WO2007137808A1 - Compositions comprenant des oligosaccharides pour traiter une maladie à prion - Google Patents

Compositions comprenant des oligosaccharides pour traiter une maladie à prion Download PDF

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WO2007137808A1
WO2007137808A1 PCT/EP2007/004727 EP2007004727W WO2007137808A1 WO 2007137808 A1 WO2007137808 A1 WO 2007137808A1 EP 2007004727 W EP2007004727 W EP 2007004727W WO 2007137808 A1 WO2007137808 A1 WO 2007137808A1
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oligosaccharide
prp
cyclodextrin
glucose
substituted
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PCT/EP2007/004727
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English (en)
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Hilary Mcmahon
Marguerite Prior
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University College Dublin, National University Of Ireland, Dublin
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Publication of WO2007137808A1 publication Critical patent/WO2007137808A1/fr

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/721—Dextrans
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to compositions for treating prion disease.
  • compositions comprising oligosaccharides for treating prion disease.
  • Prion diseases also known as transmissible spongiform encephalopathies (TSEs)
  • TSEs transmissible spongiform encephalopathies
  • Human prion diseases include kuru, Gerstmann-Straussler Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and Creutzfeldt- Jakob Disease (CJD).
  • GSS Gerstmann-Straussler Scheinker syndrome
  • FFI Fatal Familial Insomnia
  • CJD Creutzfeldt- Jakob Disease
  • animal prion diseases are scrapie of sheep and goats and Bovine Spongiform Encephalopathy (BSE) of cattle.
  • Prion diseases are so-called because the prion protein is considered to be central to the development of the disease.
  • the prion protein is the product of a single gene located on chromosome 20.
  • the prion protein exists in at least two conformational forms with distinct physiochemical properties.
  • the normal cellular form of the prion protein (PrP c ) is expressed at high levels in neuronal cells. It is a glycosylphosphatidylinositol (GPI) anchored cell surface protein and, during prion disease, it is thought to be converted to an abnormal pathological form known as PrP Sc .
  • GPI glycosylphosphatidylinositol
  • PrP Sc is detergent insoluble and partially proteinase resistant, and its tendency to aggregate in the brain leads to the characteristic neuropatho logical features such as spongiform degeneration of the brain.
  • PrP c and PrP Sc have the same amino acid sequence but differ dramatically in conformation. PrP consists of 42% ⁇ -helix and 3% ⁇ -sheet, while P PrrPP SScc ppoossesses a much higher ⁇ -sheet content of 43% and a lower ⁇ -helix content of 30%.
  • Treating prion diseases presents the challenge of developing a drug which is effective against prion diseases and which is capable of crossing the blood-brain- barrier of subjects. This challenge has not so far been overcome, with the result that there is no known cure for prion diseases.
  • oligosaccharide ⁇ - cyclodextrin
  • ⁇ - cyclodextrin may be useful in the treatment of Alzheimer's disease, which is characterised pathologically by the classical senile plaque in the human brain, and is not a disease generally associated with prion proteins.
  • a substituted or unsubstituted linear, branched or cyclic oligosaccharide for the preparation of a medicament for treating prion disease.
  • a pharmaceutical composition for treating prion disease comprising a pharmaceutically effective amount of a substituted or unsubstituted linear, branched or cyclic oligosaccharide, with the exception of unsubstituted ⁇ - cyclodextrin, and a pharmaceutically acceptable excipient.
  • a method for treating prion disease comprising administering to a subject in need thereof, a therapeutically effective amount of a substituted or unsubstituted linear, branched or cyclic oligosaccharide.
  • oligosaccharide is intended to mean a saccharide oligomer comprising from 2 to 10 monosaccharide units.
  • the term "subject" is intended to mean a mammal, including human and non-human mammals.
  • the oligosaccharides used in the invention preferably comprise from 5 to 9 monosaccharide units, more preferably from 6 to 8 monosaccharide units.
  • the oligosaccharides used in the invention comprise monosaccharides which may be selected from dioses such as glycolaldehyde; trioses such as glyceraldehyde and dihydroxyacetone; tetroses such as erythrose, threose and erythrulose; pentoses including aldo-ketoses such as arabinose, lyxose, ribose, deoxyribose and xylose, and keto-pentoses such as ribulose and xylulose; hexoses including aldo-hexoses such as allose, altrose, galactose, glucose, gulose, idose, mannose and talose, and keto-hexoses such as fructose, psicose, sorbose and tagatose; heptoses including aldo-heptoses, and keto-heptoses such as mannoheptulose and
  • the oligosaccharides used in the invention may comprise a plurality of the same monosaccharide unit, or a combination of two or more different types of monosaccharide units.
  • a preferred oligosaccharide comprises a combination of no more than three different types of monosaccharide units, preferably a combination of no more than two different types of monosaccharide units.
  • An especially preferred oligosaccharide comprises a plurality of the same monosaccharide unit.
  • a preferred oligosaccharide comprises at least six monosaccharide units, which are preferably independently selected from glucose, galactose and mannose, preferably a combination of glucose and galactose or a combination of glucose and mannose, or glucose alone.
  • the oligosaccharide which may be linear or cyclic, comprises 6 or 7 or 8 monosaccharide units, which are each glucose. These include maltohexaose, maltoheptaose, ⁇ -cyclodextrin, ⁇ -cyclodextrin and ⁇ -cyclodextrin.
  • the oligosaccharides used in the invention are preferably cyclic oligosaccharides which may be substituted or unsubstituted.
  • a preferred cyclic oligosaccharide comprises at least six monosaccharide units, which are preferably independently selected from glucose, galactose and mannose.
  • a cyclic oligosaccharide comprising 6 or 7 or 8 monosaccharide units which are each glucose, the cyclic oligosaccharide containing six glucose units being a substituted or unsubstituted ⁇ -cyclodextrin, the cyclic oligosaccharide containing seven glucose units being a substituted or unsubstituted ⁇ -cyclodextrin, and the cyclic oligosaccharide containing eight glucose units being a substituted or unsubstituted ⁇ -cyclodextrin.
  • unsubstituted ⁇ -cyclodextrin is particularly preferred.
  • Substituted oligosaccharides may be formed by the replacement of one or more hydrogen atoms or hydroxyl groups with one or more different atoms or groups of atoms or functional groups.
  • each monosaccharide unit in an oligosaccharide may be mono- to poly-substituted.
  • Mono-, di- or tri-substituted monosaccharide units are preferred.
  • Suitable replacement functional groups include, but are not limited to, alkyl, acetylalkyl, hydroxyalkyl, carboxyalkyl, acyl including carboxyl, amino, thio, tosyl, and combinations thereof.
  • Suitable replacement functional groups include saccharides such as glucosyl and maltosyl, and combinations thereof.
  • Alkyl groups or acyl groups comprising 1 to 4 carbon atoms are preferred.
  • Alkyl groups selected from methyl, ethyl and propyl are preferred.
  • Methyl groups are particularly preferred.
  • Preferred substituted oligosaccharides include methyl- ⁇ -cyclodextrin, dimethyl- ⁇ - cyclodextrin, heptakis (2,6-di-O-methyl)- ⁇ -cyclodextrin, trimethyl- ⁇ -cyclodextrin, heptakis (2, 3, 6-tri-O-methyl)- ⁇ -cyclodextrin, acetylated dimethyl- ⁇ -cyclodextrin, acetylated trimethyl- ⁇ -cyclodextrin, hydroxyethyl- ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin. Methyl- ⁇ -cyclodextrin is especially preferred.
  • the oligosaccharides used in the invention may encapsulate cholesterol.
  • Preferred oligosaccharides used to encapsulate cholesterol are ⁇ -cyclodextrin and methyl- ⁇ - cyclodextrin (chol-M ⁇ -CD). Methyl- ⁇ -cyclodextrin is particularly preferred.
  • composition or medicament according to the invention is preferably formulated for oral or parenteral administration.
  • suitable oral dosage forms include tablets, capsules, pills, powders, granules and the like.
  • Suitable parenteral dosage forms include sterile injectable aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.
  • Alpha- ( ⁇ ), beta- ( ⁇ ) and gamma- ( ⁇ ) cyclodextrins (CDs), methyl- ⁇ -CD (M ⁇ - CD), maltohexaose (G6), maltohepatose (G7) and water-soluble cholesterol complex were purchased from Sigma- Aldrich, Dublin, Ireland.
  • Cell culture reagents such as Opti-MEM, Foetal Bovine Serum (FBS), glutamine and penicillin/streptomycin were Gibco (Trade Mark) products supplied by Biosciences, Dublin, Ireland.
  • Secondary antibody anti-mouse peroxidase conjugate and cholera toxin B subunit type inaba 569B peroxidase conjugate were purchased from Calbiochem, La Jolla, California, USA.
  • Epoxy activated sepharose 6B was obtained from Amersham Pharmacia, New Jersey, USA. Pefabloc was purchased from Boehringer Manheim, Indianapolis, Indiana, USA. G418 was purchased from Promega, Mannheim, Germany while all other reagents were from Sigma- Aldrich.
  • the primary antibody 3F4 (1 : 10000 dilution) used is characterised as recognising the peptide epitope 109-112 of human PrP and was purchased from Signet Laboratories, Dedham, Massachusetts, USA.
  • the primary antibody 8H4 used is characterised as recognising the peptide epitope 175-185 of PrP and was obtained from Dr. Man-Sun Sy, Case Western Reserve, University School of Medicine, Cleveland, Ohio, USA.
  • Neuroblastoma cells (N2a) transfected with mouse PrP (N2a58) and N2a58 infected with the 22L scrapie strain (N2a22L), were obtained from Dr Sylvain Lehmann, adjoin, France. To obtain cells producing high levels of PrP Sc , the N2a22L cell line was subcloned and is referred to as N2a22L20. 3F4 tagged mouse PrP c (MoPrP 0 ) cDNA in the pcDNA3 vector was obtained from Dr Sylvain Lehmann. N2a cells were transfected with the cDNA using fugene transfection reagent according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany).
  • Stably transfected cells were screened and maintained using G418 resistance.
  • the N2a22L, N2a22L20, N2a58 and 3F4MoPrP c N2a cells were cultured in Opti-MEM supplemented with 10% FBS, penicillin/streptomycin, 2mM glutamine, and 300 ⁇ g/ml geneticin (G418 sulphate) in a humidified atmosphere.
  • lysates were adjusted to 18 ⁇ g of protein in gel loading buffer and boiled for 5 mins prior to loading onto 12% SDS-Polyacrylamide Gel
  • PrP c and PrP Sc were detected by incubating immunoblots with antibody 8H4 (3F4 tagged MoPrP was detected with antibody 3F4) followed by a horseradish peroxidase secondary antibody, and developed by enhanced chemiluminesecence (ECL). Densitometry was performed with the BioRad IMAGER Analysis Software.
  • N2a22L20 cells from 3 x 60mm dishes were lysed in 400 ⁇ l of ice-cold sucrose gradient (SG) lysis buffer (25mM Tris-HCl, pH 7.5, 15OmM NaCl, 5mM EDTA and 1% Triton X-100) on ice for 30 min. Lysates were then spun at 4,850 rpm for 10 min and the 400 ⁇ l supernatant was adjusted to 40% (w/v) sucrose prepared in SG lysis buffer without detergent. ImI 25% (w/v) sucrose and 0.5ml 5% (w/v) sucrose, both in SG lysis buffer without detergent, were then added. The gradient was then spun at 37,000 rpm for 18h at 4°C. 10 x 230 ⁇ l fractions were collected from the top of the tube and processed for PrP c , PrP Sc and GMi as follows.
  • SG ice-cold sucrose gradient
  • PrP Sc PrP Sc
  • Fractions were then spun at 45,000 rpm for 2h at 4°C and the supematants were methanol (MeOH) precipitated.
  • the precipitated pellet was suspended in gel loading buffer and PrP was visualised using 12% SDS-PAGE and western blotting with 8H4 antibody.
  • PrP Sc detection 120 ⁇ l from each fraction was protein determined and treated with 16 ⁇ g pK/mg protein for 15 min at 37°C followed by centrifugation at 70,000 rpm for 45 min. PrP Sc was visualised using SDS-PAGE and western blotting with 8H4 antibody.
  • GM detection 120 ⁇ l from each fraction was dotted onto nitrocellulose membrane, which was then blocked in 3% bovine serum albumin (BSA) for 3 hours and then incubated with cholera toxin B (CTXB) (1:4,000 dilution) for 1 hour. Following four 10 min washes in 0.1% TTBS, blots were developed by enhanced chemiluminiscence (ECL).
  • BSA bovine serum albumin
  • CTL cholera toxin B
  • Freeze dried epoxy-activated sepharose 6B was reconstituted in water and activated with 0.1M NaOH according to the manufacturer's instructions (Pharmacia). The gel was incubated with or without 0.02M ⁇ -CD, in 0.1M NaOH for 16h at 40°C with shaking. The gel was then washed in sequence with 0. IM
  • the gel was then washed four times with 500 ⁇ l of 0.5M HEPES pH 7.5 with a 15 min mixing period between each wash.
  • 500 ⁇ l of 0.5M HEPES pH 7.5 containing 500 ⁇ M ⁇ -CD was then added and the solution mixed as before for 15 min and then spun at 1 ,000 rpm for 1 min. The supernatant was removed and MeOH precipitated (Eluent 1 (El).
  • 500 ⁇ l of 0.5M SEPES pH 7.5 containing ImM ⁇ -CD was then added to the gel and the solution was mixed as before for 15 min and then spun and the supernatant removed and MeOH precipitated (E2).
  • the remaining 800 ⁇ l gel was boiled in 400 ⁇ l IX loading buffer for 10 min. The mixture was spun at 1,000 rpm for 1 min and the supernatant taken and analysed for PrP c (E3).
  • the chelex resin was removed and CaCl 2 to a concentration of l ⁇ M was added to the suspension and the suspension was then incubated in the presence or absence of ⁇ -CD or CR as indicated herein at 37°C for 72h. During the 72h period the solution was vortexed regularly to ensure equal mixing.
  • the suspension was then treated on ice with 2 x volumes of lysis buffer containing a protease inhibitor cocktail (0.5% (w/w) Na Deoxycholate 0.5% (v/v) Triton X-100, 15OmM NaCl and 5OmM Tris, pH 7.5 and proteinase inhibitors pepstatin A, Leupeptin and EDTA) the solution was vortexed for 3 min before a 20 min incubation on ice. During the 20 min incubation on ice the suspension was vortexed every 5 min. The solution was then methanol precipitated.
  • a protease inhibitor cocktail (0.5% (w/w) Na Deoxycholate 0.5% (v/v) Triton X-100, 15OmM NaCl and 5OmM Tris, pH 7.5 and proteinase inhibitors pepstatin A, Leupeptin and EDTA
  • the pellet was resuspended in 50 ⁇ l ice-cold lysis buffer containing protease inhibitors (0.5% (w/w) Na Deoxycholate 0.5% (v/v) Triton X-100, 15OmM NaCl and 50 mM Tris, pH 7.5 and pepstatin l ⁇ g/ml A, Leupeptin l ⁇ g/ml and EDTA (2mM) and vortexed.
  • the samples were then assayed for solubility by centrifuging at 70,000 rpm for 40 min in an ultracentrifuge.
  • the pellet (insoluble fraction) was resuspended in loading buffer.
  • the supernatant (soluble fraction) was methanol precipitated and the pellet obtained after centrifugation at 14,000 rpm for 10 min was resuspended in loading buffer. All samples were vortexed for 5 min in their loading buffer and samples were then analysed by 12% SDS-PAGE and immunoblotting with antibody 3F4.
  • Figure 1 ⁇ -CD reduces PrP Sc to undetectable levels in the 22L infected scrapie cell line.
  • Figure IA ⁇ -CD at concentrations of lOO ⁇ M and 500 ⁇ M was added to N2a22L cells at the time of passage and the cells were then passaged in the presence of the drug for 1 and 2 weeks.
  • Figure ⁇ B representative bands from
  • Figure ⁇ A were quantified by densitometry and were expressed as a % of the control (untreated). Black bars represent week 1 and grey bars represent week 2. * P ⁇ 0.005 (Student's t-test).
  • Figure 1C ⁇ -CD at a concentration of 500 ⁇ M was added to the N2a22L20 cells at the time of passage and the cells were passaged in the presence of the drug for 2 weeks.
  • the cells were lysed and the lysate was then analysed for PrP Sc by 12% SDS-PAGE and immunoblotting with 8H4 antibody. Cell lysates were left untreated (-pK) or digested with pK (+pK) for detection of PrP Sc . Where indicated as a drug control for PrP Sc clearance, congo red (CR) was added to the cells at 5 ⁇ g/ml for 2 weeks. Results are representative of three independent experiments. Molecular weight markers are kDa are on the left of the panels.
  • Figure 2 Structure activity studies.
  • Figure 2A Structures of ⁇ -, ⁇ -, and ⁇ -CD and their corresponding linear sugars.
  • Figure 25 ⁇ -, ⁇ -, and ⁇ -CD and the linear sugars maltohexaose and maltoheptaose, were added to the N2a22L cells at a concentration of 500 ⁇ M at the time of passage and the cells were then passaged in the presence of the drugs for 2 weeks. The cells were lysed and the lysate was then analysed for PrP Sc by 12% SDS-PAGE and immunoblotting with 8H4 antibody. Cell lysates were left untreated (-pK) or digested with pK (+pK) for detection of PrP Sc . Where indicated as a drug control for PrP Sc clearance, congo red (CR) was added to the cells at 5 ⁇ g/ml for 2 weeks. Results are representative of three independent experiments. Molecular weight markers in kDa are on the left of the panel.
  • Figure 3 IC 50 determination for ⁇ -, ⁇ - and ⁇ -CD.
  • Various concentrations of ⁇ -, ⁇ - and ⁇ -CD were added to the N2a22L cells at the time of passage and the cells were then passaged in the presence of the compounds for two weeks. Cells were then lysed and the lysate was then analysed by 12% SDS-PAGE and immunoblotting with 8H4 antibody for the effect of ⁇ -CD (Figure 3A) ⁇ -CD ( Figure 3B) and ⁇ -CD ( Figure 3 Q on PrP Sc levels. Representative bands from immunoblots were quantified by densitometry and were expressed as a % of the control (untreated). Results are representative of three independent experiments.
  • Figure 4 ⁇ -CD does not act through altering PrP c levels or through an acidic compartment.
  • Figure AA ⁇ - and ⁇ -CD were added to non-infected N2a cells at 500 ⁇ M each time the cells were passaged and the cells were then lysed at two weeks. The lysate was prepared to 18 ⁇ g of protein as described in materials and methods before SDS-PAGE and western blotting and PrP c was detected with 8H4 antibody.
  • Figure AB representative bands from Figure AA were quantified by densitometry and were expressed as a % of the control (untreated).
  • Figure AC The compounds NH 4 Cl (3OmM) and ⁇ -CD (500 ⁇ M) were added separately at the time of passage to N2a22L cells for 2 weeks ( Figure AC, lanes 2 and 3, respectively), hi addition, 3OmM NH 4 Cl was added to the N2a22L cells at the time of passage and ⁇ -CD (500 ⁇ mM) was added an hour later and the cells were passaged in this manner for a period of 2 weeks ( Figure AC, lane 4). The cells were lysed and the lysate was then analysed for PrP Sc by 12% SDS-PAGE and immunoblotting with 8H4 antibody. Cell lysates were digested with pK (+pK) for detection of PrP Sc . *P ⁇ 0.05 (Students t-test). Results are representative of three independent experiments. Molecular weight markers in kDa are on the left of the panels.
  • Figure 5 Effect of ⁇ -CD on PrP c , PrP Sc and GMi association with DRMs.
  • the N2a22L20 cell line was either treated with 500 ⁇ M ⁇ -CD a the time of passage (PrP c , PrP Sc , GMi each + ⁇ -CD) or was left untreated (PrP c , PrP Sc , GM,) and the cells were grown for 72h.
  • the cells were then lysed as for density gradients, adjusted to 40% sucrose, a density gradient applied and spun at 37,000 rpm for 18h at 4°C. 10 x 230 ⁇ l fractions were collected from the top of the tube and processed for PrP c , PrP Sc and GMi detection as described in materials and methods.
  • Fraction 1 is the top of the gradient and fraction 10 is the bottom.
  • Fractions for PrP c ( Figure 5A) and PrP Sc Figure 52?) detection were prepared according to materials and methods and analysed by 12% SDS-PAGE and immunoblotting with 8H4 antibody.
  • Fractions for GMi detection Figure 5 Q were blotted onto nitrocellulose membrane and visualised using cholera toxin B conjugated to horseradish peroxidase. Representative bands from Figures SA and 5B, and dots from Figure 5 C were quantified by densitometry and were expressed as a % of the control (the fraction presenting with the highest signal (100%)) ( Figures 5D, 5E and 5F respectively). Results are representative of three independent experiments. Molecular weight markers in kDa are on the left of the panels. Control results are plotted as ( ⁇ ) and ⁇ -CD results are plotted as ( ⁇ ).
  • Figure 6 Reintroduction of cholesterol encapsulated in methyl- ⁇ -CD does not reverse PrP Sc clearance but facilitates it.
  • Figure 6A ⁇ -CD and methyl ⁇ -CD (M ⁇ - CD) at a concentration of 500 ⁇ M were added to N2a22L20 cells at the time of passage and the cells were then passaged in the presence of the drugs for 1 week ( Figure 6A, lanes 3 and 4, respectively).
  • N2a22L20 cells were also treated with a combination of M ⁇ -CD (500 ⁇ M) and a cholesterol encapsulated form of methyl ⁇ - CD (chol-M ⁇ -CD) (46 ⁇ M) and the cells were then passaged in their presence for 1 week (Lane 5).
  • Figure 6B Representative bands from Figure 6A were then quantified by densitometry and were expressed as a % of the control (untreated).
  • Figure 6C N2a22L20 cells were treated with M ⁇ -CD or ⁇ -CD at a concentration of 500 ⁇ M at the time of passage and the cells were passaged in the presence of the drugs for 1 week and then passaged in the absence of the drugs for 3 days ( Figure 6C, lanes 3 and 5, respectively) or passaged in the presence of chol-M ⁇ -CD (46 ⁇ M) alone for 3 days (lanes 4 and 6, respectively).
  • Figure 6D M ⁇ -CD, ⁇ -CD and chol-M ⁇ -CD were added to N2a22L20 cells, with CDs at a concentration of 500 ⁇ M and chol-M ⁇ -CD at 46 ⁇ M, at the time of passage and the cells were passaged in the presence of the drugs for 2 weeks (Lanes 3, 4 and 5, respectively).
  • the cells were lysed and the lysate was then analysed for PrP Sc by 12% SDS-P AGE and immunoblotting with 8H4 antibody. Cell lysates were left untreated (-pK) or digested with pK (+pK) for detection of PrP Sc .
  • Results are representative of three independent experiments. Molecular weight markers in kDa are on the left of the panels.
  • Figure 7 Methyl- ⁇ -CD does not alter the Triton X-100 insolubility of PrP c .
  • Figure IA N2a cells were grown to confluence and the media was then replaced with fresh media containing 500 ⁇ M M ⁇ -CD for a period of Ih or cells were left untreated ( Figure IA, lanes 1-4). N2a cells were also treated with 500 ⁇ M M ⁇ -CD at the time of passage for a period of 72h or cells were left untreated ( Figure IA, lanes 5-8). The cells were then lysed and the lysate was prepared for the Triton X- 100 solubility assay and soluble (S) and insoluble (IS) fractions were separated.
  • S soluble
  • IS insoluble
  • Figure IB Representative bands from Figure IA were quantified by densitometry and were expressed as % soluble and insoluble. Black bars represent % soluble PrP c and white bars represent % insoluble PrP c . Results are representative of three independent experiments. Molecular weight markers in kDa are on the left of the panels.
  • Figure 8 PrP c binding to sepharose gel containing ⁇ -CD.
  • Figure 8 ⁇ 4 800 ⁇ g lysate from N2a cells expressing 3F4 tagged MoPrP c was loaded onto epoxy activated sepharose 6B gel prepared with ⁇ -CD. The gels were processed as described. The following were analysed for PrP c , 2.25% (18 ⁇ g) of cell lysate (control), equivalent level (2.25%) of protein in unbound material (Sl), 100% S2, S3 and S4, 100% ⁇ -CD eluents (El and E2) and 12% of final eluent (E3).
  • PrP c in these fractions was analysed by 12% SDS-PAGE and immunoblotting with 3F4 antibody
  • Figure 8B Representative bands from Figure 8A: were quantified by densitometry and PrP levels were adjusted to the level that would be present if 100% samples were loaded. %PrP was then plotted relative to the control (the level of PrPC prior to binding to the gel). Results are representative of three independent experiments. Molecular weight markers are kDa are on the left of the panels.
  • Figure 9 ⁇ -CD blocks the conversion of PrP c to PrP Sc .
  • Figures 9 A and 9B are ⁇ -CD blocks the conversion of PrP c to PrP Sc .
  • the scrapie infected cell line employed for this study was N2a infected with 22L. A subclone possessing six times the PrP Sc level was also employed (N2a22L20).
  • ⁇ -CD was added to the N2a22L cell line at lOO ⁇ M and 500 ⁇ M at the time of passage and the cells were cultured in the presence of the compound for a period of one and two weeks. Cells were then lysed ( Figure IA) and analysed for PrP Sc content by immunoblotting with 8H4 antibody. After one week of treatment, 500 ⁇ M ⁇ -CD reduced PrP Sc to 30% of the control ( Figure IA, lane 3 and Figure 15).
  • ⁇ -CD concentrations of ⁇ -CD used in this study were non-cytoxic as demonstrated by the MTT cell viability assay (data not shown).
  • ⁇ -CD was added to scrapie infected cell lysate before addition of pK and PrP Sc detection carried out as normal. It is hypothesised that the results from this experiment revealed that ⁇ -CD had no effect on pK activity (data not shown).
  • the linear counterparts for ⁇ -CD and ⁇ -CD were maltohepatose (G7) and maltohexaose (G6), respectively ( Figure 2A).
  • the corresponding linear sugar for ⁇ -CD, maltooctaose was not commercially available.
  • the compounds were added at 500 ⁇ M to the cells at the time of passage and the cells were cultured in this manner for a period of 2 weeks after which the cells were lysed and analysed for PrP Sc levels.
  • G6 and G7 displayed less anti prion activity over ⁇ - and ⁇ - CD, respectively, the importance of the cyclic nature of the CDs for enhancing anti prion activity is demonstrated.
  • CDs differ in size with ⁇ -CD possessing 6, ⁇ -CD possessing 7 and ⁇ -CD possessing 8 glucopyranose units ( Figure 2A), leading to differences in the cavity size of the compounds from 8 A for ⁇ -CD, 6 A for ⁇ -CD and 4.5 A for ⁇ -CD.
  • NH 4 Cl is a lysomotropic agent, which alkanises endosomes and lysosomes without influencing PrP Sc levels.
  • N2a22L cells were treated as before with 500 ⁇ M ⁇ -CD for 2 weeks. Cells were also treated with NH 4 Cl (3OmM) and a combination OfNH 4 Cl and ⁇ -CD for 2 weeks. Cells were also treated with NH 4 Cl (3OmM) and a combination OfNH 4 Cl and ⁇ -CD for the 2 week period and cells were then lysed and analysed for PrP Sc content.
  • Lipid rafts also known as detergent resistant microdomains (DRMs) are enriched in cholesterol and sphingolipids. The formation of rafts is dependent on cholesterol, which is believed to function as a spacer between hydrocarbon chains of sphingolipids thereby holding the raft together. Cyclodextrins are powerful extractors of cholesterol, but of the CDs, ⁇ -CD has been shown to have the highest capacity to sequester lipids. The accumulation of proteins in rafts is altered or abolished on cholesterol depletion. For PrP this holds true, and altering cholesterol levels interferes with PrP Sc production in cell culture. So, to investigate how ⁇ -CD acts against PrP Sc its effect on flotation properties was determined.
  • PrP and PrP are insoluble in cold Triton X 100. This is due to their association with membrane domains or rafts that are enriched in cholesterol, sphingolipids and glycolipids, these domains are also referred to as detergent resistant microdomains (DRMs). The association of PrP with such domains is linked with the conversion to PrP Sc . The solubility of DRMs is thought to be associated with their lipid content affecting their rigidity and consequently the ability of cold Triton X 100 to penetrate and solubilise. Consequently, PrP c and PrP Sc float in buoyant fractions in density gradients and float in similar fractions to the raft resident, cell surface ganglioside GM 1 .
  • the GMi marker similarly shifted from fraction 5 to 6 on ⁇ - CD treatment ( Figure 5 C, compare lane 5, upper panel, and lane 6, lower panel and Figure 5F).
  • PrP c on treatment with ⁇ -CD floated in lighter fractions (fractions 3-4) ( Figure 5 A, lanes 3-4, lower panel and Figure 5D) to the control (fraction 5) ( Figure 5A, lane 5, upper panel and Figure 5D).
  • N2a22L20 cells were grown for 1 week in the presence of M ⁇ -CD (500 ⁇ M) in combination with chol-M ⁇ -CD (46 ⁇ M) and cells were analysed for PrP Sc ( Figure 6A, lane 4 and Figure 6B). It has been found that that the presence of cholesterol did not inhibit but enhanced the clearance of PrP Sc , as levels fell to 20%, indicating that replenishment with cholesterol could not inhibit the M ⁇ -CD anti prion activity. Thus, it has surprisingly been found that a cyclodextrin, in particular M ⁇ -CD, in combination with cholesterol, is suitable for use as a medicament for treating prion disease.
  • N2a22L20 cells were treated with M ⁇ -CD or ⁇ -CD (each at 500 ⁇ M) for 1 week. The compounds were then removed and the cells were allowed to grow in their absence for 72h or they were replaced by chol-M ⁇ -CD (46 ⁇ M) for the 72h period. Cells were then lysed and analysed for PrP Sc levels by immunoblot with antibody 8H4 ( Figure 6Q.
  • PrP c may be directed by these compounds to naturally existing high lipid domains as a consequence of drug presence. Due to the hydrophobic core of CDs, they possess the ability to encapsulate hydrophobic moieties. If they bound PrP, this binding could aid in the anti-prion mechanism. Consequently the ability of ⁇ -CD to bind PrP c was determined.
  • ⁇ -CD was bound to epoxy activated sepharose 6B as described in materials and methods.
  • N2a cells expressing 3F4 tagged MoPrP 0 were lysed into cold lysis buffer (0.5% (w/v) Sodium Deoxycholate, 0.5% (v/v) Triton X-100, 15OmM NaCl and 5OmM Tris, pH 7.5) and the lysate was then added to gel containing ⁇ -CD that has been equilibrated to pH7.5 with 0.5M HEPES buffer and the binding process was carried out as described in materials and methods. Samples were analysed by 12% SDS-PAGE and immunoblotting with 3F4 antibody.
  • cyclodextrins have a surprising anti prion effect. This surprising effect demonstrates the potential use of cyclodextrins in a pharmaceutical composition or medicament for treating prion disease.

Abstract

La présente invention propose l'utilisation d'un oligosaccharide linéaire, ramifié ou cyclique, substitué ou non substitué, comprenant de 2 à 10 unités de monosaccharide, dans la préparation d'un médicament pour traiter une maladie de type prion. La présente invention fournit également une composition pharmaceutique pour traiter une maladie de type prion, la composition comprenant une quantité pharmaceutiquement efficace d'un oligosaccharide linéaire, ramifié ou cyclique, substitué ou non substitué, comprenant de 2 à 10 unités de monosaccharide, à l'exception de la β-cyclodextrine non substituée, et un excipient pharmaceutiquement acceptable.
PCT/EP2007/004727 2006-05-29 2007-05-29 Compositions comprenant des oligosaccharides pour traiter une maladie à prion WO2007137808A1 (fr)

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US8663729B2 (en) 2001-09-12 2014-03-04 The Iams Company Pet food compositions
US9404162B2 (en) 2005-05-31 2016-08-02 Mars, Incorporated Feline probiotic bifidobacteria and methods
US9415083B2 (en) 2004-05-10 2016-08-16 Mars, Incorporated Method for decreasing inflammation and stress in a mammal
US9427000B2 (en) 2005-05-31 2016-08-30 Mars, Incorporated Feline probiotic lactobacilli composition and methods
US9580680B2 (en) 2003-12-19 2017-02-28 Mars, Incorporated Canine probiotic bifidobacterium pseudolongum
US9771199B2 (en) 2008-07-07 2017-09-26 Mars, Incorporated Probiotic supplement, process for making, and packaging
US9821015B2 (en) 2003-12-19 2017-11-21 Mars, Incorporated Methods of use of probiotic bifidobacteria for companion animals
US10104903B2 (en) 2009-07-31 2018-10-23 Mars, Incorporated Animal food and its appearance
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8563522B2 (en) 1997-07-08 2013-10-22 The Iams Company Method of maintaining and/or attenuating a decline in quality of life
US8663729B2 (en) 2001-09-12 2014-03-04 The Iams Company Pet food compositions
US8728559B2 (en) 2001-09-12 2014-05-20 The Iams Company Pet food compositions
US9580680B2 (en) 2003-12-19 2017-02-28 Mars, Incorporated Canine probiotic bifidobacterium pseudolongum
US9821015B2 (en) 2003-12-19 2017-11-21 Mars, Incorporated Methods of use of probiotic bifidobacteria for companion animals
US9415083B2 (en) 2004-05-10 2016-08-16 Mars, Incorporated Method for decreasing inflammation and stress in a mammal
US9404162B2 (en) 2005-05-31 2016-08-02 Mars, Incorporated Feline probiotic bifidobacteria and methods
US9427000B2 (en) 2005-05-31 2016-08-30 Mars, Incorporated Feline probiotic lactobacilli composition and methods
US9771199B2 (en) 2008-07-07 2017-09-26 Mars, Incorporated Probiotic supplement, process for making, and packaging
US10709156B2 (en) 2008-07-07 2020-07-14 Mars, Incorporated Pet supplement and methods of making
US10104903B2 (en) 2009-07-31 2018-10-23 Mars, Incorporated Animal food and its appearance
EP3603649A1 (fr) * 2018-07-31 2020-02-05 Medday Pharmaceuticals Procédé de traitement de maladies à prion
WO2020025598A1 (fr) 2018-07-31 2020-02-06 Medday Pharmaceuticals Procédé de traitement de maladies à prions

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