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    • 专利摘要:
    Use of efavirenz for the treatment of lipid storage diseases. The present invention relates to the use of efavirenz to manufacture a medicament for the treatment of lipid storage diseases such as Niemann-Pick diseases. By activating CYP46, which stimulates the elimination of cholesterol in neurons, among other lipids, efavirenz is able to normalize the levels of these lipids and neuronal function. Efavirenz can also be used in combination with another active substance considered effective for the treatment of these diseases, to improve therapeutic effects. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2732498A1
    申请号:ES201830486
    申请日:2018-05-21
    公开日:2019-11-22
    发明作者:Muñoz María Dolores Ledesma；Daniel Mitroi
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] The invention relates to the use of the drug efavirenz and similar compounds for the treatment of lipid storage diseases, especially Niemann Pick's disease. Therefore, the present invention belongs to the field of medical chemistry or pharmacology.
    [0005]
    [0006] STATE OF THE TECHNIQUE
    [0007]
    [0008] Cholesterol is a major component of neuronal cell membranes vital to normal brain function. This lipid is particularly abundant in synaptic membranes where cholesterol-rich microdomains influence a variety of protein complexes. Niemann-Pick type C1 (NPC1) is an endolysosomal protein that mediates intracellular cholesterol transport. Mutations in the NPC1 gene cause Niemann-Pick type C disease (NPC), a fatal lysosomal storage disorder characterized by the accumulation of cholesterol in late / lysosomal endosomal compartments. NPC1 deficiency affects all cells in patients with NPC and has an impact on peripheral organs such as liver and spleen. However, neurons are the most vulnerable and the brain is the main target organ. NPC causes cognitive and psychiatric disorders that point to synaptic defects. Synaptic activity induces both loss and redistribution of cholesterol. The loss of cholesterol appears to be mediated by the cholesterol hydroxylase Cytochrome P450 46A1 (CYP46), which is an enzyme responsible for the renewal of cholesterol in neurons and moves to the plasma membrane after synaptic stimuli.
    [0009]
    [0010] Currently, NPC has no cure. The only treatment available is the administration of the synthetic iminoazúcar Miglustat, which inhibits glucosylceramide synthase by reducing the accumulation of gangliosides. Miglustat treatment delays the neurological progress of patients with NPC but does not cure the disease and may have side effects. Therefore, the search for new therapeutic strategies for NPC is an urgent need. Several are being considered Compounds today. Histone deacetylase inhibitors reduced cholesterol accumulation and increased NPC1 levels in cellular and animal models of NPC. A reduction in cholesterol and a moderate improvement in life expectancy were achieved in animal models of NPC by activation of the HSP70 thermal shock protein with the drug arimoclomol or by administration of the cholesterol-sequestering drug p-cyclodextrin. Clinical trials with these compounds have been approved in patients with NPC but their broad targets, potential toxicity or invasive administration due to inaccessibility through the blood brain barrier increase the concern of their application to patients.
    [0011]
    [0012] DESCRIPTION OF THE INVENTION
    [0013]
    [0014] The present invention discloses the benefits of the use of the CYP46 efavirenz activator compound (EFV) for the treatment of NPC. EFV is an approved drug for human use that is already prescribed for HIV treatment. The dose that shows efficacy in the mouse model of NPC is much lower than that used in patients with HIV. The examples of the invention show a notable increase in plasma levels of 24-hydroxycholesterol in NPC1nmf164 mice treated with EFV which provides a suitable biomarker to follow the treatment. The benefits of EFV treatment not only concern synaptic function and cognitive performance, it also produces an improvement in the lysosomal phenotype in brain tissue and the prolongation of life expectancy. The use of a combined EFV treatment with other compounds proposed to treat NPCs that are currently in clinical trials is also appropriate.
    [0015]
    [0016] By activation of CYP46, which stimulates the elimination of cholesterol in neurons, efavirenz is able to reduce abnormally high cholesterol levels discovered in the synapses of NPC1nmf164 mice and normalize neuronal function.
    [0017]
    [0018] Thus, a first aspect of the present invention relates to the use of a compound of formula (I):
    [0019]
    [0020] in which:
    [0021] X is halo,
    [0022] XI is selected from trihalomethyl, pentahaloethyl, C2-5 alkyl, C2-5 alkynyl, C3-5 cycloalkyl; or aryl;
    [0023] Z is selected from O or S;
    [0024] R is selected from:
    [0025] (a) C1-8 alkyl, unsubstituted or substituted with A, and A is selected from halo, C3-6 cycloalkyl, CN, hydroxy, C1-4 alkoxy, C2-4 alkynyl-C1-4 alkoxy, aryloxy, C1 alkylcarbonyl -4, nitro, di (C1-2 alkyl) amino, C1-4 alkyl-C1-2 alkyl, heterocycle, or arylthio;
    [0026] (b) C2-4 alkenyl, unsubstituted or substituted with
    [0027] (i) A, or
    [0028] (ii) aryl, unsubstituted or substituted with A;
    [0029] (c) C2-5 alkynyl, unsubstituted or substituted with
    [0030] (i) A, or
    [0031] (ii) aryl, unsubstituted or substituted with A; or
    [0032] (d) C3-4 cycloalkyl, unsubstituted or substituted with
    [0033] (i) A, or
    [0034] (ii) aryl, unsubstituted or substituted with A,
    [0035] or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a lipid storage disease.
    [0036]
    [0037] In a preferred embodiment, the compound of formula (I) is selected from the following group:
    [0038] (-) 6-Chloro-4-cyclopropyletinyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one, (-) 6-chloro-4-phenylethynyl-4-trifluoromethyl-1, 4-dihydro-2H-3,1-benzoxazin-2-one, (+/-) 6-chloro-4- (2-cyanophenyl) ethynyl-4- (1,1,1-trifluoromethyl) -1,4- dihydro-2H-3,1-benzoxazin-2-one,
    [0039] (+/-) 4- (1-Chloro-1,1-difluoromethyl) -4- (2-phenylethynyl) -6-chloro-1,4-dihydro-2H-3,1 benzoxazin-2-one, or
    [0040] (+/-) 4- (2- [dimethylaminomethyl] ethynyl) -4-trifluoromethyl-6-chloro-1,4-dihydro-2H-3,1-benzoxazin-2-one,
    [0041] or a pharmaceutically acceptable salt thereof.
    [0042]
    [0043] In a more preferred embodiment, the compound of formula (I) is (-) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one or a pharmaceutically salt acceptable of it.
    [0044]
    [0045] In another preferred embodiment, the lipid storage disease is selected from Niemann-Pick diseases, Gaucher disease, Fabry disease, Farber disease, Tay-Sachs disease, Sandhoff disease, Krabbe disease, metachromatic leukodystrophy, Wolman
    [0046]
    [0047] In the present invention, Niemann-Pick disease refers to all currently known disease variants, in particular types A, B and C, although in a more preferred embodiment Niemann-Pick disease refers to the type C.
    [0048]
    [0049] In another preferred embodiment, the use of a compound as described above is in combination with another active ingredient. In a more preferred embodiment, this active ingredient is selected from miglustat, histone deacetylase, arimoclomol, p-cyclodextrin, ursodeoxycholic acid, acetyl leucine or recombinant acid sphingomyelinase inhibitors.
    [0050]
    [0051] The compounds of the present invention may have asymmetric centers and may appear, except when specifically indicated, in the form of racemates, racemic mixtures or in the form of diastereomers, or enantiomers, individual, including all isomeric forms in the present invention. The term (+/-) is intended to include optical isomers (+) or optical isomers (-) or mixtures thereof.
    [0052]
    [0053] When any variable (for example, R) appears more than once in any constituent or in formula (I), its definition in each occurrence is independent of its definition in any other occurrence. In addition, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.
    [0054] As used herein except where indicated, "alkyl" is intended to include both branched and linear chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; "alkenyl" is intended to include both branched and linear chain alkyl groups with at least one carbon-carbon double bond; "Alkynyl" is intended to include both branched and linear chain alkyl groups with at least one carbon-carbon triple bond.
    [0055]
    [0056] "Halogen" or "halo", as used herein, means fluoro, chloro, bromo and iodo.
    [0057]
    [0058] As used herein, with exceptions indicated, "aryl" is intended to mean phenyl, naphthyl, tetrahydronaphthyl, biphenyl, phenanthryl, antryl or acenaphthyl.
    [0059]
    [0060] The term "heterocyclyl" or "heterocyclic", as used herein except where indicated, represents a stable 5- to 7-membered monocyclic heterocyclic or 8 to 11-membered bicyclic heterocyclic ring that is saturated, partially unsaturated or unsaturated, and consisting of carbon atoms and one to four heteroatoms selected from the group consisting of N, O and S, and in which the nitrogen and sulfur heteroatoms may be optionally oxidized, and which includes any bicyclic group in which any of the heterocyclic rings defined above is condensed to a benzene ring. The heterocyclic ring may be attached to any heteroatom or carbon atom that results in the creation of a stable structure. Examples of such heterocyclic elements include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl , pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, benzofuranyl, tetrahydropyranyl, thienyl , benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl.
    [0061]
    [0062] For these purposes, the compounds of the present invention can be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by spraying inhalation, or rectally, in unit dosage formulations containing conventional non-toxic pharmaceutically acceptable excipients, adjuvants and vehicles.
    [0063]
    [0064] Another aspect of the present invention relates to a compound of formula (I) for use in the treatment of a lipid storage disease.
    [0065]
    [0066] Thus, according to the present invention, a method of treating a lipid storage disease, such as Niemann-Pick disease, is also provided. The treatment involves the administration to a patient in need of such treatment of a pharmaceutical composition comprising a pharmaceutical vehicle and a therapeutically effective amount of a compound of the present invention.
    [0067]
    [0068] The present pharmaceutical compositions may be in the form of orally administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, in the form of sterile injectable aqueous or oleaginous suspensions or suppositories.
    [0069]
    [0070] When administered orally in the form of a suspension, the present compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose to impart mass, alginic acid or sodium alginate as suspending agent, methylcellulose as a viscosity enhancer, and sweetening / flavoring agents known in the art. In the form of immediate-release tablets, the present compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, expanders, disintegrants, diluents and lubricants known in the art.
    [0071]
    [0072] When administered by aerosol or nasal inhalation, the present compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared in the form of solutions in saline solution, using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.
    [0073] Injectable solutions or suspensions may be formulated in accordance with known art, using suitable non-toxic parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or dispersing agents or suitable humectants and suspensions, such as sterile, non-volatile tasteless oils, including mono or diglycerides, and fatty acids, including oleic acid.
    [0074]
    [0075] When administered rectally in the form of suppositories, the present compositions can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at the usual temperatures, but they liquefy or dissolve in the rectal cavity to release the drug.
    [0076]
    [0077] The compounds of the present invention can be administered orally to humans in a dosage range of 1 to 100 mg / kg body weight in divided doses. A preferred dosage range is 0.01 to 10 mg / kg body weight orally in divided doses. Another preferred dosage range is 0.01 to 20 mg / kg body weight orally in divided doses. For combination therapy with nucleoside analogs, a preferred dosage range is 0.01 to 20 mg / kg body weight for the compounds of the present invention administered orally in divided doses, and 50 mg to 5 g / kg of body weight for nucleoside analogues administered orally in divided doses. However, it is to be understood that the specific dose level and dosage frequency for any particular patient may vary and will depend on a variety of factors that include the activity of the specific compound employed, the metabolic stability and duration of action of that compound. , age, body weight, general state of health, sex, diet, mode and timing of administration, excretion rate, combination of drugs, the severity of the particular condition, and the therapy the host experiences.
    [0078]
    [0079] Unless defined otherwise, all the technical and scientific terms used herein have the same meaning as usually understood by the person skilled in the art to which the present invention belongs. Methods and materials similar or equivalent to those described herein may be used in the practice of the present invention.
    [0080] In the description and claims, the word "comprises" and its variations are not intended to exclude other technical characteristics, additives, components or steps. The objectives, advantages and additional features of the invention will be apparent to those skilled in the art upon examination of the description or can be learned through the practice of the invention. The following examples and figures are provided by way of illustration and are not intended to be limiting of the present invention.
    [0081]
    [0082] BRIEF DESCRIPTION OF THE FIGURES
    [0083]
    [0084] Figure 1. Location and level of NPC1 in wt and NPC1nmf164 mice. (A) Individual and fused images of immunofluorescences against PSD-95, synaptophysin-1 (SYP1) and NPC1 proteins in a mouse hippocampal neuron wt. The graphs show the mean value ± SEM of the colocalization of NPC1 with PSD95 or SYP1 (n = 12 neurons from 3 different cultures). (B) Image of electron microscopy of immunological labeling with gold against NPC1 in the CA1 hippocampus region of a wt mouse. The black arrows show the 15 nm gold particles bound to NPC1 (d-dendrite, synaptic sv-vesicles, m-mitochondria). (C), (D), Western blots versus NPC1 and actin-p (ACTB) in brain and total synaptosomes of wt and NPC1nmf164 mice. The graphs show the mean value ± SEM of the NPC1 levels normalized for ACTB as percentages of the control (wt values) (n = 4, Psinaptosomes = 0.0037).
    [0085]
    [0086] Figure 2. Morphology and cholesterol level of synapses in NPC1nmf164 mice. (A) Representative electron micrographs of synapses in the CA1 hippocampus region in wt and NPC1nmf164 mice (black arrows indicate postsynaptic densities, d-dendrite, synaptic sv-vesicles, m-mitochondria, MLB-multilamellar bodies). (B) Mean value ± SEM of synapse density (n = 3, P = 0.0377). (C) Mean value ± SEM of synaptic vesicle density (n = 3, 3088 synapse vesicles, 101 wt and 121 NPC1nmf164, P <0.0001). (D) Synaptic vesicle diameter (n = 3, vesicles, 528 wt and 507 NPC1nmf164). (E) Mean value ± SEM of postsynaptic density length (n = 3; postsynaptic densities, 101 wt and 121 NPC1nmf164, P <0.0001). (F) Mean value ± SEM of postsynaptic density thickness (n = 3, P <0.0001). (G), (H) Mean value ± SEM of cholesterol levels in synaptosomes (n = 4, P = 0.0476) and total brain homogenates (n = 4) in wt and NPC1nmf164 mice expressed as a percentage of wt mice.
    [0087] Figure 3. Synoptic function and behavior in wt and NPC1nmf164 mice. The following electrophysiological events were recorded in Schaffer collateral synapses of wt and NPC1nmf164 mice: (A) Basal synaptic transmission (n = 5, P = 0.0138); (B) Fiber volley amplitude (n = 5); (C) Paired pulse facilitation (n = 5, P = 0.0011); (D) LTP (n = 5, P <0.0001 in the last 10 min of registration); (E) LTD. The following behavioral tests were evaluated in wt and NPC1nmf164 mice: (F) Object location recognition test showing the scanning time of objects in new and familiar locations (n = 10, P = 0.0167); (G) Y labyrinth test showing the percentage of entries in the new arm (n = 10; P = 0.0015). (H) Contextual fear conditioning test that shows the percentage of immobility time (n = 10; P = 0.0002); (I) Open field test showing the distance covered for 5 min.
    [0088]
    [0089] Figure 4. Redistribution of cholesterol and surface distribution of the GluA1 receptor after induction of cLTP in wt and NPC1nmf164 mice. (A) Mean value ± SEM of cholesterol levels in plasma membrane fractions of hippocampal cut cultures of wt and NPC1nmf164 mice with or without cLTP induction (n = 6, Pwt = 0.0402). (B) Representative images of the mCherry-D4 cholesterol binding probe before and after cLTP induction in cultured hippocampal neurons of wt mice infected with sh-scr or sh-NPC1 RNA. (C) Western blot versus NPC1 and ACTB in hippocampal neuron extracts cultured from wt mice not infected or infected with sh-scr or sh-NPC1 RNA. The graph shows the mean value ± SEM of the NPC1 levels normalized for ACTB as a percentage of the control conditions (wt not infected). (D) Representative images of immunofluorescences against GluA1 before and after induction of cLTP in cultured hippocampal neurons not permeabilized (surface) and permeabilized (total) expressing sh-scr or sh-NPC1 RNA. The graph shows the mean value ± SEM of surface staining of GluA1 with respect to the total in cLTP conditions as a percentage of the baseline in the control situation (n = 20 by condition of 2 different experiments, Psh-Scr <0.001).
    [0090]
    [0091] Figure 5. Effects of CYP46 activation by EFV on synaptic redistribution of cholesterol and on synaptic plasticity in wt and NPC1nmf164 neurons. (A) Western blots versus CYP46 and ACTB in total brain extracts and synaptosomes of wt and NPC1nmf164 mice as a percentage of control (wt values). The graphs show the mean value ± SEM of the standardized CYP46 levels for ACTB in arbitrary units. (B) Mean value ± SEM of cholesterol levels in synaptosomes untreated and treated with EFV of wt mice (n = 5, P = 0.0233) and NPC1nmf164 (n = 4, P = 0.0188). (C) Baseline synoptic transmission (n = 5, P = 0.0117); (D) LTP (n = 5, P <0.0001) and (E) Paired pulse facilitation in non-treated and EFV treated hippocampal sections of wt and NPC1nmf164 mice (n = 5, P = 0.0229).
    [0092]
    [0093] Figure 6. Effects of CYP46 activation by EFV on the surface distribution of GluA1 induced by cLTP under wt and NPC1 deficit conditions. Representative images of immunofluorescences against GluA1 before and after induction of cLTP in cultured hippocampal neurons not permeabilized (surface) and permeabilized (total) expressing sh-Scr or sh-NPC1 RNA and treated or not with EFV. The graph shows the mean value ± SEM of surface staining of GluA1 with respect to the total after induction of cLTP as a percentage of the baseline in the control situation (n = 20 by condition of 2 separate experiments, Psh-Scr <0.001, Psh -Scr EFV <0.01, Psh-NPC1 EFV <0.001).
    [0094]
    [0095] Figure 7. Effects of EFV treatment in vivo. (A) Mean value ± SEM of plasma levels of 24 (S) -hydroxycholesterol after 6 and 8 weeks of oral treatment with EFV in wt mice (n = 6, weeks P6 = 0.0023) and NPC1nmf164 (n = 5, weeks P6 <0.0001, weeks P8 <0.0001). The following behavioral tests were evaluated in wt and NPC1nmf164 mice treated or not with EFV: (B) Object location recognition test showing the discrimination index in the form of a normalized proportion of time spent with the new object and the family object (n = 6, PWT / NPC1nmf164 = 0.0065, PNPC1nmf164 / NPC1nmf164 EFV = 0.0008). (C) Y labyrinth test showing the percentage of entries in the new arm (n = 10, P <0.0001). (D) Contextual fear conditioning that shows the percentage of immobility time (n = 10, PNPC1 <0.0025, PNPC1 EFV <0.0181). (E) Specific fear conditioning test showing the percentage of immobility time (n = 10, PNPC1 <0.0453, PNPC1 EFV <0.0054). (F) Mean value ± SEM of cholesterol levels in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV (n = 4, PNPC1 <0.0476, PNPC1 EFV <0.0118). (G) Fluorescence images representative of the CA1 hippocampus region of wt and NPC1nmf164 mice treated or not with EFV stained with filipin and an antibody against LAMP1. White arrows indicate accumulation of lysosomes in the untreated hippocampus of NPC1nmf164 mice.
    [0096] (H) Survival graph for wt and NPC1nmf164 mice treated or not with EFV (n = 5).
    [0097]
    [0098] EXAMPLES
    [0099]
    [0100] NPC1 is present at synapses and its levels are reduced in NPC1nmf164 mice
    [0101]
    [0102] Although the presence of NPC1 in synaptosomes has been reported, its distribution at synapses has not been addressed in detail. For this purpose, confocal and electronic microscopes were used together with biochemical techniques in primary neuronal cultures and in mouse brain tissue. Immunofluorescence analyzes of mature hippocampal neurons of wild-type (wt) mice indicated the presence of NPC1 in both presynaptic and postsynaptic terminations with relative enrichment in the latter. While NPC1 colocalization with the synaptophysin presynaptic marker was 22.5%, the percentage of colocalization with the PSD95 postsynaptic marker increased to 59% (Figure 1A). The postsynaptic location of NPC1 was confirmed by electron microscopy in the hippocampus of wt mice (Figure 1B). The transfer of Western versus NPC1 in total and synaptosome extracts from wt mice also showed the predominant presence of this protein in synaptic membranes (Figures 1C, D). Interestingly, although NPC1 levels were not significantly altered in total brain extracts (Figure 1C), synaptic levels were drastically reduced (70%) in NPC1nmf164 mice compared to wt mice (Figure 1D).
    [0103]
    [0104] Altered morphology and elevated cholesterol levels at the synapses of NPC1nmf164 mice
    [0105]
    [0106] Reduced levels of NPC1 at the synapses of NPC1nmf164 mice led to the analysis of synaptic characteristics in the CA1 hippocampus region. Morphological analyzes by electron microscopy showed an average reduction of 25% in the number of synapses per unit area in 3-month-old NPC1nmf164 mice compared to wt mice of the same age (Figure 2A). The number of synaptic vesicles was also reduced by 51.4% while their size increased by 62% (Figures 2B, C). Alterations in the postsynaptic compartment were also discovered. The length of postsynaptic density was 35.8% shorter while its thickness increased by 42.9% in mice. NPC1nmf164 (Figures 2D, E). To have a better understanding of the relationship between NPC1 and cholesterol, the levels of this lipid were measured at the synapses of wt and NPC1nmf164 mice. Quantification by enzymatic assays showed a 16.5 % increase in cholesterol levels in synaptosomes but no significant difference in total brain extracts of NPC1nmf164 mice compared to wt mice (Figures 2F, G).
    [0107]
    [0108] Altered synaptic function and behavior in NPC1nmf164 mice
    [0109]
    [0110] The morphological alterations discovered in the synapses of the NPC1nmf164 mice prompted to analyze their function. Electrophysiology was used in hippocampal sections to monitor Schaffer collateral synapses in the CA1 region. Basal synaptic transmission increased in NPC1nmf164 mice while fiber volley amplitude was not altered compared to wt mice (Figures 3A, B). Presynaptic plasticity events such as paired pulse facilitation increased (Figure 3C). Specific alterations in postsynaptic plasticity events were also discovered. LTP was significantly decreased (Figure 3D) while LTD was retained (Figure 3E).
    [0111]
    [0112] Given the alterations in the events that control memory and learning, several trials were conducted to monitor these abilities in live mice. In the object location recognition test, the NPC1nmf164 mice recognized the displaced object very poorly, indicating that the hippocampal spatial learning and memory are impaired (Figure 3F). Consequently, NPC1nmf164 mice entered less frequently (12%) in the new arm in the Y labyrinth test than wt mice (Figure 3G). Associative learning and memory in the contextual fear conditioning trial were also impaired in NPC1nmf164 mice compared to wt mice as evidenced by a 62% reduction in immobility time (Figure 3H). To rule out that an impaired locomotive activity could have influenced the outcome of the aforementioned trials, exploratory activity was analyzed in the open field trial. No significant difference was found in the distance covered by the NPC1nmf164 and wt mice (Figure 3I).
    [0113]
    [0114] NPC1 mediates the redistribution of cholesterol and the AMPA receptor supply required for LTP
    [0115] Recent evidence has shown that redistribution of cholesterol triggered by LTP is necessary for the synaptic supply of AMPA receptors, which in turn allows the progress of LTP. It was postulated that NPC1 mediates cholesterol changes triggered by LTP. To test this hypothesis, cholesterol changes were analyzed in cultures of organotypic sections of the hippocampus of wt and NPC1nmf164 mice after induction of chemical LTP (cLTP), which activates synapses similar to physiological LTP. The sections were subsequently centrifuged to separate different cholesterol populations with the plasma membrane enriched sediment. cLTP induced a 30.4 % reduction in cholesterol in the plasma membrane fraction of the wt mouse sections. On the contrary, no significant change was observed in the cuts of the NPC1nmf164 mice (Figure 4A). To better monitor the redistribution of cholesterol after induction of LTP, this lipid was visualized in primary cultures of hippocampal neurons of wt mice in which NPC1 expression had been silenced by lentiviruses carrying NPC1 shRNA. Cholesterol was labeled by transfecting neurons with the cDNA of the cholesterol-binding domain (D4) of perfringolysin O fused to mCherry. mCherry-D4 showed a diffuse distribution of plasma membrane type in neurons infected with the lentivirus that carries nonspecific sh-scr (Figure 4B). Unlike cells expressing sh-scr, mCherry-D4 showed a mottled pattern compatible with microsomal fractions in cells expressing sh-NPC1 (Figure 4B) in which NPC1 expression was reduced by 64.2% (Figure 4C). The induction of cLTP caused an evident redistribution of mCherry-D4 of the plasma membrane fractions into microsomal-like fractions in sh-scr-expressing neurons while not inducing significant changes in sh-NPC1-expressing neurons (Figure 4B). To determine whether this was also accompanied by a deficient AMPA Rc supply in the membrane after induction of LTP, total and superficial immunofluorescence staining was performed in cultured neurons, permeabilized or not, using an antibody against the subunit. Gluc1 of AMPA Rc (Figure 4D). As expected, induction of cLTP increased the surface localization of GluA1 (95.5% increase) without affecting the total levels in neurons expressing sh-scr. In contrast, the surface supply of GluA1 in response to cLTP was blocked in sh-NPC1 neurons (Figure 4D).
    [0116]
    [0117] In vitro pharmacological activation of CYP46 restores cholesterol levels, LTP and the surface supply of GluA1 at synapses deficient in NPC1
    [0118] The previous results indicated the lack of redistribution of cholesterol after LTP and the increase in the synaptic levels of this lipid when there is a deficit of NPC1. The CYP46 hydroxylase cholesterol is the enzyme responsible for the renewal of cholesterol in neurons and moves to the plasma membrane after synaptic stimuli mediating the loss of cholesterol during excitatory neurotransmission. Therefore, it was proposed that activation of CYP46 could counteract excess cholesterol and blockade after NPC1 deficiency. First, it was confirmed that CYP46 levels were not altered in total brain extracts and in synaptosomes of NPC1nmf164 mice compared to wt mice (Figure 5A) supporting their pharmacological activation as an appropriate strategy. To carry out this strategy, anti-HIV medication efavirenz (EFV) was used, since it causes a robust CYP46 stimulation. Incubation with EFV 20 | jM reduced cholesterol in the synaptosomes of both wt and NPC1nmf164 mice (reduction of 16.1% and 18.5%, respectively) (Figure 5B). Incubation with EFV 20 j M also restored synaptic function as basal synaptic transmission and LTP, but not paired pulse facilitation, in sections of hippocampus of mice NPC1nmf164. This treatment had no significant effect on the wt mice cuts (Figures 5C-E). To test whether the EFV treatment also restored the supply of AMPA Rc to the membrane after induction of LTP in neurons deficient in NPC1, a superficial and total antiGluA1 staining was performed in neurons expressing sh-scr and sh- NPC1 where cLTP was induced in presence or absence of EFV 20 j M (Figure 6). The induction of cLTP in sh-scr neurons increased the surface presence of GluA1 in a similar manner in the neurons treated with EFV and untreated (increase of 66.6% and 71.3%, respectively). Notably, the EFV treatment counteracted the blockade of superficial supply of GluA1 in response to cLTP in sh-NPC1 neurons, taking it to levels similar to those of sh-scr neurons (Figure 6).
    [0119]
    [0120] Oral EFV treatment prevents cholesterol accumulation and memory impairment and prolongs life expectancy in NPC1nmf164 mice
    [0121]
    [0122] The positive effects of in vitro EFV treatment on synaptic cholesterol levels and plasticity led to testing this drug in vivo. EFV has the ability to cross the blood brain barrier and stimulates the degradation of brain cholesterol in mice at doses 300 times lower than those used for patients with HIV. This dose, 0.09 mg / kg / day, was administered in the drinking water at 1.5 month old wt and NPC1nmf164 mice and treatment was maintained for 8 weeks. To follow the treatment, plasma levels of the CYP46 24 (S) -hydroxycholesterol metabolite were measured at different times throughout the treatment (6 and 8 weeks). This metabolite had similar levels in the plasma of the untreated wt and NPC1nmf164 mice. In wt mice, the EFV treatment caused a slight increase in the levels of 24 (S) -hydroxycholesterol only at 6 weeks of treatment. In contrast, levels of this metabolite increased dramatically in NPC1nmf164 mice treated with EFV at both 6 and 8 weeks of treatment (355.7% and 223.7%, respectively) (Figure 7A). Immediately before sacrifice, functional effects were determined by different behavioral tests. EFV increased interest in the object located in the new location and improved by 7.8% the number of entries in the new arm of the Y labyrinth in mice NPC1nmf164, indicative of improved spatial learning and memory of the hippocampus (Figures 7B, C ). EFV also improved contextual and specific learning and memory in the fear conditioning paradigm (Figure 7D, E). The EFV treatment had no significant effects on the behavior of the wt mice (Figures 6B-E). Synaptosomes from the brains of mice were isolated to monitor the effects on synaptic cholesterol levels. EFV reduced by 38.1% and 17.2% cholesterol levels in the synaptosomes of NPC1nmf164 and wt mice, respectively (Figure 7F) as determined by enzymatic assays. In addition to these biochemical means, Filipina, a fluorescent antibiotic that binds cholesterol, was used to examine cholesterol levels in the hippocampal tissue. Since the accumulation of this lipid in lysosomes is a hallmark of NPC1 deficiency, joint staining with the LAMP1 lysosomal marker was carried out. While staining with Filipina in wt mice treated or not with EFV was barely detectable, a strong signal was evident in the hippocampus of untreated NPC1nmf164 mice consistent with increased lipid levels (Figure 7G). EFV significantly reduced staining with Filipina in the hippocampus of NPC1nmf164 mice. Staining with LAMP1 in the NPC1nmf164 mice showed the accumulation of usual enlarged lysosomes of the NPC1 deficit. This phenotype was not evident in the hippocampus of NPC1nmf164 mice treated with EFV (Figure 7G). Given the positive effects of EFV on cholesterol behavior and accumulation after 8 weeks of treatment, administration of EFV in a group of wt and NPC1nmf164 mice was prolonged to determine the effects on survival. All untreated NPC1nmf164 mice died after 16 weeks of age with a Average survival time of 100 ± 2 days. None of the NPC1nmf164 mice treated with EFV died before 16 weeks of age. The average survival time of the NPC1nmf164 mice treated with EFV was 129 ± 2 days (Figure 7H).
    [0123]
    [0124] DISCUSSION
    [0125]
    [0126] NPC1 is well known for its contribution to cholesterol transport through the endolysosomal compartment of all cells that facilitates cholesterol excretion by direct lipid binding. Despite the ubiquitous presence of NPC1, the impact of its deficit is particularly significant in neurons. In fact, cognitive and psychiatric disorders are frequent consequences of NPC1 mutations in patients with NPC. The results presented here, which reveal a fundamental role of NPC1 in synaptic function, contribute to explaining the neuronal vulnerability in the NPC1 deficit. Using mice that carry a mutation in the NPC1 gene (NPC1nmf164), similar to the usual in patients with NPC, it was shown that NPC1 defects have a profound impact on synapses. In addition to the morphological alterations in the synaptic vesicles, the relative enrichment of the protein in postsinapsis and its contribution to synaptic plasticity were revealed. NPC1 mediates the redistribution of cholesterol and the dynamism of AMPA Rc necessary for LTP, which leads to the postsynaptic plasticity required for learning and memory. The deterioration of these events after NPC1 mutations may explain the progressive cognitive deficits in patients with NPC. These results highlight the relevance of cholesterol dynamics in LTP and the cooperative action of the two cholesterol-related proteins for their effective progress. While the NPC1 cholesterol-binding protein mediates the redistribution of LTP-induced cholesterol, the CYP46 cholesterol hydroxylation enzyme is responsible for the elimination of cholesterol. Notably, the pharmacological activation of CYP46 prevents LTP deficits in NPC1nmf164 mice. In these mice it was shown that cholesterol accumulates in synaptosomes in which NPC1 is present although at reduced levels compared to wt mice. It is proposed that by improving the elimination of cholesterol from synapses, the activation of CYP46 promotes the function of residual NPC1, which would allow a redistribution of cholesterol.
    [0127]
    [0128] MATERIALS AND METHODS
    [0129] Mice and ethical statement
    [0130]
    [0131] A breeding colony of NPC1nmf164 mice carrying the D1005G-Npc1 mutation in the CBMSO was established from C57BL / 6J-Npc1nmf164 / J mice acquired from Jackson Laboratories. Male and female homozygous wt and NPC1nmf164 offspring from the same litter were identified by PCR carried out in DNA isolated from the tail, and randomized to experimental groups. No gender-dependent difference was observed in any of the results. The CBMSO and CSIC internal review boards approved all procedures that involved the use of mice that were carried out in accordance with the specific guidelines of the European Union for the protection of animal welfare (Directive 2010/63 / EU).
    [0132]
    [0133] Antibodies
    [0134]
    [0135] Antibodies against the following proteins were used for Western blots and immunofluorescences: NPC1 (rabbit polyclonal, Novus Biologicals, United Kingdom, NB 400-148), PSD-95 (mouse monoclonal, NeuroMab, Ca, USA. , 75-028), Synaptophysin1 (guinea pig polyclonal, Synaptic Systems, Germany, 101004), CYP46A1 (rabbit polyclonal, Proteintech, USA, 12486-1-AP), GluR1 C terminal (rabbit polyclonal, (Abcam , ab31232), GluR1 N terminal (mouse monoclonal, Merck-Millipore, MAB2263), LAMP1 (rat monoclonal, DSHB), and anti-p-actin (mouse monoclonal, Sigma-Aldrich, A2228). secondary antibodies of goat anti-rabbit, rabbit anti-mouse, and donkey anti-rat conjugated with Alexa Fluor or HRP (Life Technologies).
    [0136]
    [0137] Neuronal cultures
    [0138]
    [0139] Primary cultures of hippocampal neurons were prepared from 18-day (E18) mouse embryos. Neurons were maintained in a 5 % CO2 atmosphere at 37 ° C in Neurobasal medium (Thermo Fisher Scientific, 21103-049) supplemented with B27 (Thermo Fisher Scientific, 17504044) and GlutaMAX (Thermo Fisher Scientific, 35050061). On the day in vitro (DIV) 7, the culture medium was replaced with medium without GlutaMAX. The cultures were used in DIV 14.
    [0140]
    [0141] Organotypic cut cultures
    [0142] Cultures of organotypic hippocampal cuts were prepared as is known in the art. In summary, the hippocampi of 7-day postnatal mice were dissected and 400 pm cuts were prepared with a tissue microtome (Leica Biosystems). The cuts were placed in a cell culture insert (Merck) in a medium containing 20% horse serum, 1 mM L-glutamine, 21 mM CaCl, mSO MgSO42, 1 mg / l insulin, 0.0012% of ascorbic acid, 30 mM HEPES, 13 mM D-glucose and 35.2 mM NaHCO. The cuts were maintained at 35.5 ° C and the culture medium was changed to a recent one every 2-3 days. The cuts were used after DIV 7.
    [0143]
    [0144] Synaptosome Isolation
    [0145]
    [0146] The brains of the mice were homogenized in 0.32 mM sucrose, 1 mM EDTA, 1 mg / ml BSA, 5 mM HEPES at pH 7.4) and centrifuged at 3000 g for 12 min at 4 ° C. The supernatant was further centrifuged for 12 min at 14000 g at 4 ° C. The pellet was resuspended in Krebs-Ringer buffer (140 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM EDTA, 10 mM Hepes at pH 7.4) and mixed with Percoll (45% v / v). The resulting solution was centrifuged for 2 min at 14000 rpm at 4 ° C. Synaptosomes were collected from the surface with a syringe and resuspended in Krebs-Ringer buffer, followed by centrifugation at 14000 rpm for 30 s at 4 ° C. Synaptosomes were obtained in the sediment that was resuspended in Hepes-Krebs buffer (147 mM NaCl, 3 mM KCl, 10 mM glucose, MgSO42 mM, 2 mM CaCl2, 20 mM Hepes at pH 7.4).
    [0147]
    [0148] Electron microscopy and immunological marking with gold
    [0149]
    [0150] NPC1nmf164 and wt mice of the same litter (n = 3 per genotype) were perfused with phosphate buffered saline (PBS) and fixative (4% paraformaldehyde [PFA] and 2% glutaraldehyde [GTA] in phosphate buffer) 0.1 M at pH 7.4 or 2% PFA and 0.2% GTA in the same buffer for immunological labeling with gold). Brains were fixed in 4% PFA overnight (ON) and sectioned in cuts 200 pm thick or 300 pm thick for immunological marking cuts with gold. The hippocampus sections were subsequently fixed in 1% osmium tetraoxide (in 0.1 M phosphate buffer at pH 7.4), dehydrated in ethanol and embedded in Epon (TAAB 812 Resin, TAAB Laboratories). The ultra-thin serial sections of the CA1 region were collected in hole or single slot grids, coated with Formvar, and stained with acetate of uranyl (Electron Microscopy Sciences) and lead citrate. The samples for immunological gold marking were inactivated for free aldehydes with 0.05 M NH4Cl. The sections were cryopreserved in glycerol, frozen by immersion in liquid propane at -180 ° C and transferred to a Leica freezing replacement unit. AFS Freezing substitution was carried out at -90 ° C in methanol containing 0.5% uranyl acetate for 80 h. Sections of the ultra-thin 70 nm CA1 hippocampus were incubated with the primary antibody against NPC1 (1:20) followed by a secondary antibody coupled to 15 nm gold particles. Finally, the sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (JEM1010, jeol, Akishima, Tokyo, Japan). The CA1 synapses were identified by position and randomly sampled and photographed with a 20,000 x magnification with a 4k TemCam-F416 CMOS camera (TVIPS, Gauting, Germany). The images were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The density of SV was calculated as the number of vesicles per pm2 within 10 nm of the presynaptic membrane and no more than 300 nm from the limit of the active zone.
    [0151]
    [0152] Immunofluorescence
    [0153]
    [0154] Neurons with 4% PFA and 4% sucrose were fixed in PBS at RT. Non-specific binding was blocked with 0.2% gelatin and 1% BSA (Sigma-Aldrich, A9418) in PBS. For surface GluA1 labeling, the neurons were incubated for 2 h with an antibody against the N-terminal end of GluR1. Total GluA1 was then immunologically detected after a permeabilization step (30 minute incubation in 0.1% Triton X-100 and 0.2% gelatin and 1% BSA in PBS) with an antibody against to the C-terminal end of GluR1. The corresponding secondary antibodies conjugated with Alexa Fluor 555 and Alexa Fluor 647 (Life Technologies) were incubated for 1 h. The coverslips were mounted in Permount (Life Technologies). Images of the cells were acquired using an inverted confocal microscope (LSM800, Carl Zeiss). Quantification was carried out using ImageJ software. The regions of interest in individual dendrites were manually selected in the total GluA1 channel. Therefore, the quantification of the images was blind with respect to the superficial GluA1 channel.
    [0155]
    [0156] Immunohistofluorescence
    [0157] Mice were perfused intracardially with PBS and 4% PFA, fixed with 4% PFA in PBS ON at 4 ° C and then cryopreserved in 30% sucrose (Merck Millipore, 107687) in PBS for 48 h . The samples were then frozen in Optimum Cut Temperature (Tissue-Tek; Thermo Fisher Scientific, 23-730-571). Sagittal sections (30 mm) were obtained with a CM 1950 Ag Protect freezing microtome (Leica, Solms, Germany). Sections were blocked with 0.2% gelatin and 1% BSA in PBS and incubated with the primary antibody ON at 4 ° C in blocking solution. Sections were incubated in donkey secondary antibody conjugated with Alexa Fluor (Thermo Fisher Scientific, A-21206 or AP180SA6MI) and 100 pg / ml filipine (a fluorescent polienic antibiotic with high cholesterol affinity, Sigma-Aldrich) for 1 , 5 ha TA. Finally, the sections were washed and mounted with Prolong Gold Antifade (Thermo Fisher Scientific, P36930). Images were taken with an LSM800 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
    [0158]
    [0159] Induction of chemical LTP (cLTP)
    [0160]
    [0161] The cLTP treatment was carried out in hippocampal sections. These were transferred to an immersion housing chamber containing artificial cerebrospinal fluid (ACSF; 119 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 11 mM D-glucose, NaHCO326 mM, MgCl21.25 mM and CaCl22.5 mM) saturated with 95% O2 / 5% CO2 for 5 minutes at RT. CLTP was induced by transferring the cuts to a separate chamber containing 0.1 pM Rolipram, 50 pM Forskolin and 100 pM Picrotoxin in ACSF lacking MgCl 2 for 15 minutes at RT. After the induction period, the cuts were washed in cold PBS and immediately homogenized in MES buffer (25 mM MES, 2 mM EDTA and a protease and phosphatase inhibitor cocktail (Roche)). The homogenized sections were centrifuged at 100,000 g for 2 hours at 4 ° C to separate the plasma membrane (sediment, resuspended in PBS-0.1% SDS) and the microsomal fraction (supernatant). In neuronal cultures, the medium was changed to ACSF in DIV 17. For redistribution of cholesterol with mCherry-D4, baseline images were acquired after 15 min and then the solution was exchanged for ACSF containing Rolipram 0.1 pM, Forskolin 50 pM, and Picrotoxin 100 pM without MgCl2. After a 15 min treatment, fluorescence images were acquired in the form of z-cells using a Nikon A1R + confocal microscope. For recycling of GluA1 receptors cLTP was induced as described above, followed by fixation. The images are they took an LSM800 confocal microscope (Cari Zeiss AG, Oberkochen, Germany).
    [0162]
    [0163] Production of lentiviral particles, neuronal infection and transfection of mCherry-D4 and image acquisition
    [0164]
    [0165] Packaging plasmids and short hairpin RNA plasmids (shRNA) against NPC1 or a mixed control plasmid (pGFP-C-shLenti) were obtained in Origene (Rockville, MD, USA). Plasmids were amplified and the Maxi prep protocol was followed (Macherey-Nagel, NucleoBond® Xtra Maxi, 740414.10). PCMV and PMD2.G (packaging plasmids), NPC1 mRNA plasmid or mixed shRNA together with PEI were added to HEK295T cells. The medium was changed to Opti-MEM and the lentiviral particles were isolated by ultracentrifugation. The sediment was resuspended in PBS and stored at -80 ° C. The lentiviral particles were added to the neuron medium in DIV 6 ON and after that the medium was completely changed by recent means. Neurons were transfected with mCherry-D4 plasmid in DIV 13 with Lipofectamine 3000 (Life technologies). Images of z batteries were acquired using a microscope with Nikon A1R + focal point.
    [0166]
    [0167] Quantification of cholesterol and 24 (S) -hydroxycholesterol
    [0168]
    [0169] Before the quantification of cholesterol, the amount of protein was measured by the Bicinconinic Acid Protein Assay Kit (BCA) (Thermo Fisher Scientific). Cholesterol content was measured by the Amplex Red Cholesterol Test Kit (Thermo Fisher Scientific) and referred to the amount of protein. 24 (S) -hydroxycholesterol levels were measured using the enzyme-linked immunosorbent assay kit (ELISA) of 24 (S) -hydroxycholesterol (Abcam, ab204530, Cambridge, UK) according to the manufacturer's protocol.
    [0170]
    [0171] EFV treatment in vitro, ex vivo and in vivo
    [0172]
    [0173] EFV was dissolved in DMSO at a working concentration of 20 mM and kept at -20 ° C protected from light. Synaptosomes and neuronal cultures were treated with 20 pM EFV for 2 h at 37 ° C. The brain sections were incubated with 20 pM EFV in the light-protected equipment for 1 h before the field records that lasted another 2 h during which the EFV was maintained. The in vivo treatment began in mice at 6 weeks of age. EFV was administered in the water of the light protected beverage at a dose of 0.9 mg / kg / day. The EFV solutions were changed every 3 days. Groups of control mice received only vehicle (DMSO). The treatment lasted 8 weeks or until death in the case of survival experiments.
    [0174]
    [0175] Electrophysiological records in hippocampal sections
    [0176]
    [0177] Mice were decapitated and the brain was rapidly removed and placed in an ice-cold oxygenated dissection solution (233 mM sucrose, 4 mM KCl, 25 mM MgCl, 10 mM NaHCO326 and glucose, saturated with 95% O2 / 5% of CO2). Coronal cuts (300 pm thick) were prepared with a vibratome (Leica, VT1200S) and kept in a chamber containing ACSF (11 mM glucose, 119 mM NaCl, 2.5 mM KCl, 1 mM NaH 2 PO 4 , NaHCO 3 26 mM, 1.25 mM MgCl2 and 22.5 mM CaCl, saturated with 95% O2 / 5% CO2) at 32 ° C for at least 1 h before registration. After this recovery time, the cuts were maintained at 25 ° C. For electrophysiological records, the cuts were placed in a submerged chamber and perfused with ACSF at 25 ° C. Schaffer collateral CA1 synaptic responses were recorded as extracellular fEPSP from stratum radiatum CA1 using a concentric bipolar platinum-iridium stimulation electrode and a low resistance glass recording microelectrode filled with ACSF. The pClamp9 (Molecular Devices) software was used for the acquisition. To measure the input-output ratio, fEPSP was evoked at different increasing stimulation intensities (from 50 to 100 every 10 pA, 150 and 200 pA) and the slope of the response was calculated. This curve was also used to establish the baseline of fEPSP at ~ 20% (for PPF experiments), ~ 30% (for LTP experiments) or ~ 50% (for LTD experiments) of the maximum slope. PPF was measured over a range of interesting intervals (ISI; six traces for each ISI, ISI 50-400 ms) at 0.067 Hz. Baseline stimulation was provided every 15 s (pulse duration 0.05 ms ) for at least 20 min before induction of LTP or LTD to ensure the stability of the response. LTP was induced by theta burst stimulation (4 pulses at 100 Hz, with repeated bursts at 5 Hz and each tetanus including three 10-burst trains separated by 15 s). LTD was induced using 900 pulses at 1 Hz. Responses were recorded for 1 h after induction of LTP or LTD. All records were carried out in the presence of the GABAA picrotoxin receptor antagonist (0.1 mM).
    [0178] Behavior analysis
    [0179]
    [0180] Open field exploration test. The locomotive activity was measured in a transparent plexiglass box measuring 43.2 cm x 43.2 cm, equipped outside with photoelectric beam detectors to monitor horizontal and vertical activity. Activity levels were recorded with a MED Associates activity monitor (MED Associates, St. Albans, VT). Locomotive activity data was collected through a PC and analyzed with the activity monitoring data analysis software of MED Associates. The mice were placed in the center of the open field apparatus and allowed to move freely. Data were collected individually for each animal for 5 min.
    [0181]
    [0182] Object location recognition test. After manipulation and habituation to the test stage, the mice underwent 3 6-minute training sessions, during which they were allowed to freely explore 2 identical objects (small glass bottles) that were placed in defined locations on the stage of the essay. The next day, a 6-minute trial session was held, during which the location of one of the objects was changed, while the location of the other remained unchanged. A video camera was used to monitor and record the behavior of the animals. The time that the animals spent exploring the object in the new location and in the known location during the test was manually scored by an observer from the videotape.
    [0183]
    [0184] Y labyrinth test. The Y labyrinth test was carried out using a symmetrical Y labyrinth made of black plastic and the wall at the end of each arm was marked with a different paper shape. The mice were trained in the Y maze with one of the arms closed. The mice were placed at the end of the exit arm, away from the center, and allowed to explore freely for 5 min. After scanning, the mice were placed back in the cage. An hour later, the originally locked arm was opened and defined as the "new arm." The mice were placed in the Y maze on the start arm and allowed to move freely for 5 min. The percentage of entries in the new arm was measured.
    [0185]
    [0186] Contextual and specific fear conditioning test. The system was used combined StarFear (Panlab-Harvard Apparatus) for contextual and specific fear conditioning. The conditioning was carried out in a chamber with a stainless steel grid floor in a sound attenuation box. The grid floor was connected to a discharge generator and an auditory signal was supplied from a speaker on the wall of the chamber. In order to assess the sensitivity of the mice, individual mice were placed in the fear conditioning apparatus and electric shocks were supplied in the legs (2 s) of increasing intensity until an audible vocalization response was heard from the observer . The intensity of the discharge began at 0.05 mA and was increased in increments of 0.05 mA; 15 s elapsed between each download. The two groups of mice analyzed, wt and NPC1nmf164, showed the same sensitivity to electric shocks. On the day of conditioning, the mice were placed individually in the conditioning chamber. After a scan period of 3 min, each mouse was exposed to three tone-discharge pairings on the legs (tone (CS), 30 s; discharge on the legs, 2 s, 0.2 mA at the end of the tone ; separated by an interval between tests of 1 min). One minute after the third discharge on the legs, the mouse was returned to its housing cage. In this session, the walls of the cameras were black, any sound, light or fan was disconnected, the mice were placed directly on the grid floor and 70% ethanol was used for cleaning between animals. The next day (rehearsal day), the contextual test was conducted for conditioned response to fear. The conditioning of fear in the context of the training chamber was evaluated by returning each mouse to the same conditioning chamber and measuring immobility as fear behavior for 6 min without any tone or discharge on the supplied legs. On the third day, the context and manipulation of the mice were changed to assess the fear conditioned solely by the tone: lemon extract was used to perfume the cameras, the floor and the walls of the cameras were replaced with colored plastic covers white, the ventilation fan and the camera light were connected, a background noise of 67 dB was established, CR-36 wall disinfectant was used for cleaning between animals and the test was carried out by a different experimenter . The mice were put in the chambers for 6 min. The immobility was evaluated during a baseline period of 3 min (pre-CS) followed by an additional period of 3 min during the conditioning tone (CS) during which the tone was persistently presented for 3 min. Freezing time and average movement were automatically recorded using commercial software (FREEZINGV1.3, Panlab, Harvard Apparatus). To assess conditioned fear, the percentage of freezing / immobility time in the two test days was calculated. The immobility episode was defined as a complete lack of movement in addition to breathing for at least 2 s.
    [0187]
    [0188] Statistic analysis
    [0189]
    [0190] Statistical analyzes were carried out using GraphPad Prism 5 software (GraphPad Software, USA). Graphics were represented using GraphPad. For comparisons between genotypes and experimental groups, the unpaired two-tailed Student's t-test was used for parametric distribution data. For multiple comparisons, data with a normal distribution were analyzed by two-way ANOVA by a Bonferroni post hoc trial. All statistical comparisons were based on biological replicates and all values were presented as the mean value ± SEM. P values below 0.05 were considered significant and the statistical tests and sample size (n values) that the experiments were used are specified in the legends of the figures. In the figures, asterisks indicate P values as follows: * <0.05; ** <0.01; *** <0.001.
    权利要求:
    Claims (7)
    [1]
    1. Use of a compound of formula (I):

    [2]
    2. Use of a compound according to claim 1 wherein said compound is selected from the following group:
    (-) 6-Chloro-4-cyclopropyletinyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one, (-) 6-chloro-4-phenylethynyl-4-trifluoromethyl-1, 4-dihydro-2H-3,1-benzoxazin-2-one, (+/-) 6-Chloro-4- (2-cyanophenyl) ethynyl-4- (1,1,1-trifluoromethyl) -1,4-dihydro-2H-3,1-benzoxazin-2-one,
    (+/-) 4- (1-Chloro-1,1-difluoromethyl) -4- (2-phenylethynyl) -6-chloro-1,4-dihydro-2H-3,1-benzoxazin-2-one, or
    (+/-) 4- (2- [dimethylaminomethyl] ethynyl) -4-trifluoromethyl-6-chloro-1,4-dihydro-2H-3,1-benzoxazin-2-one,
    or a pharmaceutically acceptable salt thereof.
    [3]
    3. Use of a compound according to claim 2 wherein said compound is (-) 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one or a pharmaceutically acceptable salt thereof.
    [4]
    4. Use of a compound according to any one of claims 1 to 3 wherein the lipid storage disease is selected from Niemann-Pick diseases, Gaucher disease, Fabry disease, Farber disease, Tay disease. Sachs, Sandhoff disease, Krabbe disease, metachromatic leukodystrophy, Wolman disease.
    [5]
    5. Use of a compound according to claim 4 wherein the lipid storage disease is Niemann-Pick type C disease.
    [6]
    6. Use of a compound according to any one of claims 1 to 5 in combination with another active ingredient.
    [7]
    7. Use of a compound according to claim 6 wherein the active ingredient is selected from miglustat, histone deacetylase, arymoclomol, P-cyclodextrin, ursodeoxycholic acid, acetyl-leucine or recombinant acid sphingomyelinase inhibitors.
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    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2008144458A1|2007-05-15|2008-11-27|Z & Z Medical Holdings, Inc.|Dissolution of arterial cholesterol plaques by pharmacologically induced elevation of endogenous bile salts|
    WO2011131943A2|2010-04-20|2011-10-27|Cipla Limited|Pharmaceutical compositions|
    WO2015059466A1|2013-10-25|2015-04-30|Cipla Limited|Pharmaceutical compositions comprising efavirenz|
    US10610564B2|2015-02-26|2020-04-07|Stc.Unm|IRGM and precision autophagy controls for antimicrobial and inflammatory disease states and methods of detection of autophagy|
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