CN101618214A - Medicine target point for treating friable X syndrome and application thereof in pharmacy - Google Patents

Abstract

The invention relates to a novel medicine target point for treating friable X syndrome and an application thereof in pharmacy. The research discovers that the function of the FMR1 gene of a friable X syndrome model animal for knocking and removing a rat central nerve dopamine 1 type receptor (D1R) is lowered, and simultaneously discovers that the distribution of G-protein coupled receptor kinase-2 (GRK2) is abnormal, the expression of the GRK2 on a cell membrane is increased, and the GRK2 in cytoplasm is reduced. These pathological changes participate in the onset process of the friable X syndrome. A pharmacological method is adopted, an abdominal cavity injection D1R receptor excitant and a GRK2 suppressor are solely used or united, and the praxiology symptom of the friable X syndrome model animal can be obviously improved. The invention provides the novel medicine target point for treating the friable X syndrome and can develop novel treating medicines according to the target point, and the developed novel medicines not only can be solely used but also can be united to use.

Description

The drug target of treatment fragile X syndrome and the application in pharmacy thereof

Technical field

The present invention relates to treat the drug target of fragile X syndrome and the application in pharmacy thereof, specifically, nervus centralis dopamine 1 receptor (D1 receptor) function reduces and/or the active unusual increase of G-albumen Rhizoma Nelumbinis connection receptor kinase-2 (GRK2) can be used as the drug target of treatment fragile X syndrome, and can be used for preparing the medicine for the treatment of fragile X syndrome.

Technical background

Fragile X syndrome is modal heritability mental retardation disease.Among per approximately 2000 male of its sickness rate or 4000 women people morbidity is arranged, clinical manifestation mainly is mental retardation (IQ is 40-60).According to conservative estimation, China has 400,000 fragile X syndrome patients at least.Fragile X syndrome is to be caused lacking in the body its coded product FMRP protein to cause by the sudden change of FMR1 (Fragile X Mental Retardation 1) gene, lack FMRP in the body and finally cause mental retardation, thereby FMRP is regarded as a kind of intelligence albumen.Protein synthesis synaptic plasticity particularly after-stage plasticity (late-phaseLTP) play a very important role in forming, FMRP is that a kind of mRNA relevant with ribosome is conjugated protein, the gathering of participation mRNA is also regulated and control the efficient of transcribing of target gene.The long time journey strengthens (LTP) and is widely regarded as the cytology basis that Learning and Memory forms.Studies show that,, can induce normal LTP, obviously strengthen but the long time journey suppresses (LTD) in fragile X syndrome model FMR1 knock out mice Hippocampus CAi zone.Have research to think, 1 group of receptor of metabotropic glutamate (Gp1 mGluR) is owing to the inhibition that loses FMRP is regulated and control, and activity obviously increases and causes LTD enhancing, enhanced LTD to cause the synapse growth to slow down.Therefore, Gp1 mGluR signal excessively or unusually may be cognitive disorder, top epilepsy, hypoevolutism and the stunted reason of synapse that causes fragile X syndrome.Yet, still be in the starting stage as the research of drug target.Up to the present, the treatment fragile X syndrome does not still have active drug, and the medicine of seeking efficient high specificity becomes the urgent task of current medicine circle.

Summary of the invention

The object of the present invention is to provide a kind of drug target of the treatment fragile X syndrome that actual application value is arranged newly, and use it for the medicine of development of new treatment fragile X syndrome.

The invention provides a kind of treat fragile X syndrome drug target, this target spot is that nervus centralis d1 dopamine receptor function reduces and/or active unusual the increasing of G-albumen Rhizoma Nelumbinis connection receptor kinase-2.

Can develop the medicine of treatment fragile X syndrome according to this target spot.This medicine is central dopamine D1 receptor stimulating agent and/or G-albumen Rhizoma Nelumbinis connection receptor kinase-inhibitor 2, can be micromolecular compound, Chinese medicine, plant amedica extract, effective site or effective ingredient, nucleic acid class such as RNAi, polypeptide class or protein drug can use or adopt composition forms to comprise that the associating other drug uses separately.

Central dopamine D1 receptor stimulating agent of the present invention and G-albumen Rhizoma Nelumbinis connection receptor kinase-inhibitor 2 can use separately separately, also can the medication combined use of two classes, all have the effect of treatment fragile X syndrome.

Treatment of the present invention is formed, the pharmacokinetics, pharmacodynamics, mode of administration, the route of administration that comprise certain drug according to different situations, character, degree and the treatment time etc. of receptor's age, body weight, hepatic and renal function state, disease are with the appropriate dosage administration.

The invention has the beneficial effects as follows, utilize central dopamine D1 receptor stimulating agent or G-albumen Rhizoma Nelumbinis connection receptor kinase-inhibitor 2, can improve the behavior performance of fragile X syndrome animal effectively, demonstrate the using value of treatment fragile X syndrome.Both further illustrated the pathogenesis of fragile X syndrome, the treatment for fragile X syndrome provides new treatment target spot again.

Description of drawings:

In order to make the present invention more clear, further specify as follows in conjunction with the accompanying drawings:

Fig. 1 FMR1 knock out mice synaptic plasticity reduces.Figure 1A, 1B shows: the effect of strengthening synapse long time journey enhancing (LTP) behind the exciting D1 receptor of SKF81297 (all is abbreviated as Fmr1 at the FMR1 knock out mice among the figure ^-/-) disappear.Fig. 1 C, 1D shows: the ACC neuron of cultivating, adopt the exciting D1 receptor of SKF81297, relatively glutamic acid AMPA GluR1 recipient cell after birth transhipment level and GluR1 receptor serine site phosphorylation level find that FMR1 knock out mice neuron significantly reduces than normal mouse neuron.

Fig. 2 adenylate cyclase activity reduces.The ACC neuron of cultivating, the exciting D1 receptor of SKF81297, cause that adenylate cyclase activity significantly raises at intact animal's cell, but at the FMR1 knock out mice, adenylate cyclase activity and the proteic ability of activation G α s reduce at the FMR1 knock out mice.

Fig. 3 cell membrane GRK2 obviously increases.At the ACC neuron of cultivating, the GRK2 in the FMR1 knock out mice cytoplasm shifts unusually to cell membrane.

Fig. 4 striatal neuron D1 receptor and GRK2 expression.At the striatal neuron of cultivating, behind the exciting D1 receptor of SKF81297, FMR1 knock out mice neuron AC is active and activation G α s is proteic can significantly reduce by force rate intact animal cell, and the GRK2 in the cytoplasm shifts unusually to cell membrane.

The effect of Fig. 5 D1 receptor stimulating agent and GRK2 inhibitor.ACC neuron cultivating uses D1 receptor stimulating agent SKF81297 or GRK2 inhibitor heparin separately, or unites use, all can make phosphorylation AMPAGluR1 expression of receptor increase on the FMR1 knock out mice cell membrane.

The therapeutical effect of Fig. 6 D1 receptor stimulating agent.At the FMR1 knock out mice, lumbar injection D1 receptor stimulating agent SKF81297 (1mg/kg) can significantly suppress FMR1 knock out mice over-activity symptom, and at normal mouse, medicine does not then make significant difference to motion.

The specific embodiment:

Further specify the present invention in the following embodiments, this does not limit the scope of the invention.

Embodiment 1: with D1 receptor and GRK2 is the research of medicine novel targets

Materials and methods

1. laboratory animal

Grow up (6-8 week) FMR1 knock out mice and wild type normal control Mus (FVB.129P2-Fmr1tm1Cgr kind system) available from U.S. Jacksen laboratory.Raise in constant temperature, constant humidity, illumination in 12 hours, freely drink water in the animal housing pellet nursing.All experimental arrangements meet the The Fourth Military Medical University animal and use the regulation with administration committee.

2. the full cell patch of brain slice of growing up clamps record

Mice is adopted the 1-2% isoflurane anesthesia, and vibratome laterally cuts 300 μ m ACC brain sheets, places gaseous mixture (95%O under the room temperature ₂And 5%CO ₂) in the saturated artificial cerebrospinal fluid (ACSF), recover to experimentize behind the 1h.ACSF forms (in mM): NaCl 124; NaHCO ₃25; KCl 2.5; KH ₂PO ₄1; CaCl ₂2; MgSO ₄2; Glucose 10.The brain sheet is positioned among the record Xiao Chi during record, ACSF circulation perfusion, and gaseous mixture is saturated, record under the room temperature.Full cell excitement postsynaptic currents (EPSC) and LTP adopt U.S. Axon 200B amplifier record, and medicine adds from perfusate.When inducing LTP, add 100 μ M picrotoxin in the ACSF perfusate and be used to block GABA _AThe inhibition transmission of mediation.Clamp down on voltage-70mV, record excitatory postsynaptic current (EPSC), intensity is 50-80pA, frequency 0.02Hz, the beginning electricity irritation is induced behind the record 10min baseline.The LTP abductive approach: stimulating electrode is positioned over ACC V layer, stimulates for 80 times, and frequency 2Hz, neuron is clamped down in+30mV simultaneously, returns to the baseline logging mode then, continuous record 30min.

3. former generation neuron cultivation

Be born back (P0) mice 3-5 on the same day only, get brain after the crymoanesthesia of ice face, isolate frontal lobe (prefrontal cortex), cut ACC zone cortex, use 0.125%trypsin (Invitrogen, CA) digestion 15min after the mechanical dispersion.Cell is pressed 3-4 * 10 ³Cells/cm ²Density is planted in poly and is relied on the acid-treated coverslip of peace, uses the Neurobasal-A culture medium, and adding B27 and 2mM GlutaMax (Invitrogen, CA).37 ℃ in CO ₂Cultivate in the incubator, changed liquid once in per 4 days, 2-3 experimentizes after week.

4.Western trace detects

The Tris buffer cracking of neuron of cultivating or cerebral tissue, the Tris buffer consists of 10mM Tris-HCl, 2mM EDTA, 1%SDS, 1X protease inhibitor, pH=7.4.The Bradford method is extracted total protein, respectively gets protein extract after quantitatively to transfer to same concentrations, carries out the SDS-polyacrylamide gel electrophoresis, nitrocellulose filter after a resistive connection closes 1 hour, washs 3 times after shifting, again with two anti-labellings 1 hour, ECL system colour developing then, the exposure of X sheet.

5. behavioristics's experiment

According to traditional research method, adopt the spontaneous activity in mice instrument to detect spacious autonomic activities behavior of mice.The prologue portable laboratory is open active box (43.2 * 43.2 * 30.5cm; MED-associates, St.Albans, VT), and after the mouse peritoneal administration 45 minutes, be put in one by one in the experimental provision, take the photograph and write down operating range in each mice 30 minutes, computer automatic analysis mutually automatically.

The result

1.FMR1 knock out mice ACC area L TP and cell membrane GluR1 receptor

Brain slice's patch-clamp detects cingule gyrus cortex LTP and finds, the intact animal, obviously strengthen the effect that synapse long time journey strengthens (LTP) behind the exciting D1 receptor of SKF81297, and at the FMR1 knock out mice, exciting D1 receptor then do not have obvious LTP occur (accompanying drawing 1A, 1B); The ACC neuron of cultivating, adopt the exciting dopamine D 1 receptor (D1 receptor) of SKF81297, relatively glutamic acid AMPA GluR1 recipient cell after birth transhipment level (accompanying drawing 1C) and GluR1 receptor serine site phosphorylation level (accompanying drawing 1D) find that FMR1 knock out mice neuron significantly reduces than normal mouse neuron.Think at present, the ability of the height indirect reaction synaptic plasticity (as LTP) of GluR1 receptor serine site phosphorylation level and film transhipment, and LTP is considered to one of cytological mechanism of most important brain learning and memory.

2.FMR1 knock out mice ACC zone adenyl cyclase (AC) activity

The D1 receptor is the G-protein coupling receptor, and behind the exciting D1 receptor of SKF81297, adenyl cyclase (AC) is active significantly to raise at intact animal's cell, but at the FMR1 knock out mice, AC activity then raise not significantly (accompanying drawing 2A); Further experiment is found, mainly is because the proteic ability of D1 receptor activation G α s reduces (accompanying drawing 2B) at the FMR1 knock out mice.Therefore, D1 receptor activation AC ability reduces at the FMR1 knock out mice.

3.GRK2 expression at ACC

G-albumen Rhizoma Nelumbinis connection receptor kinase-2 (GRK2) is present in the cytoplasm, and it can cause the D1 receptor phosphorylation after cell membrane shifts, thereby regulates the D1 function of receptors.Experiment finds that the GRK2 Tot Prot does not have significant difference (accompanying drawing 3A) in FMR1 knock out mice and the normal mouse cell.But owing to lack the proteic inhibitory action of FMRP, GRK2 is active to be occurred unusually, and the GRK2 that shows as in the cytoplasm shifts (accompanying drawing 3B) unusually to cell membrane.On the cell membrane too much GRK2 cause the excessive phosphorylation of D1 receptor, thereby cause that FMR1 knock out mice D1 function of receptors is unusual.

4. adenyl cyclase (AC) and GRK2 are in striatal expression

In another important area striatum relevant with the dopamine receptor function, similar variation is found in experiment in brain, and promptly behind the exciting D1 receptor of SKF81297, FMR1 knock out mice neuron AC specific activity intact animal cell is significantly reduced (accompanying drawing 4A).Behind the exciting D1 receptor of SKF81297, activate the proteic ability of G α s and significantly reduce (accompanying drawing 4B) at the FMR1 knock out mice.In addition, the GRK2 in the FMR1 knock out mice striatum neurocyte slurry shifts (accompanying drawing 4C) unusually to cell membrane.On the cell membrane too much GRK2 cause the excessive phosphorylation of D1 receptor, cause that the D1 function of receptors is unusual, may be the unusual reason of fragile X syndrome animal movement.

5. pharmaceutical research result

At the discovery of above-mentioned pathological research, adopt pharmacology's means, design has the medicine of therapeutical effect targetedly, by the cell signalling process that dysregulation changes, reaches the purpose of Drug therapy.At first, on the isolated cells model, use D1 receptor stimulating agent SKF81297 or associating GRK2 inhibitor heparin to act on the FMR1 knock out mice neuron of cultivation separately, all certain degree improve the D1 function of receptors, make phosphorylation AMPA GluR1 expression of receptor increase (accompanying drawing 5A) on the cell membrane, GluR1 receptor total protein is expressed on the cell membrane also increases (accompanying drawing 5B).AMPA GluR1 expression of receptor increases on the cell membrane, is the committed step that LTP expresses, and prompting has the improvement effect to the Learning and Memory ability.

6. mice behavioristics experimental result

At last, in FMR1 knock out mice integral level, observe the symptom (accompanying drawing 6) whether medicine can treat FMR1 knock out mice over-activity.Experiment is found, inject D1 receptor stimulating agent SKF81297 (1mg/kg) and GRK2 inhibitor heparin (1mg/kg) simultaneously at mouse peritoneal, can significantly suppress FMR1 knock out mice over-activity symptom, as seen mice total move distance in different time obviously reduces, and at normal mouse, medicine does not then make significant difference to motion.

Conclusion

Adopt transgenic animal to experimental studies have found that nervus centralis d1 dopamine receptor dysfunction and GRK2 abnormal activation may be one of fragile X syndrome pathomechanisms; Behavioristics discovers, unites and uses D1 receptor stimulating agent and GRK2 blocker, can improve the clinical manifestation of fragile X syndrome.

Embodiment 2: be the research of medicine novel targets with the D1 receptor

Research method is with embodiment 1.D1 receptor stimulating agent SKF81297 lumbar injection is selected in the research of mice behavioristics.

The result: mouse peritoneal injection D1 receptor stimulating agent SKF81297 (1mg/kg) can significantly suppress FMR1 knock out mice over-activity symptom, and visible mice total move distance in different time obviously reduces, and normal mouse movement is not made significant difference.

Conclusion: behavioristics discovers, adopts the D1 receptor stimulating agent, can improve the clinical manifestation of fragile X syndrome.

Embodiment 3: be the research of medicine novel targets with GRK2

Research method is with embodiment 1.GRK2 inhibitor heparin lumbar injection is selected in the research of mice behavioristics.

The result: mouse peritoneal injection GRK2 inhibitor heparin (1mg/kg) can significantly suppress FMR1 knock out mice over-activity symptom, and visible mice total move distance in different time obviously reduces, and normal mouse movement is not made significant difference.

Conclusion: behavioristics discovers that unusual GRK2 activity in the block nerves cell can be improved the clinical manifestation of fragile X syndrome.

Claims (5)

1. Nervus centralis dopamine 1 receptor and G-albumen Rhizoma Nelumbinis the connection receptor kinase-the 2nd, the treatment fragile X syndrome the medicine novel targets, be used for the treatment of fragile X syndrome.
2. 2. according to claims 1, the medicine of treatment fragile X syndrome is dopamine 1 receptor agonist and G-albumen Rhizoma Nelumbinis connection receptor kinase-inhibitor 2.
3. 3. according to claims 2 described central dopamine 1 receptor agonist, can be micromolecular compound, Chinese medicine, plant amedica extract, effective site or effective ingredient, nucleic acid class such as RNAi, polypeptide class or protein drug.
4. 4. according to claims 2 described G-albumen Rhizoma Nelumbinis connection receptor kinase-inhibitor 2, can be micromolecular compound, Chinese medicine, plant amedica extract, effective site or effective ingredient, nucleic acid class such as RNAi, polypeptide class or protein drug.
5. 5. according to claims 2 described central dopamine 1 receptor agonist and G-albumen Rhizoma Nelumbinis connection receptor kinase-inhibitor 2, can use separately, also can the medication combined use of two classes.

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