipids screened
(cholesterol and gangliosides are not shown) only sphingosine was capable of inducing the reduction in the calcium pool this quickly (and did not affect lysosomal pH, not shown) (Figure 1, graph F). Each trace is representative of 3-5 independent experiments.
Example 2 - Ul 8666a induces a rapid reduction in the late endosome/lysosomal calcium pool correlating with accumulation of sphingosine
Treatment of RAW macrophages with Ul 8666a over a 24h timecourse leads to a blockage in transport of BODIPY labelled lactosylceramide (BODIP Y-LacCer) from late endosomes (punctate appearance) to the Golgi (perinuclear crescent staining) at 2h post-treatment which persists up to 24h (Figure 2 A, upper panels). Cholesterol levels (visualized by filipin, Figure 2 A, lower panels) and GSL levels (measured by HPLC, Figure 2, graph D) are normal for the first 4h of treatment but are elevated after 8h and 24h. Lysosomal calcium levels (measured with GPN) are decreased following only Ih treatment (Figure 2, graph B) which correlates with elevations in sphingosine levels at 30 min and Ih post Ul 8666a treatment (Figure 2, graph C). This suggests that sphingosine storage is the primary event in NPCl pathology and leads to the lysosomal calcium defect which ultimately results in downstream lipid storage.
Example 3 - Correction of several NPCl defective cellular phenotypes using calcium modulating drugs
Treatment of NPCl null CHO cells with lμM thapsigargin for Ih leads to a correction in defective transport of BODIP Y-LacCer from the endocytic system to the Golgi (Figure 3.1, A, panels I to IV). This is associated with a gradual reduction in GSL levels back to wild- type levels after 24h treatment with thapsigargin (Figure 3.1, graph B). Cholesterol localization (filipin, Fig. 3.2, A, panels I to IV) and cholesterol levels (amplex red cholesterol oxidase assay, Fig. 3.2, graph B) also return to normal at Ih post thapsigargin treatment. To assess whether this correction correlated with delivery of cholesterol to the ER we measured the levels and localization of neutral lipids using nile red (Fig. 3.3, A, panels I to IV) and the levels of cholesterol esters using the amplex red cholesterol esterase assay (Fig. 3.3, graph B). Both showed elevations in cholesterol esters and correct localization to the perinuclear ER illustrating that the stored cholesterol in NPCl cells can be delivered to the ER for utilization following elevation in cytosolic calcium. Example 4 - Multiple calcium modulating drugs are capable of reversing the defective NPCl cellular transport defect
In addition to thapsigargin, other calcium modulating agents that elevate intracellular calcium levels, curcumin and lα, 25-dihydroxyvitamin D3 (lα,25(OH)2VD3) have been tested. All three agents were capable of correcting defective BODIPY-LacCer transport in NPCl null glial cells (Figure 4), indicating that any agent which elevates intracellular calcium may correct several aspects of NPCl cellular pathology.
Example 5 - Improvement in the NPCl mouse model following administration of a curcumin supplemented diet
NPCl knock-out mice were fed a diet of pelleted mouse chow with or without curcumin amounting to a dosage of 150mg/kg/day from weaning at 3 weeks of age until death.
Curcumin fed mice had superficial improvements in coat condition and also improved gait compared to ataxic untreated NPC mice (Figure 5, A, panels I to IV). Curcumin fed NPCl mice also exhibited improved weight gain and a delay in subsequent weight loss accompanied by an increase in life expectancy (Figure 5, B). Additional functional benefits were seen in motor function as measured by the open field test (Figure 5, C), curcumin fed NPCl mice remained functional for an additional 3 weeks (~33% improvement) compared to untreated NPC mice.
Example 6 - Altered endocytosis in diseases with associated NPC-like phenotypes can also be corrected following curcumin treatment
Smith-Lemli-Opitz (SLO) null (DHCR7-/-) mouse embryonic fibroblasts have been shown to have an NPCl -like cellular phenotype (cholesterol storage in enlarged lysosomes, Wassif et al. MoI. Genet. Metab. 2002). This defect is believed to be caused by elevations in 7-dehydrocholesteroI (caused by mutations in DHCR7, the enzyme that catalyses the conversion of 7-dehydrocholesterol to cholesterol) which inhibits the NPCl protein. Addition of curcumin to these fibroblasts (Figure 6, micrographs), to overcome the observed calcium defects (Fig. 6, graphs) corrects the defective transport of BODIPY- LacCer that is observed under cholesterol null growth conditions (LPDS, Figure 6 micrographs) illustrating that increasing intracellular calcium is a potential therapy for any disorder where the function of the NPCl protein is inhibited.
Example 7 - Curcumin reduces NPCl-null CHO cell lysosomal storage
Figure 7 shows that NPCl-null CHO cells have elevated lysotracker staining (measured using a 96-well plate fluorimeter) indicative of lysosomal storage (control CHO cells = 7300 ± 250). Treatment with 30μM curcumin (an inhibitor of the sarco/endoplasmic reticulum calcium ATPase) for the indicated time leads to a reduction in lysotracker staining indicative of a reduction in lysosomal storage. Images (micrographs labelled "CHO" and "Curcumin") are representative of NPCl-null CHO cell lysotracker staining (top) and 24h curcumin treated NPCl-null CHO cells (bottom): a reduction in fluorescence is clearly visible.
Example 8 - increased life expectancy of NPCl mice fed curcumin and further benefit upon combination of curcumin with NB-DNJ
Untreated NPCl knock-out mice (see triangular data points in Figure 8) loose weight rapidly between 7-11 weeks age and are all dead by week 11. NPCl knock-out mice fed a diet of mouse chow supplemented with 150mg/kg/day curcumin (see circular data points in Figure 8) have a similar decrease in body weight compared to untreated NPCl mice but survive until 13.5 weeks of age (23% increase). Combination treatment (see diamond- shaped data points in Figure 8) of curcumin and NB-DNJ (NB-DNJ alone leads to a 1 week increase in lifespan of NPCl mice, Zervas et al. Current Biology 2001) leads to a reduction in decline of NPCl knock-out mice body weight and an increase in survival to 16 weeks (45% increase) indicative of synergy between the two drugs (see Figure 8).
Example 9 - ISP-I partially corrects the reduction in NPCl-null CHO cell lysosomal calcium levels
Wild-type (see upper, black trace in Figure 9), NPCl-null (see middle, light grey trace in Figure 9) and 15OnM ISP-1/myriocin treated NPCl-null (see lower, dark grey trace in Figure 9) CHO cells were labeled with Calcium Green 1-AM and Fura Red- AM to measure intracellular calcium. Lysosomal calcium was released following addition of ionomycin to permeabilise all membranes to calcium (apart from the lysosomal membrane) and then GPN (a substrate of cathepsin C which upon cleavage induces lysosomal osmotic lysis). NPCl -null cells have a characteristic reduction in lysosomal calcium compared to wild-types. Following 3-day treatment with 15OnM ISP-I (an inhibitor of serine palmitoyltransferase, the first sphingolipid biosynthetic enzyme) NPCl lysosomal calcium is elevated almost to normal levels (see Figure 9).
Example 10 - ISP-I (myriocin) reduces NPCl-null CHO cell lysosomal storage
Figure 10 (A), first two micrographs, and Figure 10 (B), first two bars, show that NPCl- null CHO cells have elevated lysotracker staining compared to controls (measured using a 96-well plate fluorimeter), indicative of lysosomal storage. Treatment with 15OnM ISP- 1 /myriocin (an inhibitor of serine palmitoyltransferase the first enzyme in sphingolipid biosynthesis) for the indicated time leads to a reduction in lysotracker staining indicative of a reduction in lysosomal storage (see Figure 10 (A), third and fourth micrographs, and Figure 10 (B), third and fourth bars of the graph). Images (A) are representative microscopy images of lysotracker staining similar to the 96 well plate assay. In (B) a reduction in fluorescence is clearly visible at 3 and 5 days post-treatment.
Example 11 - Live mycobacteriutn bovis (BCG) and mycotic acid lipids (M.A.) from mycobacterium tuberculosis (Tb) induce an NPCl phenotype in RAW mouse macrophages
RAW cells were infected overnight with either live BCG, live Mycobacterium smegmatis (M. smeg) or mycolic acid lipids isolated from Tb, fixed and stained with the indicated probes for sphingomyelin (lysenin), ganglioside GMl and endocytosis (cholera toxin) or cholesterol (filipin). Punctate accumulation of all lipids is indicative of an NPCl storage phenotype and dysfunctional endocytosis, this combination of phenotypes is specific for NPCl. As can be seen from the micrographs in Figure 11, this NPCl phenotype was observed with live BCG and lipids from Tb but not from the non-pathogenic M. smeg. Live mycobacterium bovis (BCG) and mycolic acid lipids (M.A.) from Mycobacterium tuberculosis (Tb) therefore induce an NPCl phenotype in RAW mouse macrophages.
Example 12 - Live BCG infection depletes lysosomal calcium which can be overcome using calcium agonists (curcumin) to clear the infection
RAW cells, grown in the presence of the calcium indicator Rhod-dextran for 24h to label lysosomes, were infected overnight with either live or heat killed (HK) BCG. Figure 12 A shows that infection with live-BCG depletes calcium in the lysosome (reduced Rhod- dextran signal, representative images of each experiment are shown below the graph) whereas infection with dead HK-BCG cannot induce this phenotype. Figure 12B shows that treatment of live BCG infected cells with curcumin for 4h to elevate cytosolic calcium levels leads to clearance of infectious mycbacteria (reduction in GFP-labelled BCG signal) and a reduction in cholesterol storage (filipin stained). Pre-treatment with the specific cytosolic calcium chelator BAPTA-AM inhibits this clearance and prevents reduction in cholesterol storage. The experiments therefore demonstrate that live BCG infection depletes lysosomal calcium (an NPC phenotype), and that this can be overcome using calcium agonists (curcumin) to clear the infection.
Example 13 - Depletion of sphingolipids with miglustat (NB-DNJ) reverses the accumulation of sphingomyelin induced by BCG and Tb secreted lipids
RAW macrophages were pre-treated with either vehicle (water) or 5OmM MB-DNJ for 24h prior to addition of either BCG or Tb secreted lipids (mycolates) for a further 48h at a concentration of 5mg/ml to induce the NPCl -like phenotype. Cells were subsequently fixed and stained with lysenin (blue, or dark grey shading in Figure 13) for sphingomyelin or Hoechst (green, or light grey shading in Figure 13) for the nuclei. The micrographs in Figure 13 show that under normal conditions sphingomyelin can be found as punctate structures on the plasma membrane, but following treatment with MB-DNJ there is an elevation in sphingomyelin levels as a result of increased flux through that biosynthetic pathway. Addition of BCG or Tb mycolates induces intracellular storage of large quantities of sphingomyelin in compartments that surround the nucleus and spread out through the cytosol (lysosomes). Following treatment with M3-DNJ, the elevated levels of sphingomyelin remained in the mycolate treated cells but were correctly localised to the plasma membrane, indicating a correction in endocytic recycling of lipids and as such normalised phago-lysosome function. (N=2, bar = 5mm.) Figure 13 therefore shows that depletion of sphingolipids with the inhibitor of sphingo lipid biosynthesis miglustat (NB- DNJ) reverses the accumulation of sphingomyelin induced by BCG and Tb secreted lipids.
Example 14 - Tablet composition
Tablets, each weighing 0.15 g and containing 25 mg of a compound which elevates intracellular calcium or a compound which reduces sphingosine storage, for use in accordance with the invention, are manufactured as follows:
Composition for 10,000 tablets
Active compound (250 g)
Lactose (800 g) Corn starch (415g)
Talc powder (30 g)
Magnesium stearate (5 g)
The active compound, lactose and half of the corn starch are mixed. The mixture is then forced through a sieve 0.5 mm mesh size. Corn starch (10 g) is suspended in warm water (90 ml). The resulting paste is used to granulate the powder. The granulate is dried and broken up into small fragments on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium is added, carefully mixed and processed into tablets.
Example 15 - Injectable Formulation
Formulation A
Active compound 200 mg
Hydrochloric Acid Solution 0.1M or
Sodium Hydroxide Solution 0. IM q.s. to pH 4.0 to 7.0 Sterile water q.s. to 10 ml
The compound which elevates intracellular calcium or the compound which reduces sphingosine storage, for use in accordance with the invention, is dissolved in most of the water (35° 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch is then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.
Formulation B
Active Compound 125 mg
Sterile, Pyrogen-free, pH 7 Phosphate
Buffer, q.s. to 25 ml Active compound 200 mg
Benzyl Alcohol 0.10 g
Glycofurol 75 1.45 g
Water for injection q.s to 3.00 ml
The active compound is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).
Example 16 - Syrup Formulation
Active compound 250 mg
Sorbitol Solution 1.50 g
Glycerol 2.00 g
Sodium benzoate 0.005 g
Flavour 0.0125 ml Purified Water q.s. to 5.00 ml
The compound which elevates intracellular calcium or the compound which reduces sphingosine storage, for use in accordance with the invention, is dissolved in a mixture of the glycerol and most of the purified water. An aqueous solution of the sodium benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavour. The volume is made up with purified water and mixed well.

Claims

1. A compound which elevates intracellular calcium for use in the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype.
2. A compound as claimed in claim 1 which is a SERCA inhibitor, a Ryanodine receptor modulator, an IP3 receptor modulator, a NAADP receptor modulator, an EDG receptor modulator, a P2X receptor modulator or a Glutamate receptor modulator.
3. A compound as claimed in claim 1 or claim 2 which is:
a compound of formula (A):
Figure imgf000084_0001
wherein: RZ1 is selected from hydrogen, hydroxyl, carboxyl, amino, thiol, halo, substituted or unsubstituted Ci-1O alkyl, substituted or unsubstituted Ci-I0 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C1-Io alkylamino, di(C1-io)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said Ci-I0 alkyl is optionally interrupted by N(R'), O, S or arylene; RZ2 is selected from hydrogen, substituted or unsubstituted C MO alkyl and acyl, wherein said C1-I0 alkyl is optionally interrupted by N(R'), O, S or arylene; and either XZ1 is CH and Xz2 is CHR' wherein Xzl and XZ2 are bonded by a C-C single bond, or XZ1 is C and XZ2 is CR' wherein XZ1 and XZ2 are bonded by a C=C double bond; and
R' is H, Ci-6 alkyl or aryl; or a pharmaceutically acceptable salt thereof;
a compound of formula (B):
Figure imgf000085_0001
wherein:
RX1 is a substituted or unsubstituted pyrrole ring; R5", RX3, Rx4, RX5, RX6, RX7 and Rx8 which are the same or different, are independently selected from hydrogen, halo, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted Ci-I0 alkyl, aryl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted C3-20 heterocyclyl, substituted or unsubstituted C1-10 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C]-10 alkylamino, di(C1-i0)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said C1-10 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C1-6 alkyl or aryl; and either Zxl is CH and lP is CH2RX9 wherein ZX1 and Z*2 are bonded by a C-C single bond, or Z zXi is C and Z rX^2 is CHR' wherein Z ?X\ and Z τX2 are bonded by a C=C double bond; and
Rx9 is H, C1-6 alkyl or aryl; or a pharmaceutically acceptable salt thereof;
a compound of formula (C):
Figure imgf000085_0002
wherein: RM1, RM2, RM3, RM4 and RM5, which are the same or different, are independently selected from hydrogen and substituted or unsubstituted C1-10 alkyl, provided that at least one of RM1, RM2, RM3, RM4 and RM5 is substituted or unsubstituted C1-10 alkyl, wherein said C1-10 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C1-6 alkyl or aryl; or a pharmaceutically acceptable salt thereof;
a compound of formula (D):
Figure imgf000086_0001
wherein: E1, E2, E3 and E4, which are the same or different, are independently selected from
C(R05) and N provided that no more than two OfE1, E2, E3 and E4 are N;
R01 is selected from hydrogen, substituted or unsubstituted Ci-I0 alkyl, aryl, substituted or unsubstituted C3-25 cycloalkyl, and substituted or unsubstituted C3-20 heterocyclyl, wherein said C1-I0 alkyl is optionally interrupted by N(R'), O, S or arylene, wherein R' is H, C1-6 alkyl or aryl; and
R02, R03, R°4 and each R05, which are the same or different, are independently selected from hydrogen, halo, hydroxyl, carboxyl, amino, thiol, substituted or unsubstituted C1-10 alkyl, aryl, substituted or unsubstituted C3-25 cycloalkyl, substituted or unsubstituted C3-20 heterocyclyl, substituted or unsubstituted C1-10 alkoxy, substituted or unsubstituted aryloxy, acyl, ester, acyloxy, C1-10 alkylamino, di(Ci.]o)alkylamino, amido, acylamido, -0-C3-25 cycloalkyl and -0-C3-20 heterocyclyl, wherein said Cj.io alkyl is optionally interrupted by N(R'), O, S or arylene; or a pharmaceutically acceptable salt thereof;
a compound of formula (E):
Figure imgf000087_0001
wherein:
RN1 is a substituted or unsubstituted C14-20 alkyl group, wherein said C14-20 alkyl is optionally interrupted by N(R'), O, S or arylene; or a pharmaceutically acceptable salt thereof;
a compound of formula (F):
Figure imgf000087_0002
wherein n is O or 1 ; and
RY1 is a substituted or unsubstituted aryl or heteroaryl group; or a pharmaceutically acceptable salt thereof;
or any one of the following compounds: lα,25-dihydroxyvitamin D3; curcumin; cyclopiazonic acid; Cyclic ADP-Ribose; an adenine nucleotide; adenosine; L-thyroxine; sulmazole, isomazole; sulmazole analogs lacking the methylsulfinyl oxygen of sulmazole; an anthraquinone; digoxin, digitoxin; ouabain; 1,6- dihydro-2-methyl-6-oxo-(3 ,4-bipyridine)-5-carbonitrile); 1 , 1 '-diheptyl-4,4'-bipyridinium bromide; 5>>w-bis(w-aminobenzoyl-m-amino-p-methylbenzoyl- 1 -naphthyl-amino-4,6,8- trisulfonate)carbamide; a C1-20 linear, branched or cyclic alkane which is substituted with at least one halo group and which is otherwise unsubstituted or substituted; phenol which is substituted with a C1-I0 alkyl group and with at least one halo group; biphenyl which is substituted with at least two halo groups; 4-(C2-Cg alkyl)phenol; tacrolimus; rapamycin; quinolidomicin Al; bastadin 5; bastadin 7; bastadin 19; a RyR-modulating peptide; a sulfhydryl reagent; mesotetra-(4-N-methylpyridyl)-porphine tetraiodide (TMPyP), tetrasodium-mesotetra-(4-sulfonatophenyl)-porphine; a disulfonic stilbene derivative; acetic anhydride; maleic anhydride; diethylpyrocarbonate; quercetin; miconazole; clotrimazole; ketonazole; 1-heptanol; 1-octanol; DPI 201-106; cyclosporine A; TMB-8; 2,3-butanedione monoxime; methylenedioxyndenes; inositol 1,4,5-trisphosphate 2-aminoethoxydiphenyl borate; nicotinic acid adenine dinucleotide phosphate; sphingosine-1 -phosphate (SEP); lyso- phosphatidyl-choline (LPC) and glutamate; or a pharmaceutically acceptable salt thereof.
4. A compound as claimed in any one of claims 1 to 3 which is: lα,25-dihydroxyvitamin D3; curcumin, thapsigargin, cyclopiazonic acid, Cyclic ADP- Ribose, ryanodine, 9,21-didehydroryanodine, guanidinopropionyl, /?-alanil-ryanodine, caffeine, 3,7-dimethylxanthine, 1,7-dimethylxanthine, 1,3-dimethylxanthine, 3,9-dimethylxanthine, 9- methyl-7-bromoeudistomin D, palmitoyl carnitine, adenosine 5'-(β,y-methylene)triphosphate, cyclic AMP, adenosine diphosphate, adenosine monophosphate, adenosine, L-thyroxine, sulmazole, isomazole, doxorubicin, mitoxantrone, daunorubicin, rubidazone, doxorubicinol, digoxin, digitoxin, ouabain, l,6-dihydro-2-methyl-6-oxo-(3,4-bipyridine)-5-carbonitrile, 1,1 '- diheptyl-4,4'-bipyridinium bromide, 5ym-bis(w-aminobenzoyl-m-amino-p-methylbenzoyl-l- naphthyl-amino-4,6,8-trisulfonate)carbamide, 2-bromo-2-chloro- 1,1,1 -trifluoroethane, 2- chloro- 1 -(difluoromethoxy)- 1 , 1 ,2-trifluoroethane, 2-chloro-2-(difluoromethoxy)- 1,1,1- trifluoroethane), 4-chloro-/«-cresol, δ-hexachloro-cyclohexane, chlorocresol, 4-(C2-C9 alkyl)phenol, tacrolimus, rapamycin, quinolidomicin Al, bastadin 5, bastadin 7, bastadin 19, imperatoxin-a, myotoxin-a, ryanotoxin, N-ethylmaleimide, thimerosal, 5,5'-dithiobis-(2- nitrobenzoic acid), 2,2'-dithiodipyridine, 4,4'-dithiodipyridine, N-succinimidyl 3-(2- pyridyldithio)propionate, mesotetra-(4-N-methylpyridyl)-poφhine tetraiodide (TMPyP), tetrasodium-mesotetra-(4-sulfonatophenyl)-porphine, 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid, 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, acetic anhydride, maleic anhydride, diethylpyrocarbonate, quercetin, miconazole, clotrimazole, ketonazole, 1- heptanol, 1-octanol, DPI 201-106, cyclosporine A, TMB-8, 2,3-butanedione monoxime, inositol 1,4,5-trisphosphate, 2-aminoethoxydiphenyl borate, nicotinic acid adenine dinucleotide phosphate, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, sphingosine- 1 -phosphate, lyso-phosphatidyl-choline, or glutamate; or a pharmaceutically acceptable salt thereof.
5. A compound as claimed in any one of the preceding claims wherein the compound which elevates intracellular calcium is thapsigargin, curcumin or lα,25-dihydroxyvitamin D3.
6. A compound as claimed in any one of the preceding claims wherein the disease which has a Niemann-Pick disease type C like cellular phenotype is selected from Niemann-Pick disease type C, Smith-Lemli-Opitz Syndrome, Tangier disease, Huntington's disease, Cystic Fibrosis, Pelizaeus-Merzbacher disease, Mucolipidosis II (Icell) and a disorder which alters the activity of an enzyme involved in cholesterol synthesis or homeostasis.
7. A compound as claimed in any one of the preceding claims wherein the disease which has a Niemann-Pick disease type C like cellular phenotype is Niemann-Pick disease type Cl, Niemann-Pick disease type C2 or Smith-Lemli-Opitz Syndrome.
8. A compound as claimed in any one of the preceding claims wherein the disease which has a Niemann-Pick disease type C like cellular phenotype is a pathogenic infection, which pathogenic infection is capable of blocking lysosome-phagosome fusion via induction of a NPC like cellular phenotype.
9. A compound as claimed in claim 8 wherein the pathogenic infection is selected from an infection caused by Mycobacteria, Salmonella, Brucella, Coxiella, or Anaplasma phagocytophilum.
10. A compound as claimed in claim 8 or claim 9 wherein the pathogenic infection is a mycobacterial infection.
11. A compound as claimed in any one of claims 8 to 10 wherein the pathogenic infection is an infection caused by Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacterium africanum, Mycobacterium cannetti, and/or Mycobacterium microtti.
12. A compound as defined in any one of the preceding claims for use in the treatment of tuberculosis.
13. A compound as claimed in claim 12 wherein the compound is other than curcumin.
14. A compound as claimed in any one of the preceding claims which is for use in said treatment of said disease by coadministration with an inhibitor of sphingolipid biosynthesis.
15. A compound as claimed in claim 14 wherein the inhibitor of sphingolipid biosynthesis is N-butyldeoxynoj irimycin; N-nonyldeoxynojirimycin; N- butyldeoxygalactonojirimycinj N-S-adamantane-l-yl-methoxypentyl-deoxynojirimycin; alpha-homogalactonojirimycin; nojirimycin; deoxynojirimycin; N7-oxadecyl- deoxynojirimycin; deoxygalactonojirimycin; N-butyl-deoxygalactonojirimycin; N-nonyl- deoxygalactonoj irimycin; N-nonyl-6deoxygalactonoj irimycin; N7-oxanonyl-6deoxy-DGJ; alpha-homoallonojirimycin; beta-l-C-butyl-deoxygalactonojirimycin; l,5-dideoxy-l,5- imino-D-glucitol, 1 ,5-(Butylimino)- 1 ,5-dideoxy-D-gIucitol; 1 ,5-(Methylimino)- 1,5- dideoxy-D-glucitol; 1 ,5-(Hexylimino)- 1 ,5-dideoxy-D-glucitol; 1 ,5-(Nonylylimino)- 1,5- dideoxy-D-glucitol; 1 ,5-(2-Ethylbutylimino)- 1 ,5-dideoxy-D-glucitol; 1 ,5-(2- Methylpentylimino)- 1 ,5-dideoxy-D-glucitol; 1 ,5-(Benzyloxycarbonylimino)- 1 ,5-dideoxy- D-glucitol, tetraacetate; l,5-(Phenylacetylimino)-l,5-dideoxy-D-glucitol, tetraacetate; 1,5- (Benzoylimino)-l,5-dideoxy-D-glucitol, tetraacetate;!, 5-(Butylimino)-l,5-dideoxy-D- glucitol, tetraacetate; l,5-(Ethyl malonylimino)-l,5-dideoxy-D-glucitol, tetraacetate; 1,5- (Hexylimino)-l,5-dideoxy-D-glucitol, tetraacetate; l,5-(Nonylimino)-l,5-dideoxy-D- glucitol, tetraacetate; l,5-(Benzyloxycarbonylimino)-l,5-dideoxy-D-glucitol, tetraisobutyrate; l,5-(Butylimino)-l,5-dideoxy-D-glucitol, tetrabutyrate; l,5-(Butylimino)- 1 ,5-dideoxy-D-glucitol, tetrapropionate; l,5-(Butylimino)-l,5-dideoxy-D-glucitol, tetrabenzoate; l,5-Dideoxy-l,5-imino-D-glucitol, tetraisobutyrate; 1,5- (Hydrocinnamoylimino)-l,5-dideoxy-D-glucitol, tetraacetate; l,5-(Methyl malonylimino)- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-D-glucitol, tetraisobutyrate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-4R,6-O-(phenylmethylene)-D-glucitol, diacetate; l,5-[(Phenoxymethyl)carbonylimino]-l,5-dideoxy-D-glucitol, tetraacetate; 1,5- [(Ethylbutyl)imino]-l,5-dideoxy-D-glucitol, tetraacetate; l,5-(Butylimino)-l,5-dideoxy-D- glucitol, 2,3-diacetate; 1 ,5-(Hexylimino)- 1 ,5-dideoxy-4R,6-O-(phenylmethylene)-D- glucitol, diacetate; l,5-(Hexylimino)-l,5-dideoxy-D-glucitol, 2,3-diacetate; l,5-[(2- Methylpentyl)imino]- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-(Butylimino)- 1 ,5-dideoxy- D-glucitol, 6-acetate; l,5-[(3-Nicotinoyl)imino]-l,5-dideoxy-D-gIucitol, tetraacetate; 1,5- (Cinnamoylimino)- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-D- glucitol, 2,3-dibutyrate; 1 ,5-(Butylimino)- 1 ,5-dideoxy-4R,6-O-(phenylmethylene)-D- glucitol, 2,3-dibutyrate; l,5-(Phenylacetylimino)-l,5-dideoxy-D-glucitol, tetraisobutyrate; 1 ,5-[(4-Chlorophenyl)acetylimino]- 1 ,5-dideoxy-D-glucitol, tetraacetate; 1 ,5-[(4- Biphenyl)acetylimino]-l,5-dideoxy-D-glucitol, tetraacetate; 1,5- (Benzyloxycarbonylimino)- 1 ,5-dideoxy-D-glucitol, tetrabutyrate; 1 ,5-Dideoxy- 1 ,5-imino- D-glucitol, tetrabutyrate; 3,4,5-piperidinetriol,l-propyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, l-pentyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5- piperidinetriol, l-heptyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- butyl-2- (hydroxymethyl)-, (2S, 3S, 4R, 5S); 3,4,5-piperidinetriol, l-nonyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol,l- (1-ethyl) propyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (3-methyl) butyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (2-phenyl) ethyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (3-phenyl) propyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol,l- (1-ethyl) hexyl-2- (hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, 1- (2-ethyl) butyl-2-
(hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, l-[(2R)-(2-methyl-2-phenyl) ethyl]-2-(hydroxymethyl)-, (2S, 3R, 4R, 5S); 3,4,5-piperidinetriol, l-[(2S)-(2-methyl-2- phenyl) ethyl] -2-(hydroxymethyl)-, (2S, 3R, 4R, 5S), β-L-homofuconojirimycin; propyl 2- acetamido-2-deoxy-4-O-05-D-galactopyranosyl)-3-O-(2-(iV-0S-L- homofuconojirimycinyl))ethyl)-α-D-glucopyranoside; D,L-threo-l-phenyl-2- decanoylamino-3-morpholino-l-propanol (PDMP); D,L-threo-l-phenyl-2- hexadecanoylamino-3-morpholino-l-propanol (PPMP); D-threo-l-phenyl-2- palmitoilamino-3-pyrrolidino-l-propanol (P4); 4'-hydroxy-D-threo-l-phenyl-2- palmitoilamino-3-pyrrolidino-l-propanol (4'-hydroxy-P4); 3',4'-ethylenedioxy-P4 (EtDO- P4); 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine; cytidin-5'-yl sialylethylphosphonate; sialic acid; Soyasaponin I; Fumonisin, Myriocin; L-cycloserine; L-threo- dihydrosphingosine (Safingol); D-e-MAPP, LCL16, LCL284, LCL120, B13, LCL15, LCL204, LCL85, W-5, W-7, triflupromazine, terfenadine, suloctidil, sertraline, promethazine, paroxetine, nortriptyline, maprotiline, norfluoxetine, fluoxetine, fendiline, protriptyline, chlorprothixene, promazine, pimethixene, drofenine, doxepine, cyproheptadine, cyclobenzaprine, clomiphene, cloperastine, camylofin, bepridil, benztropine, astemizole, amlodipine, trimipramine, trihexylphenidyl, trifluoroperazin, thioridazin, thioproperazine, tamoxifen, quinacrine, propericiane, perhexiline, mianserin, prochlorperazine, imipramine, dibucaine, desipramine, clomipramine, alimenazine, amitriptyline, cyamemazine, cocaine, chlorpromazine, AY9944, manumycin A, scyphostatin or a compound of any one of the following formulae:
Figure imgf000092_0001
wherein R is CH3 or CH2C6H5; or a pharmaceutically acceptable salt thereof.
16. Use of a compound which elevates intracellular calcium in the manufacture of a medicament for the treatment of a disease which has a Niemann-Pick disease type C like cellular phenotype.
17. A compound which reduces sphingosine storage for use in the treatment of Niemann-Pick disease type C.
18. A compound as claimed in claim 17 which is an inhibitor of ceramide biosynthesis or an inhibitor of ceramide degradation.
19. A compound as claimed in claim 17 or claim 18 which is an inhibitor of serine palmitoyltransferase, an inhibitor of dihydroceramide synthase, a sphingomyelinase inhibitor or a ceramidase inhibitor.
20. A compound as claimed in any one of claims 17 to 19 which is:
an inhibitor of ceramide biosynthesis selected from L-tΛreo-dihydrosphingosine (Safingol) and an inhibitor of the following formula (IX):
Figure imgf000093_0001
in which: q is 0 or 1 ; r is 0 or 1 ;
RKa is H, COOH or an unsubstituted or substituted ester;
R1Xb is an unsubstituted or substituted Ci-6 alkyl;
R0^ and R , which are the same or different, are each independently selected from H, unsubstituted or substituted C1-6 alkyl and unsubstituted or substituted phenyl;
RKe and RIxf, which are the same or different, are each independently selected from H, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl; either (a) one of RKg and R0* is H and the other is ORIXr, wherein RKr is selected from H, unsubstituted or substituted C1-6 alkyl, unsubstituted or substituted phenyl and unsubstituted or substituted acyl, or (b) RKg and R001 together form an oxo group;
RKi is H, unsubstituted or substituted Ci-6 alkyl, unsubstituted or substituted C]-6 alkoxy and unsubstituted or substituted phenyl;
RKj is H, unsubstituted or substituted Ci-6 alkyl or a group of the following formula (X): O CORlXn in which RKn and RKo, which are the same or different, are each independently selected from OH, unsubstituted or substituted C1-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted Ci-6 alkylamino and unsubstituted or substituted di(C1-6)alkylamino;
R0^ is H, unsubstituted or substituted C1-6 alkyl or a group of the following formula (XI):
Figure imgf000094_0001
in which RKp and RKq, which are the same or different, are each independently selected from OH, unsubstituted or substituted C1-6 alkoxy, unsubstituted or substituted phenoxy, amino, unsubstituted or substituted C1-6 alkylamino and unsubstituted or substituted di(C1-6)alkylamino; and
RKm is selected from H and unsubstituted or substituted C1-2O alkyl, which Ci-20 alkyl is optionally interrupted by N(R'), O, S or phenylene, wherein R' is H, Ci-6 alkyl or phenyl; or a pharmaceutically acceptable salt thereof;
an inhibitor of ceramide biosynthesis of the following formula (XS):
Figure imgf000094_0002
in which:
RXa is H, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted CI-20 alkylene-aryl, substituted or unsubstituted Cj-20 alkylene-C3-20 heteroaryl, substituted or unsubstituted C1-20 alkylene-C3-25 cycloalkyl, substituted or unsubstituted Ci-20 alkylene-C3- 20 heterocyclyl, substituted or unsubstituted Ci-20 alkylene-O-C3-20 heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted C3-20 heteroaryl, substituted or unsubstituted C3-25 cycloalkyl or substituted or unsubstituted C3-20 heterocyclyl wherein said C1-20 alkyl and C1-20 alkylene are optionally interrupted by N(R'), O, S or arylene wherein R' is H, Cj-6 alkyl or aryl; and
R and R , which are the same or different, are independently selected from H, unsubstituted or substituted C1-I0 alkyl and unsubstituted or substituted aryl; or a pharmaceutically acceptable salt thereof;
a ceramidase inhibitor of formula (G):
Figure imgf000095_0001
wherein: RP1 is selected from hydrogen and unsubstituted or substituted Ci-6 alkyl;
RP2 and Rp3, which are the same or different, are independently selected from hydrogen, hydroxyl and unsubstituted or substituted C1-6 alkyl;
RP4 is hydrogen, -NO2, -KH2 or -N(H)C(O)(CH2)nCH2Ypl, wherein YP1 is hydrogen, aryl or heteroaryl, and wherein n is 0 or an integer of 1 to 10; RP5 is selected from unsubstituted or substituted methyl, unsubstituted or substituted ethyl, aryl and heteroaryl; YP2 is -CH2- or -C(O)-; YP3 is -CH2- or -N(H)-;
EP1 is N or N+H, provided that EP1 is N when YP2 is -C(O)-; and m is 0 or an integer of 1 to 14; or a pharmaceutically acceptable salt thereof;
a sphingomyelinase inhibitor selected from manumycin A, scyphostatin and a compound of formula (H):
Figure imgf000096_0001
wherein:
RQ1 is C1-6 alkyl, -RQ2-aryl or -RQ2-0H; RQ2 is unsubstituted or substituted C1-6 alkylene; and
RQ3 is unsubstituted or substituted C1-20 alkyl, wherein said C1-20 alkyl is optionally interrupted by N(R' ), O, S or arylene, wherein R' is H, C1-6 alkyl or aryl; or a pharmaceutically acceptable salt thereof;
a sphingomyelinase inhibitor selected from W-5, W-7, dibucaine, mianserin, perhexiline, tamoxifen, trihexylphenidyl, amlodipine, bepridil, astemizole, suloctidil, AY9944, benztropine, camylofin, cloperastine, cocaine, clomiphene, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, paroxetine, sertraline, terfenadine and a compound of formula (J):
Figure imgf000096_0002
wherein:
RG1 and RG3, which are the same or different, are independently selected from hydrogen, halo, cyano, -CF3, -S(O)2NMe2, -SMe and C1-6 alkyl;
ZG1 is selected from -S-, -CH2-CH2-, -CH=CH- and -CH2-O-;
XG1 is C and Y01 is CH and XG1 and Y01 are linked by a double bond, or XG1 is selected from N and CH, Y01 is CH2 and XG1 and Y01 are linked by a single bond; RG2 is a group -Y°2-RG4, wherein Y°2 is substituted or unsubstituted C1-4 alkyl and
R >G4 is selected from amino, C1-10 alkylamino, di(C1-io)alkylamino and unsubstituted or substituted C3-20 heterocyclyl; provided that X°\ Y01 and RG2 may together form the group:
Figure imgf000097_0001
or a pharmaceutically acceptable salt thereof.
21. A compound as claimed in any one of claims 17 to 20, which is : myriocin (ISP-I), fumonisin, L-cycloserine, L-tAreo-dihydrosphingosine (Safingol), D-e- MAPP, LCL16, LCL284, LCL120, B13, LCL15, LCL204, LCL85, W-5, W-7, triflupromazine, terfenadine, suloctidil, sertraline, promethazine, paroxetine, nortriptyline, maprotiline, norfluoxetine, fluoxetine, fendiline, protriptyline, chlorprothixene, promazine, pimethixene, drofenine, doxepine, cyproheptadine, cyclobenzaprine, clomiphene, cloperastine, camylofin, bepridil, benztropine, astemizole, amlodipine, trimipramine, trihexylphenidyl, trifluoroperazin, thioridazin, thioproperazine, tamoxifen, quinacrine, propericiane, perhexiline, mianserin, prochlorperazine, imipramine, dibucaine, desipramine, clomipramine, alimenazine, amitriptyline, cyamemazine, cocaine, chlorpromazine, AY9944, manumycin A, scyphostatin or a compound of any one of the following formulae:
Figure imgf000097_0002
wherein R is CH3 or CH2C6H5; or a pharmaceutically acceptable salt thereof.
22. A compound as claimed in any one of claims 17 to 21 which is myriocin.
23. A compound as claimed in any one of claims 17 to 22 wherein the disease is Niemann-Pick disease type Cl or Niemann-Pick disease type C2.
24. A compound as claimed in any one of claims 1 to 15 and 17 to 23 which is for administration as a dietary supplement.
25. Use of a compound which reduces sphingosine storage in the manufacture of a medicament for the treatment of Niemann-Pick disease type C.
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