WO2007073687A1 - A g protein-coupled receptor antagonist and its use for preventing and treating alzheimer’s disease - Google Patents
A g protein-coupled receptor antagonist and its use for preventing and treating alzheimer’s disease Download PDFInfo
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- WO2007073687A1 WO2007073687A1 PCT/CN2006/003595 CN2006003595W WO2007073687A1 WO 2007073687 A1 WO2007073687 A1 WO 2007073687A1 CN 2006003595 W CN2006003595 W CN 2006003595W WO 2007073687 A1 WO2007073687 A1 WO 2007073687A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
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Definitions
- G protein-coupled receptor antagonist for preventing and treating Alzheimer's disease and application thereof
- the present invention relates to the prevention or treatment of Alzheimer's disease or related neurological diseases, in particular to a method for screening for a medicament for preventing or treating Alzheimer's disease and /3-adrenergic receptor or opioid receptor The use of antagonists in the treatment of Alzheimer's disease. Background technique
- Alzheimer's disease is characterized by progressive dementia and personality changes and is the most common neurodegenerative disorder associated with aging. Alzheimer's disease affects 5-11% of people over the age of 65 and 30% of those over the age of 85. Alzheimer's disease is caused by abnormal accumulation of amyloid plaques near degenerating neurons and active astrocytes.
- Amyloid plaques are mainly composed of amyloid. It is a neuropathological marker of Alzheimer's disease and its formation is considered to be the main cause of Alzheimer's disease. In addition, recent studies have revealed that diffuse oligomerization of A/S can also be neurotoxic and associated with Alzheimer's disease (Nature 416, 535-9, 2002).
- the ⁇ precursor protein ( ⁇ ) is sequentially formed by sequential cleavage with ⁇ -secretase. As shown in Figure 1, ⁇ -secretase cleaves the ⁇ precursor protein to produce a soluble ⁇ - ⁇ fragment and a C99 fragment, which is subsequently cleaved by ⁇ -secretase to produce a C60 fragment.
- AjS has at least two forms, namely 40 amino acid forms of ⁇ 4 ⁇ and 42 amino acid forms of ⁇ 42 .
- the 42 amino acid form of A ⁇ 42 is more likely to form amyloid plaques and is thought to be more relevant to the etiology of Alzheimer's disease.
- ⁇ -secretase plays a key role in Alzheimer's disease by determining the ratio of the two major ⁇ /3 forms ( ⁇ /3 4() and ⁇ & 2 ).
- the ⁇ -secretase complex includes at least four essential components: Presenilin-1 (PS1), nicastrin (NCSTN), APH-1, and PEN-2.
- PS1 Presenilin-1
- NCSTN nicastrin
- APH-1 APH-1
- PEN-2 a commonly recognized catalytic component
- Alzheimer's disease Although the association between premature protein-1 mutations and familial Alzheimer's disease provides clues to the genetic etiology of Alzheimer's disease, familial Alzheimer's disease accounts for only a deficiency of all Alzheimer's cases. 10%. In contrast, most Alzheimer's disease is sporadic, indicating that factors other than mutations in presenilin-1 are more important in the pathogenesis of Alzheimer's disease. Therefore, it is important to study how various factors or environmental effects contribute to the pathogenesis of Alzheimer's disease.
- a ⁇ produced by in vitro cell culture can be reduced by activating intracellular signaling pathways or membrane receptors such as muscarinic acetylcholine receptors.
- membrane receptors such as muscarinic acetylcholine receptors.
- ⁇ /3 levels and amyloid plaque formation are affected by somatostatin or environmental factors.
- the shear of APP can also be regulated by neurotransmitters and synaptic activity.
- activation of a neurotransmitter receptor associated with phosphatidylinositol hydrolysis or activation of protein kinase C can increase APP metabolism and reduce ⁇ production (Ulus and Wurtman, J. Pharm. Exp. Ther, 281, 149 (1997)).
- U.S. Patent Nos. 6,187,756 and 6,043,224 disclose the use of ⁇ -adrenergic receptor antagonists to modulate cAMP levels to alleviate neuropathic lesions caused by abnormal expression of sputum.
- a ⁇ -adrenergic receptor antagonist is used to inhibit APP synthesis by modulating cAMP levels.
- regulation of APP metabolism can also be used to alleviate neuropathy associated with APP-associated amyloid plaque formation.
- U.S. Patent No. 5,385,915 the disclosure of which is incorporated herein by reference in its entirety, the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire disclosure Regulation of APP cleavage leads to regulation of the production of A ⁇ accumulated in amyloid plaques.
- U.S. Patent No. 5,242,932 discloses the use of compounds such as chlomquine and primaquine to modulate and influence intracellular transport and cleavage of proteins (including APP) in mammalian cells.
- Technical Solution 1 comprises the steps of: (a) activating a receptor and determining its initial degree of endocytosis, said receptor being a G protein-coupled receptor that binds to presenilin-1; (b) in the presence of a candidate agent , activating the receptor as described in (a), again determining the degree of endocytosis of the receptor; (c) determining the difference in the degree of endocytosis in (a) and (b); (d) selecting another if the difference is less than the threshold Repeat steps (a) through (c) for the candidate reagent.
- Technical Solution 2 includes the following steps: (a) measuring the initial degree of binding of the receptor to presenilin-1 or ⁇ -secretase, which is a G protein-coupled receptor that binds to presenilin-1; The degree of binding of the receptor to presenilin-1 or ⁇ -secretase is again measured in the presence of a candidate agent as described in (a); (c) determining the difference in the degree of binding in (a) and (b); (d) Repeat steps (a) through (c) if another difference is selected if the difference is less than a certain threshold.
- At least one of the receptors is selected from the group consisting of a beta adrenergic receptor and a delta-opioid receptor.
- the ⁇ adrenergic receptor may be a ⁇ 2 adrenergic receptor ( ⁇ 2 ⁇ ).
- the receptor is expressed in a cell that has been transfected with a gene encoding the receptor On.
- the activity of ⁇ -secretase in the number of endocytic vesicles, presenilin-1 in endocytic vesicles, late endocytic vesicles and lysosomes is detected or
- the initial degree of endocytosis and the degree of re-endocytosis were determined by the formation of amyloid/3 ( ⁇ ).
- the initial degree of binding and the degree of recombination are determined by detecting fluorescence resonance energy transfer.
- a receptor antagonist for endocytosis of a G protein-coupled receptor which inhibits binding to presenilin-1 during endocytosis is provided for the preparation or treatment of Alzheimer's disease or related Neuropathic drugs.
- an agent for interfering with the binding of a G protein coupled receptor to presenilin-1 or ⁇ -secretase for the preparation of a medicament for treating or preventing Alzheimer's disease or related neuropathy in a third aspect of the invention, there is provided an agent for interfering with the binding of a G protein coupled receptor to presenilin-1 or ⁇ -secretase for the preparation of a medicament for treating or preventing Alzheimer's disease or related neuropathy .
- an antagonist of 5-adrenergic receptor or opioid receptor is provided for the preparation of a medicament for the treatment or prevention of Alzheimer's disease or related neuropathy.
- a ⁇ 2 adrenergic receptor (P2AR) antagonist is provided for the preparation of a medicament for the treatment or prevention of Alzheimer's disease or related neuropathy.
- the antagonist is provided as at least one of ICI 118, 551, propranolol (butoxamine or naltrindole) for the preparation of a treatment or Prevention of Alzheimer's disease or related neuropathy drugs.
- the antagonist is provided as ICI 118, 551 or butoxamine for the preparation of a medicament for the treatment or prevention of "Hertzheimer's disease or related neuropathy.
- Figure 1 shows the process by which ⁇ -secretase and ⁇ -secretase act sequentially on APP to produce ⁇ .
- ⁇ is first cleaved by ⁇ -secretase to produce soluble APPs- ⁇ and C99.
- C99 is then cleaved by ⁇ -secretase to produce ⁇ and C60.
- Figure 2 shows the four major components of ⁇ -secretase: Presenilin, nicastrin (NCSTN), APH-1 and PEN-2.
- Figure 3 shows the endocytic process after endonuclear activation and the transfer of endocytic vesicles to late endosomes and lysosomes (LEL).
- Fig. 4 is a flow chart showing a method of screening for a receptor antagonist for treating or preventing Alzheimer's disease according to the present invention.
- FIG. 5 shows stimulation of G protein coupled receptor (GPCR) to increase the production of A ⁇ in cell lines and primary cultured hippocampal cells.
- GPCR G protein coupled receptor
- Figure 6 shows that stimulation & adrenergic receptors enhance ⁇ -secretase activity.
- (a) Expression substrate method The effects of Iso treatment at different times on C99 cleavage and C60 production in HE 293 cells were examined.
- Figure 7 shows the enhancement of ⁇ -secretase activity caused by ⁇ -opioid receptor activation, and an enhanced time course.
- SH-SY5Y neuroblastoma cells (a) and hippocampal slices (b) showed that the cell membrane fraction separated after treatment with DADLE or NALT for 30 minutes detected ⁇ -secretase activity by a fluorescent substrate method.
- (c) shows the time course of ⁇ 2-adrenergic receptor activation enhancing ⁇ -secretase activity.
- C6 glial cells were treated with Iso for the indicated times. The cell membrane fraction was assayed for gamma-secretase activity using a fluorogenic substrate method.
- Figure 8 shows the correlation between receptor endocytosis and enhanced ⁇ -secretase activity.
- a-e The ⁇ -secretase activity was measured by a fluorogenic substrate method.
- ⁇ 2 adrenergic receptors L339, 340A mutant (&ARLL) and ⁇ 3 adrenergic receptors were not endocytosed (f) or enhanced ⁇ -secretase activity (g) after Iso treatment.
- CTX ligament L toxin
- Fsk forskolin
- db-cAMP dybutyl cyclic adenosine monophosphate
- PTX pertussis toxin
- Dyn K44A dynamin II K44A
- Con A concanavalin A
- Sue sucrose solution
- K+ dpi potassium removal Solution
- NS RNAi non-specific RNA interference
- /3 ⁇ 4ARm /3 ⁇ 4AR
- Iso isoproterenol.
- Figure 9 shows that the enhancement of ⁇ -secretase activity caused by ⁇ -opioid receptor activation cannot be eliminated by pertussis toxin (PTX).
- PTX pertussis toxin
- Figure 10 shows that the HEK293 cells were treated with transferrin as indicated in the intercondylar cells, and the membrane fraction was assayed for gamma-secretase activity using a fluorescent substrate method.
- Figure 11 shows the increase in ⁇ -secretase activity and ⁇ in endocytic bodies.
- c Immune separation of endocytic bodies and lysosomal assays showed that Iso treatment increased A
- Figure 12 shows an increase in ⁇ -secretase activity in endocytic bodies and lysosomes after DALDE activation of ⁇ -opioid receptors.
- Figure 13 shows that enrichment of ⁇ -secretase requires endocytic transport.
- Figure 14 shows ⁇ -secretase activity, ⁇ production and amyloid plaque formation are enhanced in vivo, and ⁇ 2 adrenergic receptor selective antagonist ICI 118,551 effectively inhibits amyloid plaque formation.
- ⁇ -secretase activity (a) and secretory ⁇ 4 ⁇ and ⁇ / 3 ⁇ 4 2 levels in hippocampus after acute treatment with norepinephrine (NE) or clenbuterol (Cle) in rats ) is enhanced (*P ⁇ 0.01).
- NE norepinephrine
- Cle clenbuterol
- c ⁇ -g Amyloid plaques in the brain of APPswe/PSlAE9 transgenic mice were enhanced after chronic treatment with Iso(c), clenbuterol(d) or ICI 118,551(f).
- Panels c, d and f are representative amyloid plaques of female (left) and male (right) mice.
- Figure e is a statistical analysis of mouse amyloid plaques of panels c and d.
- Figure g is a statistical analysis of mouse amyloid plaques of Figure f.
- Figure 15 shows the experimental results of the animal model.
- (a) The results of the visible platform experiment. No effect of genotype or substance on the experiment was found ( ⁇ S.MS-P - OO S ⁇
- Figure 16 shows the experimental results of receptor subtype selective antagonists in animal models.
- Betaxolol is a selective antagonist of the beta-adrenergic receptor that is able to cross the blood-brain barrier.
- ICI 118,551 is a selective antagonist of the ⁇ 2 adrenergic receptor capable of passing the blood-brain barrier.
- FIG 17 shows the experimental results of receptor subtype selective antagonists in animal models.
- Metoprolol is a selective antagonist of the beta-adrenergic receptor that is able to cross the blood-brain barrier.
- Figure 18 shows the results of non-transgenic mice.
- Antagonists of the beta adrenergic receptor have no effect on non-transgenic mice, indicating that transgenes are required.
- (c) There was no drug effect in the test ( ⁇ 0.817).
- Figure 19 shows the results of an animal experiment with the ⁇ -opioid receptor selective antagonist naltrindole.
- Naltrindole is a selective antagonist of ⁇ -opioid receptors that pass the blood-brain barrier.
- (b) In the invisible platform experiment, Naltrindole treatment significantly ameliorated cognitive impairment ( 4.945, corpse ⁇ 0.030).
- Figure 22 shows the percentage of time the mouse stayed in the platform quadrant during the 24 hours after the last invisible platform experiment.
- Figure 23 shows the interaction of cell surface DOR and PS1.
- Figure 23 A HEK293 cells co-transfected with donor GFP-DOR and acceptor HA-PS l (Cy3 fluorescein), before and after fluorescent bleaching with a 561 imi laser (red line) and after ( Mixed emission spectrum of blue line) (excitation wavelength 488 nm). The emission spectrum shows two regions in the same cell that have been fluorescently bleached (left) and not bleached (right). The emission of donor GFP is only increased in the area where the fluorescence is bleached.
- Figure 23B Unmixed GFP-DOR and PSl-Cy3 images before and after acceptor fluorescent bleaching in HEK293 cells.
- Fluorescent bleached areas are shown with white wireframes.
- the pseudo-color magnified at the bottom shows the intensity of GFP emission from the cell surface before and after fluorescent bleaching.
- Cell surface donor GHP-DOR emission is enhanced after fluorescent bleaching of the receptor PS1-Cy3.
- the ruler is 10 /mi.
- Figure 23C shows the average energy transfer efficiency of GFP-DOR and PS1-Cy3 on the cell surface. The number is the number of cells in the experiment. The data was obtained from three independent experiments. detailed description
- the present invention relates to a method for screening for a medicament for preventing or treating Alzheimer's disease or other related neuropathy.
- the invention also relates to the use of an adrenergic receptor or an antagonist of an opioid receptor for the treatment of Alzheimer's disease, in particular an antagonist of the beta-adrenergic receptor and the delta-opioid receptor.
- an antagonist of ⁇ -adrenergic receptors in the treatment of Alzheimer's disease and a new use of an antagonist of ⁇ -opioid receptors in the treatment of Alzheimer's disease.
- the invention also relates to methods for screening for agents that may be used to treat Alzheimer's disease or other related neuropathy. As mentioned earlier, familial Alzheimer's disease accounts for only 10% of all Alzheimer's disease.
- factors other than genetic factors may play an important role in the etiology of Alzheimer's disease.
- Environmental factors such as stress, may act by activating receptors, including beta-adrenergic receptors and delta-opioid receptors in G-protein coupled receptors.
- the central nervous system expresses several G-protein-coupled receptors, especially the ⁇ 2-adrenergic receptors, which are expressed in the hippocampus and cortex, the areas mainly involved in the pathogenesis of Alzheimer's disease. In the central nervous system, these receptors act on the signaling of adrenaline, dopamine, and opioid peptides, causing regulation of a variety of neurological functions, such as stimuli, learning, memory, and pain.
- G protein guanine-binding protein
- cAMP guanine-binding protein
- clathrin-mediated endocytosis occurs in activated receptors, which not only play a key role in signal transduction but also in receptor desensitization.
- G-protein coupled receptors after endocytosis are circulated through early endocytic vesicles, late endocytic vesicles, and lysosomes (LEL).
- the transport of different endocytic vesicles is mediated by Rab GTPase, which itself can also serve as a marker for various vesicles.
- the present invention is based on the inventor's innovative discovery that activation of ⁇ -adrenergic receptors (especially ⁇ 2-adrenergic receptors) or ⁇ -opioid receptors leads to the production of ⁇ -secretase in late endocytic bodies and lysosomes.
- the accumulation has increased.
- ⁇ -secretase is the optimal reaction condition for acid ⁇ . Therefore, the accumulation of ⁇ -secretase in the late acidic endocytic bodies and lysosomes increases, resulting in an increase in activity and an increase in the production of A ⁇ .
- Figure 3 shows the pathway from activation of ⁇ -adrenergic receptors or ⁇ -opioid receptors to increased production of A ⁇ .
- activation of ⁇ -adrenergic receptors and ⁇ -opioid receptors is accompanied by clathrin-mediated endocytosis, including the formation of clathrin-coated pits (CCP) and CCP detachment.
- CCP clathrin-coated pits
- the inventors of the present invention found that the active component of ⁇ -secretase, Presenilin-1 (PS1) constitutively binds to these receptors. As a result of this endocytosis, presenilin-1 or gamma-secretase is brought into endocytic vesicles.
- endocytic vesicles are then transformed into late endocytic bodies and lysosomes (LEL) by Rab5 and Rab7-mediated vesicle trafficking, whereby gamma-secretase activity is enhanced here. Increased ⁇ -secretase activity leads to an increase in ⁇ production.
- LEL late endocytic bodies and lysosomes
- an "antagonist” includes a compound that prevents, reduces or inhibits receptor activation. Such compounds can compete with receptor activators for the same receptor binding site, or bind to different sites on the receptor and reduce the effects of the receptor activator.
- the present invention specifically relates to methods of screening for agents that treat or prevent Alzheimer's disease or related neuropathy.
- the screening method may be based on the ability of the candidate agent to inhibit endocytosis of a receptor that binds to presenilin-1 or gamma-secretase, or based on its ability to attenuate or eliminate binding of the receptor to presenilin-1 or gamma-secretase.
- the method of the invention comprises measuring the presence or absence of a candidate agent in combination with presenilin-1 or gamma-secretase
- the extent of endocytosis of the receptor, or the extent to which the receptor binds to presenilin-1 or gamma-secretase (step 41).
- These receptors include G-protein coupled receptors introduced by endogenous or vector transfection.
- the difference in the degree of endocytosis or binding in the presence or absence of the candidate agent is determined (step 42).
- the degree of endocytosis can be measured by endocytosis vesicles, endocytosed presenilin-1 or ⁇ -secretase, increase in presenilin-1 or ⁇ -secretase in LEL, and ⁇ -secretase activity in LEL. Enhancement or increase in ⁇ production is achieved.
- the extent to which the receptor binds to the presenilin-1 or gamma-secretase can be determined by any suitable method, such as fluorescence resonance energy transfer (FRET), which will be described in detail below.
- FRET fluorescence resonance energy transfer
- the candidate agent may be used to treat or prevent Alzheimer's disease or related neuropathy (as shown in step 43). ). If the difference is not significant, the previous step can be repeated with another candidate reagent (as shown in step 44). Note that the method illustrated in Figure 4 is shown sequentially, with one candidate reagent being tested each time, and those skilled in the art would be willing to test multiple reagents simultaneously, such as by using a multiwell plate or other high throughput method.
- the degree of re-endocytosis of the G-protein coupled receptor and presenilin 1 in the cell is statistically lower than the initial endocytosis (preferably significantly lower)
- the degree of re-endocytosis of G-protein coupled receptor and presenilin 1 is 60% or less of the initial endocytosis, indicating significant difference.
- the degree of initial endocytosis refers to the degree of endocytosis that is activated by the use of the candidate agent and is determined by the degree of endocytosis.
- the degree of endocytosis is the degree of endocytosis that is activated by the use of the candidate agent in the same test system.
- the present invention specifically relates to the treatment or treatment of Alzheimer by administering to the human body an effective amount of an antagonist that binds to a ⁇ -adrenergic receptor (especially a ⁇ 2-adrenergic receptor) and/or a delta-opioid receptor.
- an effective dose of the antagonist is sufficient to reduce receptor endocytosis that results in the transport of gamma-secretase to late endocytic bodies and lysosomes.
- the present invention also relates to the use of an antagonist that binds to ⁇ -adrenergic receptors (especially ⁇ 2-adrenergic receptors) and ⁇ -opioid receptors for the production of a medicament for the treatment or prevention of Alzheimer's disease and other neuropathic diseases.
- ⁇ -adrenergic receptors especially ⁇ 2-adrenergic receptors
- ⁇ -opioid receptors for the production of a medicament for the treatment or prevention of Alzheimer's disease and other neuropathic diseases.
- the effective dose of an antagonist that binds to a beta-adrenergic receptor and/or a delta-opioid receptor will depend on the mode of administration of the drug to the patient, the frequency of administration, and the components of the drug, as well as the patient's weight, sex, age and body. situation. Typically, effective doses can range from 1 ⁇ g/kg body weight to 10 mg/kg body weight per day. Although individual differences exist, one skilled in the art will be able to determine the optimal effective dose for each component. Administration to a patient can be by any suitable route of administration of a similar agent, including oral, injection, transdermal Ite, and the like.
- the compounds or components of the invention may be used to treat Alzheimer's or other related neuropathies in a mammal (human or other mammal).
- These compounds or components include pharmaceutically acceptable carriers and/or excipients such as physiological saline, buffer, dextrose, glycerol, ethanol, starch, and the like.
- these compounds or components can be prepared in dosage forms commonly used in similar pharmaceuticals, including injections, tablets, capsules, patches, and the like. The preparation of these dosage forms is known in the art.
- ⁇ 293 cells have a functional G protein-coupled receptor signaling pathway and are capable of normally secreting ⁇ /3.
- the ⁇ 293 cells used in this experiment were transfected with ⁇ 2-adrenergic receptors and sputum mutants (APPswe;).
- the mutant APPswe is a "Swedish” mutation in familial Alzheimer's disease, a mutation at the 670th and 671th codons.
- C99 ⁇ -secretase substrate
- ⁇ 2-adrenergic receptor Experiments in which HEK293 cells were co-transfected with ⁇ -secretase substrate (C99) and ⁇ 2-adrenergic receptor further confirmed that ⁇ -secretase is involved in the increase in ⁇ /3 production by ⁇ 2-adrenergic receptors.
- C99 is a product of /3-secretase-mediated sputum shear (see Figure 1).
- C99 is a direct substrate for gamma-secretase and a direct precursor for ⁇ .
- Figure 5b shows that stimulation of ⁇ 2-adrenergic receptors with Iso in co-transfected HEK293 cells resulted in an increase in A ⁇ production and was comparable to the previously described cells co-transfected with APPswe and ⁇ 2-adrenergic receptors. Again, this increase can be eliminated by Pro, which has no effect in itself. Therefore, the increase in secreted A/3 production is due to an increase in the activity of ⁇ -secretase.
- ⁇ -adrenergic receptors especially ⁇ 2-adrenergic receptors
- ⁇ -opioid receptors potentiates the production and secretion of A ⁇ , due to the ⁇ -secretase to C99 (or similar substrate) Caused by shear enhancement.
- Activation of ⁇ 2-adrenergic receptors increases ⁇ -secretase activity
- the increase in the production of A ⁇ caused by activation of the ⁇ -adrenergic receptor or the ⁇ -opioid receptor described above may be caused by an increase in the expression level or activity of the ⁇ -secretase.
- the inventors examined the effect of ⁇ 2-adrenergic receptor activation on the expression level and activity of ⁇ -secretase. As shown in Figure 6a, HEK293 transfected with C99 in Iso treatment In the cell, the production of C60 is increased. C60 is produced by C99 mediated by ⁇ -secretase. However, the same treatment did not result in any change in the expression level of PS1.
- PS1 is the active site subunit of ⁇ -secretase, present as a heterodimer of amino and carbon-based fragments (ie, PS1-NTF and PS1-CTF). This result suggests that after activation of the ⁇ 2-adrenergic receptor, the activity of ⁇ -secretase is increased and the expression of ⁇ -secretase is not changed.
- a fluorogenic substrate was used to directly measure the enzyme activity of ⁇ -secretase.
- This fluorogenic substrate is based on a ⁇ -secretase-specific substrate sequence to which a fluorescent reporter molecule is attached.
- the endogenous ⁇ -adrenergic receptor of C6 glioblastoma was stimulated for 30 minutes and the activity of ⁇ -secretase was enhanced (Fig. 6b). This effect was verified in hippocampal slices (Fig. 6c).
- the increase in ⁇ -secretase activity caused by Iso after the absence of presenilin in mouse embryonic fibroblasts was confirmed, and the specificity of this method for ⁇ -secretase activity was verified (Fig. 6d). Taken together, these results show that activation of the ⁇ 2-adrenergic receptor stimulates ⁇ -secretase activity, resulting in increased production of ⁇ /3.
- Activation of the receptor to enhance ⁇ -secretase activity is not limited to the ⁇ -adrenergic receptor. Similar results were obtained with endogenous ⁇ -opioid receptors that stimulated SH-SY5Y neuroblastoma (Fig. 7a) or cultured primary hippocampal cells (Fig. 7b). Further, detection of the activity of ⁇ -secretase showed that the activity of ⁇ -secretase reached a maximum at about 30 minutes of stimulation of the ⁇ 2-adrenergic receptor, and returned to the background level at about 60 minutes (Fig. ⁇ ).
- Example 3 Enhanced ⁇ -secretase activity independent of cAMP signal
- G protein-coupled receptors including ⁇ 2-adrenergic receptors
- ⁇ 2-adrenergic receptors once activated, induce activation of Gs protein-dependent adenylate cyclase, resulting in elevated intracellular cAMP levels.
- a mutant of the ⁇ 2-adrenergic receptor that does not activate the Gs protein 03 ⁇ 4AR T68F, Y132G, Y219A, or ⁇ , was used in subsequent experiments. 2 ⁇ ⁇ ). This mutant can not be eliminated ⁇ - enhanced secretion activity (FIG.
- the cAMP signaling pathway is not involved in enhancing ⁇ -secretase activity and may also be true for ⁇ -opioid receptors.
- the delta-opioid receptor is known to activate the pertussis toxin ( ⁇ ) sensitive Gi/o protein and to reduce cAMP levels by inhibiting adenylate cyclase.
- Pretreatment with SH-SY5Y neuroblastoma with pertussis toxin did not alter ⁇ -secretase activity induced by DADLE stimulation (Fig. 9). This result shows that ⁇ -secretase activity caused by activation of the ⁇ -opioid receptor is not regulated by cAMP.
- GPCR including beta-adrenergic receptors and opioid receptors
- Figure 8c shows that the effect of Iso on gamma-secretase activity can be abolished by pretreatment with endocytic inhibitors such as concanavalin (Con A), high osmolality (Sue) and potassium-free solution (K + dpi).
- Figure 8d shows that Iso-induced enhancement of ⁇ -secretase activity can be abolished by Dynham's dominant negative mutant Dyn K44A, which is capable of inhibiting clathrin or caveolin-mediated endocytosis. Since ⁇ 2-adrenergic receptors are mainly endocytosed by clathrin-mediated mechanisms, small interfering RNAs against clathrin heavy chains can be used to remove intracellular clathrin expression.
- Figure 8e shows that Iso-induced enhancement of ⁇ -secretase activity can be abolished by RA interference (RNAi).
- endocytic vesicles are transported to their destination through various specific endocytic pathways. These endocytic pathways involve Rab guanosine triphosphatase (Rab GTPase) regulated intracellular vesicle transport. It is now known that endocytic transport from the cell membrane to the early endocytosis and continued to endocytosis and lysosomes can be labeled with Rab5 and lysosomal-labeled Rab7, the dominant negative mutant Rab5, respectively. S34N and Rab7 T22N inhibition.
- Rab GTPase Rab guanosine triphosphatase
- the Flag antibody was used to immunoprecipitate vesicles of late endocytic bodies and lysosomes from cells transfected with Flag-Rab7, followed by early endocytosis
- the marker EEAl early endosome antigen 1
- the late endocytotoxin and lysosome-labeled LAMP-1 lysosome-associated membrane protein-1 confirmed the components.
- Figure 11c late endocytic bodies and lysosomes were found in the ⁇ 2-adrenergic receptor after 1 hour of stimulation.
- Figure 12 shows that ⁇ -secretase activity in late endocytic bodies and lysosomes is also enhanced after ⁇ -opioid receptor activation.
- the experiment in Figure 12 was performed by stimulating SH-SY5Y neuroblastoma with 1 ⁇ DADLE for 30 minutes, then separating the subcellular fractions, and then using the components for alkaline phosphatase (AP) and fluorogenic substrate. analysis. The results showed that DADLE treatment only enhanced the ⁇ -secretase activity of the alkaline phosphatase cell fraction (*P ⁇ 0.01).
- Dyn K44A and Rab5 S34N effectively prevented the localization of PS1 in late endocytic bodies and lysosomes after ⁇ 2-adrenergic receptor stimulation (Fig. 13a), suggesting that PS1 may be small from the cell membrane to late endocytosis. Body and lysosomal transport.
- 0-adaptin which is capable of labeling clathrin-coated sags and vesicles, was found to stimulate ⁇ -opioid receptors in HEK293 cells 3 minutes later; PS1 and 0-adaptin and endocytic receptors form colocalization (Fig. 13b).
- PS1 and activated ⁇ 2-adrenergic receptors or ⁇ -opioid receptors form co-endocytosis after agonist stimulation.
- This result is not surprising because PS1 is capable of constitutively binding membrane proteins, such as ⁇ and Notch, and ⁇ 2-adrenergic receptors are capable of mediating endocytosis of other transmembrane proteins by forming heterodimers.
- the binding of PS1 to the ⁇ 2-adrenergic receptor or the ⁇ -opioid receptor was examined by immunoprecipitation.
- An agent capable of eliminating or attenuating the binding of presenilin 1 to the receptor can be screened by any suitable method, such as fluorescence resonance energy transfer (FRET).
- Fluorescence resonance energy transfer is the process by which energy transfers from a close-range (O nm) donor fluorescein to a receptor luciferin. Therefore, this technique can be used to detect proteins that are physically bound together.
- energy transfer non-radioactive energy transfer reduces the fluorescence emission of the donor. Therefore, energy transfer is detected by comparing the intensity of the fluorescence emission of the donor before and after the same sample is destroyed by the receptor fluorescein (e.g., the method of fluorescent bleaching). If energy transfer occurs, the fluorescent emission of the donor is enhanced after the fluorescent bleaching receptor.
- GFP-labeled ⁇ -opioid receptor GFP-DOR
- presenilin 1-Cy3 changes in energy transfer efficiency between green fluorescent protein (GFP)-labeled ⁇ -opioid receptor (GFP-DOR) and presenilin 1-Cy3 were detected before and after fluorescent bleaching in co-transfected HEK293 cells.
- the cells were co-transfected with GFP-tagged ⁇ -opioid receptor (GFP-DOR) and HA-labeled presenilin 1 (HA-PS1).
- the expression of HA-PS1 was detected by the first antibody of HA and the second antibody (Jackson ImmunoResearch) linked to Cy3 fluorescein, and the expression of GFP-DOR was detected by GFP fluorescein.
- Figures 23A-23C show one of the results of such an experiment. Images were acquired using a Leica confocal microscope, including a mixed emission spectrum of the donor GFP-DOR and the receptor PS1-Cy3 fluorescein (488 nm laser excitation). It was first tested whether the intensity of donor (GFP-DOR) emission in HEK293 cells co-expressing two proteins was enhanced after fluorescent bleaching of the receptor (PSl-Cy3).
- Figure 23A shows images of fluorescent bleached and non-fluorescent bleached areas of cells before and after fluorescent bleaching. Selected areas were bleached with a 561 nm laser within the Cy3 absorption spectrum.
- the corresponding intensity of the GFP-DOR emitted light is greatly enhanced in the fluorescently bleached region compared to the non-bleached region of the same cell. This result shows that energy transfer occurs between GFP and Cy3, and this energy transfer can be eliminated or reduced by fluorescent bleaching of Cy3.
- Figure 23B shows representative images of GFP-DOR and PSl-Cy3 in transfected HEK293 cells before and after 561 nm laser fluorescence bleaching of the receptor.
- the GFP-DOR image shows that the fluorescent emission of the receptor (pseudo-color intensity image) is enhanced after fluorescent bleaching, and this enhancement occurs only in the intracellular fluorescent bleaching region.
- the average relative energy transfer efficiency of GFP-DOR and PS1-Cy3 near the cell surface was about 22.8 ⁇ 3.9%, showing the interaction between the two.
- cells expressing GFP-DOR were incubated with a GFP primary antibody and a secondary antibody to which Cy3 was ligated. The energy transfer efficiency of this positive control was 27.9 ⁇ 5.3%.
- Cells expressing GFP-DOR were incubated with a primary antibody against the non-specific protein actin and a secondary antibody ligated with Cy3 as a negative control. The energy transfer efficiency of this negative control was 7.8 ⁇ 4.7%.
- Example 8 Enhancement of ⁇ -secretase activity in animals, A/3 production and formation of amyloid plaques
- ⁇ 2-adrenergic receptors The effects of ⁇ 2-adrenergic receptors on these Alzheimer's disease-related molecules were further investigated in animals.
- In vivo experiments in rats showed acute injection of the adrenergic receptor endogenous ligand norepinephrine (NE) or the ⁇ 2-adrenergic receptor selective agonist d e nbuteml (Cle), the rat hippocampal ⁇ Both secretase activity (Fig. 14a) and ⁇ levels (Fig. 14b) were significantly elevated. Based on these results, long-term administration of these receptor agonists to animal models can be expected to treat pathological changes that may exacerbate Alzheimer's disease.
- NE adrenergic receptor endogenous ligand norepinephrine
- d e nbuteml the rat hippocampal ⁇ Both secretase activity (Fig.
- APPswe/PSlAE9 mice and non-transgenic (NTg) littermates were grouped by sex and age.
- the test compound is administered orally, starting from the age of 4 months and lasting for 1 or 2 months.
- the effects of various compounds on APPswe/PSlAE9 mice and non-transgenic mice were evaluated using the Morris water maze experiment.
- the Morris water maze experiment was developed by neuroscientist ichard G. Morris in 1984 and is now a commonly used method for studying the role of hippocampus in spatial memory formation.
- the labyrinth in the experiment was a circular pool (1.2 m in diameter) filled with 24-25 "C water, and milk powder was added to make the water opaque.
- a fixed space indication was placed around the pool, including the curtains with the bold pattern and the obvious The shelf of the object.
- the mouse was carefully faced to the pool wall to the water. The mouse first received a certain amount of visible platform training (ie, two consecutive days, eight times a day), learning to swim to a pole marked elevation
- the circular platform (10 cm in diameter) is visible.
- the visible platform training is divided into two groups per day (ie, four trainings per group) for statistical data analysis. It can be seen that during the platform training, the platform position of each training (Northeast, Southeast) , Southwest or Northwest) and the starting position (East, South, West or North) are pseudo-random decisions.
- the invisible platform training was also carried out for a certain number of days (i.e., 6 consecutive days, four times a day), during which the mice were allowed to find a platform placed 1.5 cm below the surface of the water. If the mouse cannot find the platform within 60 seconds, it will be directed to the platform.
- the position of the platform remained unchanged, and the mice entered the pool in a direction randomly selected from four directions (east, south, west, or north). After each training session, the mice were left on the platform for 30 seconds before being transferred from the platform to the cage.
- APPswe/PSlAE9 transgenic mice and non-transgenic mice were administered propranolol and nadolol to evaluate ⁇ -adrenergic receptor antagonists.
- the effect on amyloid plaque formation APP S we/PSlAE9 transgenic mice and non-transgenic mice were grouped by sex and age matching. The compound is administered orally, from 4 months of age to 6 months. Propranolol easily crosses the blood-brain barrier and, therefore, antagonizes the ⁇ -adrenergic receptors of the central nervous system.
- nadolol is also an antagonist of the ⁇ -adrenergic receptor, but does not pass the blood-brain barrier.
- the mice were then subjected to spatial learning and memory testing using the Morris water maze test.
- mice were then subjected to six days of invisible platform training and a set of training sessions were performed each day.
- propranolol is able to antagonize the ⁇ -adrenergic receptors of the central nervous system. Since propranolol is a non-selective ⁇ -adrenergic receptor antagonist, it is further possible to study which subtype in vivo can cause the above effects by using a subtype-selective ⁇ -adrenoreceptor antagonist.
- the subtype-selective ⁇ -adrenergic receptor antagonists used include Betaxolol (betaxolol) and ICI 118, 511. Betaxolol is a beta ⁇ -adrenergic receptor capable of passing the blood-brain barrier.
- ICI 118,511 is a ⁇ 2-adrenergic receptor capable of passing the blood-brain barrier.
- FIGs 17a-17d show animal experiment results similar to Figure 16.
- Metoprolol is a beta-adrenergic receptor antagonist that is able to cross the blood-brain barrier.
- Butoxamine is a beta2-adrenergic receptor antagonist that is able to cross the blood-brain barrier.
- the ⁇ -adrenergic receptor antagonist mainly targets the ⁇ -adrenergic receptor of the central nervous system to achieve a decrease in ⁇ -secretase activity and ⁇ production. Further, most of the required relief of spatial memory deficits can be achieved by inhibiting the ⁇ 2-adrenergic receptors, while inhibiting the ⁇ -adrenergic receptors from acting. Therefore, according to the object of the present invention, a ⁇ -adrenergic receptor antagonist for controlling or treating Alzheimer's disease should be more likely to act on the ⁇ 2-adrenergic receptor.
- Figures 19a-19c show animal experiments similar to Figure 15, but using ⁇ -opioid receptor antagonists ⁇
- Naltrindoleo Naltrindole is a ⁇ -opioid receptor antagonist that passes through the blood-brain barrier.
- mice 4 months old APPswe/PSlAE9 double transgenic mice were given normal saline (Sal), 2 mg/kg cl e nbuterol (/3 ⁇ 4-adrenergic receptor agonist) or 1 mg/kg ICI 118. , 551 (/3 ⁇ 4-adrenergic receptor antagonist) 30 days later, spatial learning and memory were examined using the Morris water maze test (described above).
- the labyrinth in the experiment was a circular pool (1.2 m in diameter) filled with water at 24-25 °C, and milk powder was added to make the water opaque. Place a fixed space indication around the pool, including a curtain with a bold pattern and a shelf with visible objects.
- the mice were carefully faced to the pool wall to the water.
- the mice were first trained on a number of visible platforms (i.e., two consecutive days, eight times a day) and learned to swim on a raised circular platform (10 cm in diameter) marked with a pole. It can be seen that the platform training is divided into two groups per day (ie, four trainings per group) for statistical data analysis. It can be seen that during the platform training, the platform position (Northeast, Southeast, Southwest or Northwest) and the starting position (East, South, West or North) are pseudo-randomly determined.
- Invisible platform training was continued for 6 days (4 trainings per day) and the mice were looking for a platform 1.5 cm below the surface of the water. If the platform cannot be found within 60 seconds, the mouse is led to the platform.
- the position of the platform remains unchanged, and the mouse enters the pool from one of the four directions (east, south, west, or north) in a pseudo-randomly selected direction.
- the mice were left on the platform for 30 seconds before being transferred from the platform to the cage.
- Figure 21 shows the escape time of the control panel, clenbuterol treatment, and ICI 118,551 treated double transgenic and non-transgenic mice in an invisible platform experiment of the Morris water maze experiment.
- Figure 22 shows the percentage of time the mouse stayed in the platform quadrant during the 24 hours after the last invisible platform experiment.
- RNAi plasmid of the human clathrin heavy chain was designed as follows: 5'-GCTGGGAAAACTCTTCAGATT-3 '.
- the control (NS) RNAi plasmid was 5 '-GGCCGCAAAGACCTTGTCCTTA-3. Animal and drug treatment
- mice purchased from The Jackson Laboratory
- cannula front-back, -0.6 mm; left-right, -1.2 mm; dorsal-ventral, -1.8 mm
- mice were perfused with normal saline after anesthesia.
- the brain was isolated and the hemisphere was fixed with 4% polycarboxylic acid for 5 hours at 4 °C.
- the half brain was sliced into 10 micrometers. Sections were incubated with A/3 antibody 6E10 and incubated with TRITC-linked secondary antibodies. Sliced After observation with a laser confocal microscope (Leica TCS SP2). The area of amyloid plaques was counted using Image-Pro Plus 5.1 software (Media Cybernetic). Hippocampus culture and acute section preparation
- Primary hippocampal culture was prepared from newborn SD rats and electroporated with Amaxa Nucleofector system.
- B27/neurobasal medium (Invitrogen) was used for agonist treatment experiments two weeks later. Acute hippocampal slices were prepared using 8 week old SD rats. Enzyme-linked immunosorbent assay (ELISA) amyloid beta
- the cells were treated with Iso or DADLE for 1 hour and continued for 6 hours.
- Medium for enzyme-linked immunosorbent assay kit
- ⁇ 293 cells or rat hippocampal slices were lysed in RIPA buffer.
- Flag-tagged receptors and endogenous ⁇ -opioid receptors were immunoprecipitated with agarose beads or ⁇ -opioid receptor antibodies (Santa Cruz Biotech) linked to the Flag antibody.
- the immunoprecipitated complex was separated by SDS-PAGE and detected by immunoblotting.
- the experimental procedure is as described in Journal of Biological Chemistry 276, 481-7 (2001). After lysis of HEK293 cells, the cells were divided into aliquots containing 50 ⁇ g of total protein and centrifuged at 13, 000 X g for 15 minutes. The cell membrane fraction was resuspended and incubated with 50 ⁇ l of the reaction buffer containing the protease inhibitors 1, 10-phenoline (plienanthroline), aprotinin and leupeptin, and incubation at 37 °C. hour. After the completion of the reaction, the C60 produced in the membrane fraction was detected by immunoblotting. Fluorescent substrate method
- HEK293 cells co-transfected with HA-C99 and DOR were starved for 2 hours with medium containing no methionine and serum (Invitrogen), and 500) tiCi [ 35 S] methionine (GE Healthcare) Pulsed for 1 hour with or without DADLE stimulation. The cells were then followed for 3 hours in medium containing excess methionine. C99 in the cell lysate was immunoprecipitated with HA antibody and analyzed by autoradiography. Immune separation of late endocytic bodies and lysosomes
- HEK293 cells transfected with HA-labeled receptors and/or GFP-Rab7 or GFP-Rab7 T22N were first incubated with HA antibody for 30 minutes, then treated with an agonist and fixed.
- cells were treated with agonists and fixed.
- the sections were first treated with Iso or DADLE and then fixed and sectioned.
- HEK293 cells were co-transfected with GFP-DOR and HA-PS1.
- HA-PS1 expression was detected with a primary antibody to HA and a second antibody to Cy3 fluorescein
- GFP-DOR expression was detected by GFP fluorescence.
- the fluorescence spectrum of the cells of GFP-DOR or PSl-Cy3 was acquired in ⁇ mode using a 488 nm laser.
- acceptor fluorescent bleaching was performed using Leica software. The selected cell surface region was fluorescently bleached with 561 nm laser for Cy3 fluorescein.
- the Cy3 signal in HEK293 cells co-transfected with GFP-PS1 and HA-PS1 decreased by an average of 84 ⁇ 5.3% (n 50) after fluorescent bleaching.
- the energy transfer is expressed as an increase in the GFP-DORCi consensus signal after PSl-Cy3 (receptor) fluorescent bleaching.
- the relative energy efficiency is calculated as (l-[Cy3 I pre-bleaching/Cy3/after bleaching]) X 100%. Energy transfer analysis was performed on the cell surface without fluorescent bleaching as a control. data analysis
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US12/159,183 US8026073B2 (en) | 2005-12-26 | 2006-12-26 | Screening G protein-coupled receptor antagonists for methods of treating Alzheimer's disease |
EP06840640A EP1967527A4 (en) | 2005-12-26 | 2006-12-26 | G PROTEIN-COUPLED RECEPTOR ANTAGONIST AND USE THEREOF IN THE PREVENTION AND TREATMENT OF ALZHEIMER'S DISEASE |
CN200680049242.3A CN101346396B (zh) | 2005-12-26 | 2006-12-26 | 预防和治疗阿尔兹海默症的g蛋白偶联受体拮抗剂及其应用 |
JP2008547831A JP2009521238A (ja) | 2005-12-26 | 2006-12-26 | アルツハイマー病を予防および治療するためのgタンパク質共役レセプターアンタゴニスト及びその使用 |
US13/214,907 US20120004254A1 (en) | 2005-12-26 | 2011-08-22 | G protein-coupled receptor antagonist and its use for preventing and treating alzheimer's disease |
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US8129334B2 (en) * | 2006-03-31 | 2012-03-06 | The Regents Of The University Of California | Methods and compositions for treating neurodegenerative disorders and Alzheimer'S disease and improving normal memory |
WO2007123680A2 (en) | 2006-03-31 | 2007-11-01 | The Regents Of The University Of California | Methods and compositions for treating neurodegenerative disorders and alzheimer's disease and improving normal memory |
US8647887B2 (en) * | 2009-01-29 | 2014-02-11 | Commonwealth Scientific And Industrial Research Organisation | Measuring G protein coupled receptor activation |
CN104220630B (zh) | 2012-02-23 | 2017-03-08 | 特来德斯通技术公司 | 耐腐蚀且导电的金属表面 |
AU2013204332B2 (en) * | 2012-04-16 | 2015-07-16 | Commonwealth Scientific And Industrial Research Organisation | Methods and systems for detecting an analyte or classifying a sample |
KR101743960B1 (ko) | 2015-07-06 | 2017-06-08 | 서울대학교산학협력단 | G단백질 결합형 수용체19 작용제를 유효성분으로 함유하는 알츠하이머 질환 또는 치매를 예방, 치료 또는 지연하기 위한 약학적 조성물 |
CN111773386A (zh) * | 2019-04-03 | 2020-10-16 | 中国科学院深圳先进技术研究院 | 阿尔兹海默症治疗靶点 |
GB201916561D0 (en) * | 2019-11-14 | 2020-01-01 | Vib Vzw | Gamma-secretase inhibitor screening assay |
JP2023507408A (ja) * | 2019-12-18 | 2023-02-22 | キュラセン セラピューティクス インコーポレイテッド | 神経系疾患および障害を改善するための方法 |
CN113332432A (zh) * | 2020-03-03 | 2021-09-03 | 复旦大学 | 防治老年性痴呆的新途径 |
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WO2003088924A2 (en) * | 2002-03-19 | 2003-10-30 | Duke University | Phosphoinositide 3-kinase mediated inhibition of gpcrs |
-
2006
- 2006-11-17 CN CNA2006101624807A patent/CN1991364A/zh active Pending
- 2006-12-26 JP JP2008547831A patent/JP2009521238A/ja active Pending
- 2006-12-26 EP EP06840640A patent/EP1967527A4/en not_active Withdrawn
- 2006-12-26 US US12/159,183 patent/US8026073B2/en not_active Expired - Fee Related
- 2006-12-26 WO PCT/CN2006/003595 patent/WO2007073687A1/zh active Application Filing
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US6043224A (en) | 1996-09-05 | 2000-03-28 | The Massachusetts Institute Of Technology | Compositions and methods for treatment of neurological disorders and neurodegenerative diseases |
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US20080312332A1 (en) | 2008-12-18 |
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US8026073B2 (en) | 2011-09-27 |
JP2009521238A (ja) | 2009-06-04 |
EP1967527A4 (en) | 2010-03-10 |
US20120004254A1 (en) | 2012-01-05 |
CN1991364A (zh) | 2007-07-04 |
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