CN108938648A - Cholesteric -3,6- diketone is used for the treatment of neure damage - Google Patents
Cholesteric -3,6- diketone is used for the treatment of neure damage Download PDFInfo
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Abstract
The invention discloses the application of cholesteric -3,6- diketone, its deuterated object or its pharmaceutically acceptable salt in the drug of the neure damage of preparation treatment patient.Present invention demonstrates cholesterics -3,6- diketone can reduce the hippocampal neurons oxidativestress damage as caused by glutamic acid, can reduce the damage of apoptosis of rat cerebellar granule neurons caused by potassium is deprived, can reduce the damage of cortical neuron nerve excitability caused by glutamic acid, so as to for treating neure damage.
Description
Technical field
The present invention relates to the new medical uses of cholesteric -3,6- diketone, and in particular to cholesteric -3,6- diketone is in neure damage
Application in treating.
Background technique
Cholesteric -3,6- diketone is a kind of cholesterol derivative, it is known that synthetic method using cholesterol as raw material, can be by more
Kind synthetic method obtains (Zhang Weiet al, 2014；R Martinez-Pascualet al, 2017；JGSC
Buchanan et al, 1969).Low in raw material price, synthesis is simple and convenient, and druggability is good.
The activity research of the compound mainly has at present: (1) AKR1B10 enzyme inhibitor acts on；(2) antibacterial activity；(3)
Reduce heart rate activity；(4) anti-tumor activity (Zhang Weiet al, 2014；Zakariaet al, 2011；Zhang
Zhaohui et al, 1999; Shi Dayong et al, 2011).So far there has been no about the compound in nerve
The report in protection field.The report of the compound granted patent is had no at present.In addition, the compound carries out on smooth muscle cell
Experiment finds no cytotoxicity (Peng Shikaung, 1979).It is expected that once developing patent medicine, toxic side effect is opposite
It is less.
The research and development of Neuroprotective Agents are always clinical and basic research emphasis, prepare and lead on neuroprotection cell model
Different Primary cultured neurons damage models is often selected according to different purposes.
HT-22 cell strain is that hippocampus of mice primary neuron is obtained by transformation, can express cholinergic neuron marker,
Have many advantages, such as that form similar with normal hippocampal neurons and physiological function, incubation are simple, can pass on.Glutamic acid lures
The HT-22 cellular damage led includes meronecrosis, apoptosis and death etc., and there are a variety of explanation (Fukui M in molecular mechanismet al, 2009), the model is a kind of generally acknowledged neuron oxidativestress damage model (Breyer A at presentet al, 2007)。
Meanwhile oxidativestress damage runs through the ischemia and reperfusion overall process of cerebral apoplexy, oxidative stress is that research and development multiple target point nerve is protected
The important target spot of agent is protected, which has been widely used for the research of anti-oxidative damage neuroprotective agent.
Neuron outer ion intracellular maintains a certain concentration gradient under normal circumstances, is to maintain neuron depolarising and repolarization
It is necessary.When ischemic, Neuron Apoptosis around a large amount of infarcts causes penumbra region and constantly becomes infarcted region.Neuron
Apoptosis is important target spot (the Alhadidi Q for studying cerebral apoplexy neuroprotective agentet al, 2016).With the KCl's containing 25mM
BME culture medium culture cultured rat cerebellar granule neuron handles 24 hours by the BME of 5mM KCl, can establish stable cerebellum
Granule Neurons Apoptosis Model, the model have been widely used for anti-apoptotic neuroprotective agent research (Dragotto Jet al,
2015).
Excitatory toxicity is one of important pathomechanism of cerebral apoplexy, and in cerebral arterial thrombosis, neuron discharges excessive paddy ammonia
Acid, simultaneously because energy supply obstacle, the function of spongiocyte reabsorption glutamic acid are obstructed, cause aminoglutaric acid concentration excessively high, cause mind
Through excitatory toxicity (Kritis A A et al, 2015).The nerve excitability of glutamate-induced is cerebral apoplexy neuroprotection
Important target spot (Chamorro áet al, 2016).Neuronal excitability toxic model has been widely used for neuroprotective agent and grinds
Study carefully, we carry out drug neuroprotective activity research on rat cerebral cortex neuron.
Summary of the invention
The present inventor has been surprisingly found that it is thin that cholesteric -3,6- diketone can reduce the hippocampal neural as caused by glutamic acid
Born of the same parents' oxidativestress damage can reduce the damage of apoptosis of rat cerebellar granule neurons caused by potassium is deprived, can reduce glutamic acid and cause
The damage of cortical neuron nerve excitability, so as to have for developing into a variety of effects for treating neure damage
The potential of the neuroprotective agent of mechanism.
An aspect of of the present present invention provides cholesteric -3,6- diketone, its deuterated object or its pharmaceutically acceptable salt and controls in preparation
Treat the application in the drug of the neure damage of patient.In some embodiments, the neure damage is by oxidative stress
The damage that damage, Neuron Apoptosis or neuronal excitability toxicity mediate.In some embodiments, the neure damage medicine
Object is degenerative disease.Preferably, the degenerative disease includes that Parkinson's disease, alzheimer's disease, Protein tau disease or flesh wither
Contracting lateral schlerosis.In some embodiments, the neure damage is headstroke or cerebral injury.In some embodiments
In, the drug also includes another therapeutic agent.In some embodiments, the patient is people.
Another aspect of the present invention provides a kind of method of neure damage for treating patient, and this method includes to the patient
Apply a effective amount of cholesteric -3,6- diketone, its deuterated object or its pharmaceutically acceptable salt or comprising cholesteric -3,6- diketone, its
The pharmaceutical composition of deuterated object or its pharmaceutically acceptable salt.In some embodiments, the neure damage is by oxygen
Change the damage that stress damage, Neuron Apoptosis or neuronal excitability toxicity mediate.In some embodiments, the neuron
Damage is degenerative disease.Preferably, the degenerative disease includes Parkinson's disease, alzheimer's disease, Protein tau disease or flesh
Amyotrophic lateral sclerosis disease.In some embodiments, the neure damage is headstroke or cerebral injury.In some embodiments
In, the patient is people.
Another aspect of the invention provides cholesteric -3,6- diketone, its deuterated object or its pharmaceutically acceptable salt for suffering from
The treatment of the neure damage of person.In some embodiments, the neure damage is withered by oxidativestress damage, neuron
Die or neuronal excitability toxicity mediate damage.In some embodiments, the neure damage is degenerative disease.It is excellent
Selection of land, the degenerative disease include Parkinson's disease, alzheimer's disease, Protein tau disease or amyotrophic lateral sclerosis.One
In a little embodiments, the neure damage is headstroke or cerebral injury.In some embodiments, the patient is people.
Another aspect of the present invention provides a kind of method of neure damage that patient is mitigated or eliminated, the method includes
A effective amount of cholesteric -3,6- diketone, its deuterated object or its pharmaceutically acceptable salt are applied to the patient or comprising cholesteric -3,
The pharmaceutical composition of 6- diketone, its deuterated object or its pharmaceutically acceptable salt.In some embodiments, the neuron damage
Wound is caused by being mediated as oxidativestress damage, Neuron Apoptosis or neuronal excitability toxicity.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range.
Fig. 1: neuroprotection of the cholesteric -3,6- diketone to Pidolidone induction HT-22 cellular oxidation stress damage.
(A) cellular morphology figure, bar=100 μm Scale；Cell survival rate histogram that (B) mtt assay measures (Glu:0.171 ±
0.014；MB:0.708 ± 0.020；Steroidal: 0.660 ± 0.036；n=4；P<0.001 VS. Glu)；(C) cholesteric -3,6- two
The EC of ketone50Figure.
Fig. 2: the neuroprotection of the damage of apoptosis of rat cerebellar granule neurons caused by cholesteric -3,6- diketone deprives potassium.
(A) cellular morphology figure, 100 μm of Scale bar；Cell survival rate histogram that (B) mtt assay measures (5K:0.326 ±
0.010, steroid:0.449 ± 0.017, * * *P< 0.001 VS.5K, n=3)；(C) Hoechst33342 colored graph, Scale
bar 50μm；(D) apoptotic cell percentage histogram (5K:0.545 ± 0.036, steroid:0.314 ± 0.040, * * *P<
0.001 Vs.5K, n=5)。
Fig. 3: the neuroprotection of cholesteric -3,6- diketone cortical neuron excitatory toxicity caused by glutamic acid.(A)
Cellular morphology figure, 100 μm of Scale bar；Cell survival rate histogram that (B) mtt assay measures (Glu:0.338 ± 0.022,
MK801:0.930 ± 0.068, steroid:0.862 ± 0.047, * * *P< 0.001 VS.Glu, n=5).
Specific embodiment
As used herein, term " composition " refers to the preparation for being suitable for reaching therapeutic purposes to expected animal target application,
It contains at least one medicine activity component, such as compound.Optionally, the composition also contains at least one pharmaceutically
Acceptable carrier or excipient.
Term is " pharmaceutically acceptable " to indicate that the substance does not have such characteristic, the i.e. disease in view of will be treated
Disease or illness and respective administration method, the Medical practitioners that it is careful which will make rationality avoid taking the object to patient
Matter.For example, usually requiring that such substance is substantially sterile for injectable objects.
Herein, the amount of term " therapeutically effective amount " and " effective quantity " expression substance and substance is for preventing, subtracting
One or more symptoms that are light or improving disease or illness, and/or it is effective for extending the survival of the object of receiving treatment.
" treatment " used herein includes the compound or its pharmaceutically acceptable salt for giving the application, to mitigate disease
Or the symptom or complication of illness, or eliminate disease or illness.Terms used herein " mitigation " are used to describe the sign of illness
Or the process that the seriousness of symptom reduces.Symptom can reduce without eliminating.In one embodiment, the medicine of the application is given
Compositions cause to eliminate sign or symptom.
Cholesteric -3,6- diketone, its deuterated object and pharmaceutically acceptable salt
Cholesteric -3,6- diketone is being herein also referred to as " steroidal ", " steroid " or " the compound of the present invention ", structural formula such as formula (I)
It is shown.
(Formulas I)
The compound of the present invention can be configured to the form of pharmaceutically-acceptable salts or the form for pharmaceutically-acceptable salts.
Expected pharmaceutically acceptable salt form includes, but are not limited to the salt such as single, double, triple, four.Pharmaceutically-acceptable salts are at them
It is nontoxic under the amount and concentration that are administered.In the case where not preventing it from playing physiological effect, by the object for changing compound
Characteristic is managed, the preparation of such salt can be in order to pharmacology application.In physical property useful change include reduce fusing point with
Just mucosal, and increase solubility to apply the drug of higher concentration.
Pharmaceutically acceptable salt includes acid-addition salts, such as those containing sulfates, chloride, hydrochloride, rhizoma corydalis
Hydrochlorate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, mesylate, second sulphur
Hydrochlorate, benzene sulfonate, tosilate, cyclamate and quinate salt.Pharmaceutically acceptable salt can be with
It is obtained from acid, acid such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, third
Diacid, methanesulfonic acid, ethanesulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid, cyclohexylsulfamic acid, fumaric acid and chinic acid.
In the presence of acidic functionality such as carboxylic acid or phenol, pharmaceutically acceptable salt also includes base addition salts, such as that
Contain tardocillin, chloroprocanine, choline, diethanol amine, ethanol amine, tert-butylamine, ethylenediamine, meglumine, Proca a bit
Cause, aluminium, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine and zinc salt.Such salt can be prepared using suitable corresponding alkali.
By standard technique, pharmaceutically acceptable salt can be prepared.For example, the compound of free alkali form is dissolved in
In suitable solvent, such as in the aqueous solution containing suitable acid or water-alcohol solution, then evaporates solution and separated.Another
In one example, salt is prepared by reacting free alkali and acid in organic solvent.
Thus, for example, institute can be prepared by any suitable method available in the field if specific compound is alkali
The pharmaceutically acceptable salt needed, for example, handle free alkali with inorganic acid or organic acid, the inorganic acid for example hydrochloric acid, hydrobromic acid,
Sulfuric acid, nitric acid, phosphoric acid and similar acid, the organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, third
Ketone acid, oxalic acid, glycolic, salicylic acid, pyranose thuja acid (pyranosidyl acid) such as glucuronic acid or galacturonic acid,
'alpha '-hydroxy acids such as citric acid or tartaric acid, amino acid such as aspartic acid or glutamic acid, aromatic acid such as benzoic acid or cinnamic acid, sulfonic acid
Such as p-methyl benzenesulfonic acid or ethanesulfonic acid or the like.
Equally, pharmaceutically acceptable needed for being prepared by any appropriate method if specific compound is acid
Salt, for example, with inorganic base or organic alkali process free acid, the inorganic base or organic bases such as amine (primary amine, secondary amine or uncle
Amine), alkali metal hydroxide or alkaline earth metal hydroxide or the like.The illustrative example of suitable salt includes organic salt,
It is derived from amino acid (such as L- glycine, L-lysine and L-arginine), ammonia, primary amine, secondary amine and tertiary amine and cyclammonium (such as
Hydroxyethyl hydroxyethyl, piperidines, morpholine and piperazine) and inorganic salts, derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminium and
Lithium.
The pharmaceutically acceptable salt of compound can be used as complex compound presence.The example of complex compound includes 8-Chlorotheophyline network
Object is closed (to be similar to, for example, dramamine: diphenhydramine 8-Chlorotheophyline (1:1) complex compound；Dimenhydrinate) and it is various comprising cyclodextrin
Complex compound.
Present invention also contemplates that including the pharmaceutically acceptable deuterated compound or other on-radiations using the compound
Substituted compound.Deuterated is that one or more of pharmaceutical activity molecular radical or whole hydrogen are substituted for isotope deuterium, because of it
Non-toxic and non-radioactive, and stablize about 6 ~ 9 times than C-H bond, the half-life period for being metabolized site and extending drug can be closed, to drop
Low therapeutic dose, while not influencing the pharmacological activity of drug again, and it is considered as a kind of excellent method of modifying.
Pharmaceutical composition
In the present invention, " pharmaceutical composition " refers to the composition comprising cholesteric -3,6- diketone and pharmaceutically acceptable carrier,
Wherein compound and pharmaceutically acceptable carrier are present in composition in hybrid form.The composition will generally be used for
The treatment of human subjects.However, they can also be used to treat the similar or identical illness in other animal targets.
Herein, term " object ", " animal target " and similar terms refer to people and non-human vertebrate, such as mammal, such as
Non-human primates, sport animals and commercial animal, for example, horse, ox, pig, sheep, rodent and pet (such as dog and
Cat).
Suitable dosage form is partly dependent on the approach of purposes or administration, for example, orally, percutaneously, transmucosal, sucking or logical
Cross injection (parenteral).Such dosage form should enable the compound reach target cell.Other factors are known in the art
, including consideration, such as toxicity and delay compound or composition play the dosage form of its effect.
Carrier or excipient can be used to produce composition.The carrier or excipient can be selected as promoting chemical combination
The administration of object.The example of carrier includes calcium carbonate, calcium phosphate, various sugared (such as lactose, glucose or sucrose) or starch
Type, cellulose derivative, gelatin, vegetable oil, polyethylene glycol and physiological compatibility solvent.The example of physical compatibility solvent
Including water for injection (WFI) sterile solution, salting liquid and glucose.
The component of composition or composition can be applied by different paths, including in intravenous, peritonaeum, subcutaneous, flesh
Interior, oral, transmucosal, rectum, percutaneous or sucking.In some embodiments, it is preferred injection or freeze drying powder injection.Counterpart
For clothes, for example, compound can be configured to conventional oral dosage formulations, such as capsule, tablet and liquid preparation, such as sugar
Slurry, elixir and inspissated drops.
The pharmaceutical preparation of oral use can be obtained, such as by combining composition or its component with solid excipient,
Optionally grinding is formed by mixture, and the mixture of processing particle (is such as needed) after suitable adjuvant is added, thus
Obtain tablet or dragee.Suitable excipient is in particular, filler is for example sugared, including lactose, sucrose, mannitol or sorb
Alcohol；Cellulose preparation, such as cornstarch, wheaten starch, rice starch, potato starch, gelatin, gum tragacanth, methyl are fine
Tie up element, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose (CMC) and/or polyvinylpyrrolidone (PVP: povidone
(povidone)).If desired, disintegrating agent can be added, for example, crosslinking polyvinylpyrrolidone, agar or alginic acid or they
Salt, such as mosanom.
Alternatively, it can be used injection (parenteral administration), such as intramuscular, intravenous, in peritonaeum and/or skin
Under.For injection, composition of the invention or its component are configured to sterile aqueous solutions, preferably in PHYSIOLOGICALLY COMPATIBLE
In buffer or solution, such as saline solution, Hank solution or Ringer solution.In addition, composition or its component can be matched
It is made as solid form, and a moment is redissolved or is suspended before the use.Freeze-dried powder form can also be produced.
Administration can also pass through transmucosal, part or transcutaneous modalities.For transmucosal, locally or percutaneously, be formulated
It is middle to use the penetrating agent for being suitble to barrier to be penetrated.Such penetrating agent is generally known in the art, including, for example,
For mucosal, bile salt and fusidic acid derivatives.In addition, detergent can be used for promoting to penetrate.Mucosal,
For example, nose spray or suppository (per rectum or vagina) can be passed through.
The effective quantity of various components to be administered, for example described compound of the factor of consideration can be determined by standardization program
IC50, biological half-life of the compound, object age, size and weight and illness related with object.These factors
Importance with other factors is well known for those of ordinary skill in the art.In general, dosage will be treated
The about 0.01mg/kg of object is between 50mg/kg, preferably in 0. lmg/kg between 20mg/kg.Multiple agent can be used
Amount.
Composition of the invention or its component can also be used in combination with the other therapeutic agents for the treatment of same disease.This knot
Conjunction use is included in different time and applies these compounds and one or more other therapeutic agents, or uses this chemical combination simultaneously
Object and one or more other therapeutic agents.It in some embodiments, can be to one or more compounds of the invention or combination
The dosage of the other therapeutic agents used is modified, for example, being reduced by methods known to those skilled in the art relative to list
The dosage of the compound or therapeutic agent that solely use.
It is to be understood that be used in combination or combination include be used together with other therapies, drug, medical procedures etc., wherein
Other therapies or program can be different from the time of composition or its component of the invention (for example, (such as several small in a short time
When, such as 1,2,3,4-24 hours) in a long time (such as 1-2 days, 2-4 days, 4-7 days, 1-4 weeks) or with group of the invention
It closes object or its component identical time is administered.Combined use further includes and therapy or medical procedures primary or infrequently apply
(as performed the operation) is used together, and short before or after other therapies or program with composition or its component of the invention
Application in phase or longer period.In some embodiments, the present invention is for delivering composition or its component of the invention
With one or more other drugs therapeutic agents, they are delivered by identical or different administration route.
The combination application of any administration route include by identical administration route by composition of the invention or its component and
One or more other drugs therapeutic agents are delivered together with any dosage form, including two kinds of compounds are chemically connected and they
The preparation of respective therapeutic activity is kept in application.In one aspect, the other drugs therapy can with composition of the invention or
Its component is co-administered.Combined use by co-administration includes applying preparation (co-formulation) altogether or chemically connecting
The preparation of the compound connect, or in a short time (for example, in hour, in 2 hours, in 3 hours, until in 24 hours) application
The compound of two or more independent formulations forms, they are administered with identical or different approach.
The co-administration of independent formulations include via the co-administration of the delivering of a device, such as identical suction apparatus,
Identical syringe etc., or applied in a short time by different device relative to each other.The change of the invention delivered by identical administration route
The total preparation for closing object and one or more additional medicinal treatments includes preparing material together to which they can be filled by one
It sets and is administered, including different compound combinations are in a kind of preparation or compound is modified so that they connect in chemistry
It is connected together but still keeps respective biological activity.This compound chemically connected may include by two active constituents point
The connector opened, the connector maintain substantially in vivo, or in vivo may degradation.
Embodiment
Protective effect of the 1. cholesteric -3,6- diketone of embodiment in glutamate induction HT-22 oxidativestress damage model
Middle ware passes on 3 HT-22 cells after taking liquid nitrogen to recover, and uses preceding observation cell state.If cell space is mellow and full full, dash forward
Touching is complete, does not almost have dead cell, then digests half a minute with the pancreatin that 0.5ml concentration is 0.25%, dispel into single cell suspension, receive
Collect 15ml centrifuge tube, add complete medium to 3ml, 1000rpm is centrifuged 3min, removes supernatant, it is complete that precipitating is dispersed in 4ml
Full DMEM culture medium (DMEM+10% FBS+1% P/S) takes 0.8ml cell suspension to be used to pass on, then appropriate suspension is taken to be added to
In complete DMEM culture medium, it is diluted to 4*104Cells/ml is planted in 48 orifice plates (200 hole μ l/), for real after 24 hours
It tests.
Cholesteric -3,6- diketone is dissolved in DMSO first, makes its final concentration of 10mM as medicine storage liquid, by the medicine
Object stock solution dilutes 1000 times with complete DMEM culture medium, makes 10 μM of drug test concentration.Do following packet transaction: a)
Control group: cell full dose changes complete DMEM culture medium；B) model group (Glu): cell full dose tape swapping has the complete of solvent (DMSO)
Full DMEM culture medium；C) medicine group (Steroid): cell full dose tape swapping has the complete DMEM culture medium of 10 μM of steroidals；D) positive
Medicine group (methylenum careuleum MB): cell full dose tape swapping has the complete DMEM culture medium of 0.2 μM of positive drug (MB).
Full dose, which is changed, states 4 groups of solution, and after being incubated for half an hour, glutamic acid (Glu), which is added, makes final concentration of 3mM.Then, 5%
CO2Concentration cultivates 24 hours under 37 DEG C of constant temperature, observes cellular morphology under phase contrast microscope, take pictures (Figure 1A).Then, every hole
The MTT solution (PBS preparation) that 20 μ l concentration are 5mg/ml is added, is incubated for 3 hours, removes supernatant, every hole adds the DMSO of 200 μ l, gently
Light concussion 5min is completely dissolved to solid, then surveys the light absorption value in each hole under 490nm wavelength with microplate reader, takes the flat of three holes
Mean value carries out cell survival rate calculating, cell survival rate=ODEach concentration/ ODcontrol* 100%, take four batches of cell data for uniting
Meter analysis (as shown in Figure 1B).And find out the EC of cholesteric -3,6- diketone50(Fig. 1 C).
The result shows that medicine group and positive drug group are compared with the control group, cytomorphology difference is little, and form
It is consistent with MTT test result to learn observation result.MTT test result shows that the cell survival rate of model group is substantially reduced, and gallbladder
Steroid -3,6- diketone significantly improves the cellular damage as caused by oxidative stress, and improves the survival rate of HT-22 cell.
Guarantor in apoptosis of rat cerebellar granule neurons damage model caused by 2. cholesteric -3,6- diketone of embodiment is deprived in potassium
Shield effect
The rat for taking out raw 7 days is cut off, then after cutting head with major operation with small cut from vertebral foramen toward left-right ear upper limb of ophthalmology
Brain upper layer above is removed with tweezers, exposes brain tissue, cerebellar tissue is taken out and is put into be equipped with and work as dissection liquid without phenol red DMEM(
With) the middle ware being placed on ice bag in.
After removing supracerebellar meninx and blood vessel with ophthalmic tweezers, cerebellar tissue is shredded with tissue shear.Tissue is transferred to dense
Degree adds full BME culture medium to 15ml inward, is and then added 100 μ l's in 0.25% pancreatin, 37 DEG C digest 15 minutes
Dnase I (8mg/ml) dispels tissue mass with dropper, and subsequent 1000rpm is centrifuged three seconds, transfer upper layer suspension to another centrifugation
Pipe, 1000rpm are centrifuged 5 minutes, and removal supernatant obtains cell, inward plus the complete BME culture medium of 5ml, gently blow and beat cell to equal
Even (about 30 times).It is counted with cell counter, adjustment cell concentration to 5*105A/ml.
By cell seeding to PLL(L- poly-D-lysine) on the culture plate that was coated with, aqueous that week makes a circle plus sterilized
Envelope, is placed in 37 DEG C, 5%CO2, 95% humidity incubator in cultivate, after 24 hours be added Ara-C make its final concentration of 10 μM.
Neuron culture for testing, does following packet transaction: 25K group: thin on the 8th day to the 7th day plus glucose to 5mM
Born of the same parents' full dose changes the complete BME culture medium of the KCl containing 25mM；5K group: cell full dose changes the complete BME culture medium of the KCl containing 5mM；Steroid
Body group (steroid): cell full dose changes+10 μM of steroidals of complete BME culture medium of the KCl containing 5mM.
After above-mentioned processing 24 hours, phase contrast microscope observe cellular morphology (Fig. 2A), with mtt assay survey cell survival rate come
The ability (Fig. 2 B) that drug promotes neuronal survival is evaluated, it is deprivation induced to resistant to potassium that drug is evaluated with Hoechst33342 dyeing
The ability (Fig. 2 C and 2D) of apoptosis of rat cerebellar granule neurons.
Mtt assay surveys cell survival rate: passing through above-mentioned processing 24 hours, after taking pictures under phase contrast microscope, 5mg/ml is added
MTT make final concentration of 0.5g/L, be incubated for 3 hours at 37 DEG C, then measure the OD of each group at 490nm with multi-function microplate reader
Value.The calculation method of cell survival rate is as follows: cell survival rate (%)=each processing group OD value/25K group OD value * 100%.With five batches
Cell data do statistical analysis, the neuroprotective activity of preliminary assessment drug.The result shows that cholesteric -3,6- diketone can be bright
The aobvious survival rate (Fig. 2 B) for improving cerebellar granule neuron cell.
Hoechst33342 dyeing: passing through above-mentioned processing 24 hours, after bat takes fluorescence photo under phase contrast microscope, adds
Entering Hoechst33342 makes final concentration of 2 μ g/ml, and 37 DEG C are clapped in the case where differing fluorescence microscope 350nm wavelength after dyeing 15 minutes
According to calculating apoptosis rate=white apoptotic signal number/total cell number * 100%.Statistics credit is done with three batches of cell data
Analysis, the neuroprotective activity of preliminary assessment drug.The result shows that cholesteric -3,6- diketone can obviously inhibit cerebellar granule neural
The apoptosis (Fig. 2 C and 2D) of first cell.
3. cholesteric -3,6- diketone of embodiment is in the cerebral cortex neurons excitatory toxicity model caused by glutamic acid
Protective effect
Before experiment, complete DMEM culture medium, 0.25% pancreatin of 5ml are put in 37 DEG C of water-bath heating, by 75% second of surgical instrument
Alcohol disinfection, dries up coated orifice plate or ware, will be put on ice bag and cool down without phenol red DMEM.
The rat in raw 12 hours is taken out, after cutting head with major operation, is cut with ophthalmology is small from vertebral foramen toward left-right ear upper limb
It cuts off, while being cut off along seam in skull, then open skull with tweezers, expose brain tissue, take out cerebral tissue and be put into dress
Whether there is or not phenol red DMEM(when dissection liquid use) the middle ware being placed on ice bag in.
The tissue such as epicerebral meninx, blood vessel, hippocampus is removed with ophthalmic tweezers, after separating cerebral cortex, is cut with tissue shear
Onal layer tissue is 1mm3Size.Then it moves on in 5ml pancreatin digestive juice (0.25%) in 37 DEG C of digestion 15min, is then added and contains
There are the dissection liquid of FBS (10%) and Dnase I (8mg/ml, 100 μ l) to terminate to digest and gently dispel as single cell suspension.With
1000rpm is centrifuged 3s, abandons precipitating, and 1200 rpm of suspension is centrifuged 5 min.Supernatant is abandoned, precipitating is scattered in the DMEM training of 10% FBS
Softly piping and druming mixing in base is supported, after 1000rpm is centrifuged 3s, diluting cells number is 3*e in complete DMEM culture medium5Left and right is thin
Born of the same parents' suspension.Suspension is planted in coated 48 orifice plate of poly-D-lysine (5mg/L).It is placed in containing 37 DEG C, 5%CO2, humidity be 95%
Cultivated in cell incubator, after 4 hours, culture medium all change into without serum Neurobasal A culture medium (2% B27,
1% GlutaMAX, 1% P/S), the cytarabine (Ara-C) that 10 μm of ol/L are added after inoculation 24 hours inhibits non-neuron thin
Intracellular growth carried out half amount every 3 days and changes liquid.Culture was tested to the 8th day.
The cerebral cortex neurons for taking culture 7 days, are observed under the microscope, and cell is mellow and full, and cynapse is obvious, are evenly distributed,
It can be used to test.Do following packet transaction: a) Control group: precoat no Mg2+Locke,S Buffer liquid；B) glutamic acid
Group Glu: precoat no Mg2+Locke,+ 200 μM of Glu of s Buffer liquid；C) it MK801 (Dizocilpine) group: precoats and contains
10 μM of MK801 and without Mg2+Locke,s Buffer + 200μM Glu；D) steroidal group: precoat steroidal and nothing containing 10 μM
Mg2+Locke,s Buffer + 200μM Glu。
The cerebral cortex neurons for taking culture 7 days, collect each hole conditioned medium, with no Mg2+Locke,s Buffer
Liquid washs cell three times, then with no Mg2+Locke,37 DEG C of drug solution of s Buffer liquid configuration are precoated 20 minutes, later
200 μM of glutamic acid is added, 10 μM of glycine are placed in 37 DEG C of incubators and handle 30 minutes, then gain original CMC model
Base continues culture 24 hours, and microscopically observation cellular morphology (Fig. 3 A) measures group of cells survival rate (Fig. 3 B) with mtt assay,
Statistical analysis, the neuroprotective activity of preliminary assessment drug are done with five batches of cell data.
The result shows that cholesteric -3,6- diketone can keep the integrality of cerebral cortex neurons cell, increase cerebral cortex
Neuronal cell survival rate is obvious compared to glutamic acid group difference.Demonstrate cholesteric -3,6- diketone brain caused by glutamic acid
Neuroprotection in cortical neuron excitatory toxicity model.
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Claims (7)
1. cholesteric -3,6- diketone, its deuterated object or its pharmaceutically acceptable salt treat the neure damage of patient in preparation
Application in drug.
2. application according to claim 1, wherein the neure damage be by oxidativestress damage, Neuron Apoptosis or
The damage that neuronal excitability toxicity mediates.
3. application according to claim 1 or 2, wherein the neure damage is degenerative disease.
4. application according to claim 3, wherein the degenerative disease includes Parkinson's disease, alzheimer's disease, tau
Albumen disease or amyotrophic lateral sclerosis.
5. application according to claim 1 or 2, wherein the neure damage is headstroke or cerebral injury.
6. application according to claim 1 or 2, wherein the drug also includes another therapeutic agent.
7. application according to claim 1 or 2, wherein the patient is people.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120316141A1 (en) * | 2003-03-11 | 2012-12-13 | Trophos | Method for providing neuroprotection from spinal muscular atrophy |
| CN108042547A (en) * | 2017-12-12 | 2018-05-18 | 中山大学 | Cholesteric -4- alkene -3,6- diketone is preparing the application in treating or preventing neure damage drug |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120316141A1 (en) * | 2003-03-11 | 2012-12-13 | Trophos | Method for providing neuroprotection from spinal muscular atrophy |
| CN108042547A (en) * | 2017-12-12 | 2018-05-18 | 中山大学 | Cholesteric -4- alkene -3,6- diketone is preparing the application in treating or preventing neure damage drug |
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