CN104902891B

CN104902891B - Treat and prevent the composition and method of traumatic brain injury nerve member infringement

Info

Publication number: CN104902891B
Application number: CN201380051162.1A
Authority: CN
Inventors: 陈锦辉
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-09-28
Filing date: 2013-09-27
Publication date: 2018-04-27
Anticipated expiration: 2033-09-27
Also published as: WO2014048370A1; CN104902891A

Abstract

This application provides for treating, suppressing and the method and compound of preventing injuries property brain damage.Method includes applying 7,8 5,7-Dihydroxyflavones and its derivative.

Description

Treat and prevent the composition and method of traumatic brain injury nerve member infringement

Governmental interests

The present invention wins National Institutes of Health subsidy, Grant No. NS072631.Government has the present invention certain Right.

Background technology

Traumatic brain injury (TBI) be the main reason for causing dead and children and disabled young man (Aitken etc., 2009；McCarthy etc., 2006).TBI induces the neuronal death of intracerebral (including hippocampus and cortical area).TBI can not only lead to Cross direct tissue defect cause primary injury (Hall etc., 2004；Hall etc., 2005b；Singh etc., 2006), also by this Primary event trigger in the cell of survival secondary lesion (Faden etc., 1989；Fujimoto etc., 2004b；Hall etc., 2005b；Marion etc., 1997).Damage is widely present in brain tissue, and such as cerebral cortex and hippocampus, this damage is in the mankind (Ariza etc., 2006；Bigler etc., 1997；Isoniemi etc., 2006；Tate and Bigler, 2000；Umile etc., 2002； Wilde etc., 2007) and experimental animal (Bonislawski etc., 2007；Hall etc., 2005b；Lowenstein etc., 1992； Saatman etc., 2006a) in as it can be seen that and cause to recognize, feel and the defect of motor function (Greve and Zink, 2009； Hall etc., 2005b；Singh etc., 2006).Not yet occurs effective treatment for symptom after TBI of FDA approvals at present.

Document has shown that cell excitability toxicity can be induced after experimental TBI, and the latter result in the fast of cell after damage Fast death (Palmer etc., 1993).Cell excitability toxicity can cause Apoptosis or necrosis to depend on the strong of starting stimulation The characteristic of degree and cell colony (Ankarcrona etc., 1995；Bonfoco etc., 1995；Martin etc., 1998).In mouse TBI In model, most neuronal death betides interior when 24 is small after TBI, and hereafter, cell death continues in a reduced levels At least two weeks other (Zhou etc., 2012).Most of dead cell does not show the morphological feature of apoptosis, and apoptosis mark Note is only positive in fraction dead cell subgroup.In contrast, most cell death is Necrotic Death, this result Prompting, the mainly quick necrosis that moderate TBI induces.

Brain-derived neurotrophic factor (Brain-derived neurotrophic factor, BDNF) is growth factor One of member of family.It is widely distributed in adult brain and active, including hippocampus and cortex (Egan etc., 2003；Huang Deng 1999；Hyman etc., 1991).BDNF plays neuron a variety of important, such as contributes to mature neuron (Barde,1994；Ghosh etc., 1994；Lindholm etc., 1996；Shulga etc., 2008) and prematurity neuron (Gao and Chen,2009；Kirschenbaum and Goldman, 1995；Liu et al., 2003) existence.In addition, no matter to ripe rodent Animals or humans cortex, BDNF be all trophic factors most abundant during hippocampus development (Maisonpierre etc., 1990； Timmusk etc., 1994；Webster etc., 2002).If bdnf genes are knocked out in the hippocampus development stage, the reduction meeting of BDNF expression Aggravate the neuronal death (Gao and Chen, 2009) after TBI.This result illustrates that BDNF has participated in the existence of neuron, And protection neuron is expected to be used for, make it after TBI from death.7,8- 5,7-Dihydroxyflavones (7,8-dihydroxyflavone, DHF) be an imitative BDNF small molecule, some researches show that the small molecule can protect wild type neuronal from apoptosis (Jang Deng 2010).Pressure (Andero etc., 2012) for rodent, depressed (Liu et al., 2010), advanced age (Zeng etc., 2012a；Zeng etc., 2012b；Zeng etc., 2011) and alzheimer disease model (Devi and Ohno, 2012), DHF processing has Help improve its nervous function.

The content of the invention

Information disclosed herein is that DHF reduces the neuronal death after TBI (based on Necrotic Death).With DHF processing TBI mouse, promote its BDNF acceptor --- tyrosine associated kinase B's (Tyrosine-related kinase B, TrkB) Phosphorylation (Yan etc., 1997), at the same significantly reduce after TBI 24 it is small when the neuronal death of interior hippocampus and the tissue of cortex lack Damage.On the other hand, the neuroprotection of DHF can be weakened by suppressing BDNF signal paths.

Present invention comprises the Treatment and composition for of traumatic brain injury.Specifically, method suppression disclosed herein The method for making and/or preventing neuronal death after traumatic brain injury.In one embodiment, treating the method for TBI includes Study subject after TBI occurs gives DHF or derivatives thereof.DHF or derivatives thereof can also be administered in a clinical setting, wherein Study subject is taken to Clinical Institutions after TBI.In one embodiment, the side that DHF or derivatives thereof passes through intraperitoneal injection Formula is administered in the damage side of study subject.

In one embodiment, treat TBI method include it is preventative give DHF or derivatives thereof, thus DHF or its Derivative is administered before the generation of TBI.TBI is very common in some movements and occupation, such as in rugby, football, boxing, ice In the movement such as ball and wushu, head injury occurs often.In many cases, TBI is due to that the shock repeatedly on head causes.Cause This, an embodiment, which is included in before movement carries out or way, gives DHF or derivatives thereof to sportsman.

In one embodiment, there is provided herein DHF or derivatives thereof or its pharmaceutically acceptable salt, esters, molten Agent compound, dynamic isomer, optical isomer, inclusion compound, hydrate, polycrystalline or pro-drug are being prepared for possible in TBI Prevent before occurring or suppress in the medicine of the neuronal death after TBI or the medicine for treating the study subject after TBI Purposes.

In one embodiment, there is provided herein DHF or derivatives thereof or its pharmaceutically acceptable salt, esters, molten Agent compound, dynamic isomer, optical isomer, inclusion compound, hydrate, polycrystalline or pro-drug, it is used to occur in TBI Prevent or suppress before neuronal death after TBI or for treating the study subject after TBI.

In one embodiment, there is provided herein for the nerve after preventing before TBI may occur or suppressing TBI First dead or for treating the composition of the study subject after TBI, it includes a effective amount of DHF or derivatives thereof or its pharmacy Upper acceptable salt, esters, solvate, dynamic isomer, optical isomer, inclusion compound, hydrate, polycrystalline or precursor medicine Thing.

Brief description of the drawings

Cell excitability toxic death of the outer DHF protection neurons of Fig. 1 display bodies from glutamic acid/glycine mediation.(a) Light field figure shows the cellular control unit of DMSO processing.(b) cell death of the cellular control unit of PI dyeing detection DMSO processing. (c) superposition of a figures and b figures.(d) light field figure show through glutamic acid and glycine processing inducing cell excitatory toxicity cell to Give DMSO pretreatments as a control group.(e) PI dyeing detection is through glutamic acid and glycine processing and DMSO pretreatment control groups Cell death.(f) superposition of d figures and e figures.(g) light field figure is shown through glutamic acid and glycine processing inducing cell excitability poison Property cell give DHF pretreatment.(h) cell of the PI dyeing detection through glutamic acid/glycine processing and DHF pretreatments is dead Die.(i) superposition of g figures and h figures.(j) light field show without glutamic acid and glycine processing inducing cell excitatory toxicity and to The cell for giving DHF to handle.(k) cell death that PI dyeing detection is handled without glutamic acid and glycine processing with DHF.(l)j The superposition of figure and k figures.(m) cell of DMSO groups, glutamic acid/glycine treatment group, glutamic acid/glycine/DHF groups and DHF groups Death counts.(n) cell death to the DHF of the various concentrations group handled counts, with assess the dosage of DHF to through glutamic acid/ The influence of the cytoprotection of glycine inducing cell excitatory toxicity.

Fig. 2 shows that DHF processing alleviates the damage of cortex after TBI.Mouse receives the DHF in DMSO before TBI modelings Pretreatment or receive the pretreatment of the DMSO (as control) without DHF.(a) the lesion center brain of the DMSO pretreatments without DHF The cresyl viollet dyeing of piece.(b) the cresyl viollet dyeing of the lesion center brain piece of DHF pretreatments.(c) lacking for side and offside cortex is damaged Damage area ratio control.

Fig. 3 shows that DHF processing reduces the cell death of hippocampus after TBI.After TBI modelings 24 it is small when, brain piece is dyed through FJB To detect the cell death in hippocampus.(a) mouse brain slices of sham-operation group, visible hippocampal dentate in piece.Brain piece is through DAPI (4', 6- diamidino -2-phenylindone) is redyed in blueness to show nucleus.(b) TBI modelings are without giving the small of DHF processing Mouse, FJB are dyed to detect the FJB positive cells (green) in this group of hippocampus of mice dentate fascia.Brain piece is redyed in blueness through DAPI To show nucleus.(c) mouse of DHF pretreatments is received before TBI modelings, FJB is dyed to detect this group of hippocampus of mice dentate fascia In FJB positive cells (green).Brain piece is redyed in blueness through DAPI to show nucleus.(d) to receive DMSO before TBI modelings pre- The mouse of processing, FJB are dyed to detect the FJB positive cells (green) in this group of hippocampus of mice dentate fascia.Brain piece is answered through DAPI Dye is in blueness to show nucleus.(e) to the count results of the FJB positive cells of different subregions in hippocampus.

Fig. 4 shows that DHF processing adds the quantity of new life prematurity neuron in hippocampus after TBI.After modeling 24 it is small when, use DCX specific antibodies carry out immunostaining, to show the newborn prematurity neuron in hippocampus of mice dentate fascia.(a) sham-operation The positive newborn prematurity neurons (red) of Dcx in the hippocampal dentate of group mouse.Brain piece is redyed in blueness with aobvious through DAPI Show nucleus.(b) the positive newborn prematurity neurons (red) of the Dcx in the hippocampal dentate of TBI damages group mouse.Brain piece passes through DAPI is redyed in blueness to show nucleus.(c) Dcx in the hippocampal dentate of the TBI damage group mouse of DHF pretreatments is positive Newborn prematurity neuron (red).Brain piece is redyed in blueness through DAPI to show nucleus.(d) the TBI damages of DMSO pretreatments The positive newborn prematurity neurons (red) of Dcx in the hippocampal dentate of wound group mouse.Brain piece through DAPI redye in blueness with Show nucleus.

Describe in detail

Definition

Term " joint " refers to one or more therapeutic agents to add in DHF or derivatives thereof and is administered, including same When administration or in any order in succession administration.

Term " 7,8- 5,7-Dihydroxyflavones " or " DHF " refer to 7,8- dihydroxy -2- phenyl -4H-1- chromenes -4- Ketone, it is following formula: compound：

DHF is a kind of tyrosine kinase receptor B (TrkB) activator, it combines the extracellular domain (Kd=of this receptor 320nM), receptor dimer and autophosphorylation are promoted.

For the purpose for handling or treating, term " mammal " refers to any animal for being defined as mammal, Include the animal of the mankind, domestic animal and zoo, sport category or pets, such as dog, horse, cat, milk cow.Preferably, lactation Animal is the mankind.

Term " processing " or " treatment " are a kind of means for being used to obtain beneficial or desired clinical effectiveness.Herein In, beneficial or desired clinical effectiveness includes the mitigation, the mitigation of disease degree, the state of an illness of (but being not limited only to) clinical symptoms Stabilization (not deteriorating), the development for postponing or slowing down the course of disease, improvement or alleviate morbid state and relax (part or all of), It is no matter detectable or undetectable.Herein, " treatment " does not include " preventing ", " avoiding " or " prevention ".

Term " compound " refers to DHF or derivatives thereof or pharmaceutically acceptable salt, esters, solvate, change Isomers, optical isomer, inclusion compound, hydrate, polycrystalline or pro-drug, while also include the derivative of its protectiveness. Compound can include one or more chiral centres and/or double bond, therefore, exist with following form：Stereoisomer is for example Double bond isomer (that is, geometric isomer), enantiomter or diastereoisomer.According to the present invention, mention herein Chemical constitution, include the compound of the present invention, including the enantiomer of all respective compounds, diastereomer and geometrical isomerism Body, that is to say, that either the pure form of spatial chemistry is (such as：Geometric isomer is pure, enantiomeric pure or diastereo-isomerism The pure form of body) and isomer mixture is (such as：Geometric isomer, enantiomter and non-enantiomer mixture).One In the case of a little, a kind of enantiomter, diastereoisomer or geometrical isomerism are known from experience has preferably activity than other isomers Or improved toxicity or dynamic characteristic.In this case, these enantiomters, the diastereomeric of compound of the invention are different Structure body and geometric isomer then become preferred.

Term " polycrystalline " refers to originally asking disclosed compound or the solid crystalline form of its compound.Same chemical combination The different polycrystal of thing can show different physics, chemistry and/or spectral quality.Different physical properties is included (but not only It is limited to) stability is (such as：To the stability of heat or light), compressibility and density (prepare and product manufacturing in it is critically important), with And dissolution rate (this can influence bioavilability).The difference of stability can be due to chemically reactive change (such as：Otherness oxygen Change so that rapider than the formulation colour fading formed by another polycrystal by the formulation that a kind of polycrystal is formed) or mechanical property (such as：As the favourable polycrystal of dynamics is transformed into the more stable polycrystal of thermodynamics, tablet is crushed in storage) or both (such as：The tablet of single polycrystal easily ruptures at high humidity) cause.The different physical properties of polycrystal can influence its processing. Such as a kind of polycrystal may than another polycrystal it is more likely to form solvate, or be more difficult to filter, wash away it is miscellaneous Matter, because their grain shapes or size distribution are different.

Term " hydrate " refers to compounds as disclosed herein or its salt, it further comprises stoichiometry or non-chemical The water combined by non-covalent intermolecular forces of metered amount.

Term " inclusion compound (clathrate) " refers to the compound or its salt disclosed herein in form crystal lattice, the crystalline substance Lattice include space (for example, passage), have the guest molecule (for example, solvent or water) of capture wherein.

Term " pro-drug " refers to the drug molecule of the compound of the present invention, it is no biological activity until by generation Journey of apologizing for having done sth. wrong activates.Pro-drug includes the derivative of compound, its can under biotic factor (external or in vivo) hydrolysis, oxidation or Otherwise react to provide the compound of the present invention.Pro-drug can have after carrying out the reaction under biotic factor Activity, or they in the form of its unreacted in the presence of can be active.The example of pro-drug for use in the present invention includes The analog or derivative of (but not limited to) compounds as disclosed herein, it includes can biological hydrolysis part, such as can Biological water Solve acid amides, can biological hydrolysis ester, can biological hydrolysis carbamate, can biological hydrolysis carbonic ester, can biological hydrolysis Uride and can biological hydrolysis phosphate analog.Other examples of pro-drug include containing for compounds as disclosed herein There is -- NO, -- NO₂, -- ONO or -- ONO₂Partial derivative.Pro-drug can usually be prepared using well-known method.

Term " TBI scenes occurred frequently " refers to event, situation or accident that wherein traumatic brain injury may occur.Movement is One TBI scene occurred frequently.For example, the TBI head impacts occurred frequently in football.Movement includes competing and practices.Separately A kind of TBI scenes occurred frequently are military operation.Also a scene occurred frequently is common in old man, and reason is usually instability of gait.

Traumatic brain injury

In most basic definition, traumatic brain injury (TBI) is due to that wound causes to damage to brain in brief. TBI is divided into penetrability wound and non-penetrative wound.Non-penetrative wound may include that head is impacted, so as to cause brain one in skull Secondary or multiple impact and damage.Following factors can cause head impact：Fall down and (such as fall off the stairs, old man slips in bathroom), Traffic accident is hit, (rugby, football (such as striving top), ice hockey (such as push and block), boxing, wushu, hockey, slide plate are hit in movement Movement etc.) and military operation (such as explosion, fight hand-to-hand).

Damage can be from slightly to severe caused by TBI.Slight damage results only in the temporary dysfunction of cell.But The damage of severe can then cause torn tissue, bleeding and neuronal death.Originally, cell death caused by TBI betides brain Cortex, then betides hippocampus (Fujimoto etc., 2004a；Hall etc., 2005a；Saatman etc., 2006b).This cell is dead It is Necrotic Death to die (including neuronal death).Dead cell after TBI significantly shows the label rather than apoptosis of necrosis Label.TBI and neuronal death can cause cognitive disorder (such as learn, remember), sensory disturbance (assist by such as vision, trick Adjust etc.), communication disorder (such as dysarthrosis, aphasia) and behavior/emotional maladjustment (such as depression and anxiety, irritability).

The medication of DHF

In one embodiment, treating the method for TBI includes DHF or derivatives thereof being administered after TBI generations.DHF Or derivatives thereof can be administered in medical institutions, wherein study subject meet with TBI after be taken to medical institutions.In an embodiment party In case, DHF or derivatives thereof can also be administered in injured place.Injured place can be in family, business scene, in-situ match or war .

In one embodiment, treat the method for TBI include it is preventative apply DHF or derivatives thereof, thus DHF or its Derivative is administered before the generation of TBI.TBI may occur in some movements and occupation.Such as in rugby, football, fist Hit, in the movement such as ice hockey and wushu, head injury occurs often.In many cases, TBI is due to the institute of shock repeatedly on head Cause.Therefore, in one embodiment, DHF or derivatives thereof is before the match or practice that head injury may occur It is administered to sportsman.Equally, army enlisted member is frequently exposed under destructor in nearest conflict.Infantry can patrol Patrol, escort or any other military operation before receive DHF or derivatives thereof administration.

In addition, DHF or derivatives thereof can also combine or alternating delivery with one or more other compounds.TBI, it is special It is not blunt trauma, inflammatory reaction can be produced.In one embodiment, DHF or derivatives thereof can with diuretics administering drug combinations, The diuretics is administered to reduce intracranial pressure.Diuretics include (but being not limited only to) Katlex, Hydrochioro, metolazone, according to Puli's ketone etc..Initial several weeks, the risk of patient's epileptic attack of moderate and severe TBI increase after damage.In a reality Apply in mode, DHF or derivatives thereof can be used in combination with antiepileptic.The example of antiepileptic includes (but being not limited only to) Gabapentin, carbamazepine, phenytoinum naticum, Oxcarbazepine, divalproex sodium, Clonazepam, Topiramate and valproic acid.

Officinal salt

There is enough acid-base values in compound to be formed to stablize, in the case of nontoxic ackd salt or basic salt, chemical combination Thing can be suitable as officinal salt administration.Officinal salt includes those salt for being derived from pharmaceutically useful inorganic or organic base and acid.Close Suitable salt includes derivative by the well-known acid in alkali metal such as potassium and sodium, alkaline-earth metal such as calcium and magnesium, and other drugs field Thing.Particularly, the example of officinal salt is the organic acid addition salt formed with acid, acceptable anion on physiology, than Such as：It is toluene fulfonate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, anti- Bad hematic acid salt, α-ketoglutaric acid and α-glycerophosphate.Suitable inorganic salts, including sulfate, nitrate, carbon can also be formed Sour hydrogen salt and carbonate.

Officinal salt can be obtained by standard, known preparation method, such as by by compound (example alkaline enough Such as amine) reacted with suitable acid, acceptable anion on physiology.Can also prepare carboxylic acid alkali metal (for example, sodium, Potassium or lithium) or alkaline-earth metal (such as calcium) salt.

Composition

Composition based on DHF or officinal salt can with for any indication described herein therapeutically effective amount with Pharmaceutically acceptable additive, with any carrier or excipient joint prepare.Therapeutically effective amount can according to illness to be treated, its The order of severity, the therapeutic scheme taken, the pharmacokinetics of medicament and individual patients situation and change.

On the one hand, DHF is preferable over is made mixture with pharmaceutical acceptable carrier.Under normal circumstances, preferably given with orally available The form of medicine applies pharmaceutical composition, but preparation can also pass through parenteral, intravenous, intramuscular, percutaneous, oral cavity, subcutaneous, bolt Agent or the administration of other approach.Intravenous and intramuscular formulations are preferably administered in Sterile Saline.It will not make the composition of the present invention Unstable or in the case of influencing its therapeutic activity, those of ordinary skill in the art can adjust preparation under the teaching of this specification To provide the several formulations for specific route of administration.More particularly, for example, desired compound is adjusted so that its It is more stable in water or in other excipient, can be by conventional adjustment (salt formula, esterification etc.) and simple realization.

The amount of compound included in pharmaceutical active preparation is the effective dose for treating the neuronal death in TBI. Under normal circumstances, therapeutically effective amount of the compounds of this invention in pharmaceutical dosage form is typically about 0.1mg/kg to about 100mg/kg Or higher, depending on used compound, the degree of traumatic brain injury and method of administration.For the object of the invention, root According to the present invention composition prevention or prevent effective dose fall into it is above-mentioned in the described same range of therapeutically effective amount simultaneously It is usually identical with above-mentioned effective dose.

The administration of DHF or derivatives thereof can be continuous (intravenous drip) to be administered orally several times daily (for example, QID, BID, etc.), and oral, part can be included, parenteral is intramuscular, and intravenously, subcutaneously, percutaneous (it can include infiltration enhancing Agent), oral cavity and suppository administration and other administration route.Enteric oral tablet can be used for enhancing and be administered by oral route Compound bioavilability and stability.Most effective formulation is by the pharmacokinetics depending on selected particular agent Learn, and in patient traumatic brain injury the order of severity.Peroral dosage form is particularly preferred, because being easy to be administered and beneficial to trouble Person's compliance.

To treat pharmaceutical composition according to the present invention, preferably by the chemical combination of the one or more present invention of therapeutically effective amount Thing is mixed with pharmaceutically useful carrier according to conventional pharmaceutical mixed technology.Carrier can take many forms, this takes Certainly in the dosage form needed for administration, for example, oral and parenteral administration.When preparing the pharmaceutical composition of peroral dosage form, appoint What common drug media can use.Therefore, it is suitable to carry for liquid oral medicine such as supensoid agent, elixir and solution Body and additive can be used including water, glycol, oil, alcohol, flavor enhancement, preservative, colouring agent and analog.For Peroral solid dosage form Preparation such as powder, tablet, capsule, and solid dosage forms such as suppository, suitable carrier and additive include starch, sugar carrier such as Portugal Grape sugar, mannitol, lactose and relevant carriers, diluent, granulating agent, lubricant, adhesive, disintegrant and analog can be made With.If desired, tablet or capsule can carry out enteric coating for being sustained by standard technique.Can using these formulations Significantly affect the bioavilability of compound in patients.

For parenteral administration, carrier generally includes sterile water or sodium-chloride water solution, although other components, including Those dispersing aiies, may also be included in that interior.When by using the germ-free condition of sterile water or holding sterile water, the combination Thing and carrier must also sterilize.Also injection suspension can be prepared to, in this case, appropriate liquid-carrier, suspend Agent etc. can also be used.

In embodiments, compound and composition are used to treating, prevent or delaying the relevant neuronal deaths of TBI.It is excellent Selection of land, to treat, preventing or delay neuronal death, composition is administered with peroral dosage form with following amount：About 250 micrograms are to about 1 gram or more, at least once a day, or one day up to four times.Compound is preferably administered orally, but can also with parenteral administration, The form such as local administration or suppository.

Embodiment

Embodiment 1：DHF can protect neuron in testing in vitro from cell excitability toxicity.

This embodiment is the neuroprotection in order to detect DHF in cell excitability toxic damages, and in body Optimal dose in outer cell.

Hippocampal cell culture and external TBI modelings.The brain of C57BL/6 mouse (same day (P0) after birth) is solved Cut open, remove meninx, isolate hippocampus.Hippocampal tissue papain digestion, it is unicellular to be collected, tile and locate in advance with lysine On the coverslip of reason, B27Neurobasal is used^TMSerum free medium (Brewer etc., 1993) is cultivated.In order to be created in parody Wound, the cell (DIV5) of in vitro 5 days gives 100 μM of glutamic acid and 20 μM of glycine are handled, and one group of cell gives same volume DMSO is as control, and processing time is 60 minutes, when then standing 24 is small.

DHF processing.In glutamic acid and glycine before processing 30 minutes, give cell DHF (be dissolved in DMSO, it is final concentration of 500nM) handle, or give DMSO processing as control.

Propidium iodide (PI) dyes and cell count.After processing, cell and PI (10 μ g/mL) be incubated 24 it is small when.At 37 DEG C In moist incubator after 30 minutes, washed with PBS, then fixed with 2% paraformaldehyde.Cell difference or fluorescence after fixation Micro- sem observation.Each slide randomly selects 5 visuals field, with the inverted microscope of connection camera gather respectively 20 times of lower light fields and The image of PI dyeing, for inverted microscope to be equipped with the Zeiss Axiovert 200M of Apotome, camera is Zeiss Axio Cam MRc5, software used are AxioVision, v4.0.The photo gathered is integrated and marked with Photoshop 7.0.Each Experimental group takes three slides, and PI staining positive cells thereon are counted and calculate the ratio of PI positive cells.

Statistical analysis.All data are described with average value ± standard error, enclose every group of sample number thereafter.Data are used Student ' s t are examined or One-way ANOVA analyses.

As a result

In order to assess influences of the DHF to neuronal survival under glutamic acid/glycine induced damage, DHF (DMSO is dissolved in, Concentration is 500nM) and DMSO 30 minutes injection cells before damage.After damage 24 it is small when, it is dead thin to detect to carry out PI dyeing Born of the same parents.(DMSO has only been given in blank control group), and 6.45% ± 1.06% neuron is marked (Fig. 1 a-c, m) by PI.Paddy ammonia Acid/glycine causes substantial amounts of neuronal death (46.92% ± 2.53%, P=0.0001vs. blank control group) (Fig. 1 d- F and m).DHF processing significantly reduces PI positive cells, it there was only 20.15% ± 1.09%, implies DHF effectively in paddy 57.05% neuron (Fig. 1 g-I and m) is protected in the cell death of propylhomoserin/glycine induction.To without glutamic acid/sweet ammonia The healthy cell of acid treatment, the short time DHF processing do not have an impact cell death (6.56% ± 1.28%) (Fig. 1 j-l and m).These results indicate that in testing in vitro, DHF can protect nerve from the cell death that glutamic acid/glycine is guided Member.

In order to detect the optimal dose of DHF neuroprotections, the neuron of DIV5 is incubated (0- together with the DHF of various concentrations 5000nM, Fig. 1 n), time point is 30 minutes of glutamic acid/glycine before processing.After processing 24 it is small when, carry out PI dyeing detection Dead cell.There is no the group that DHF is handled, the neuron for having 46.92% ± 2.53% is PI stained positives.Even in low concentration In DHF (10nM) treatment group, cell death has all been reduced to 37.11% ± 1.33%, illustrates that DHF is just shown at low concentrations Neuroprotection.With the increase of DHF concentration, neuroprotection also strengthens therewith.When DHF concentration is 100nM, The cell death that glutamic acid/glycine is induced has fallen to 23.13% ± 3.28%, DHF when being 500nM, cell mortality It is down to 17.05% ± 1.96%.However, in the concentration of 500nM, Neuroprotective effect reaches peak, when concentration continues to rise During to 1.0 μM, cell mortality is 14.26% ± 1.52%, when concentration rises to 5.0 μM, cell mortality for 12.72% ± 0.57%.In more than 500nM, cell mortality does not have because the concentration of DHF rises and conspicuousness decline, and therefore, DHF is to body The optium concentration of the neuroprotection of outer cell is 500nM.

On the other hand, under the DHF processing of high concentration (5.0 μM, 10 times of optium concentrations), neuron shows health Form：Cell space is circle, and annex is good, no swelling or atrophy.Illustrate the cytotoxicity very little of DHF.

Embodiment 2：DHF reduces the defect of cortex after TBI

DHF is a small molecule (molecular weight 254.25g/mol), can reach brain region to pass through blood-brain barrier, from And influence the damage (Andero etc., 2011) of TBI.Here DHF is administered with the mode of intraperitoneal injection, whether to detect DHF administrations The death of cortical neuron after TBI can be reduced in model in vivo.

Method

Animal.Male C57BL/6 mouse (The Jackson Laboratories, Bar Harbor, ME) give 12/ Illumination/dark cycle environment when 12- is small, and sufficient food and water can be obtained.The mouse of 8-10 week old is chosen in modeling.It is all Program all according to Indiana University animal care and using the committee agreement carry out.

TBI modelings.The C57BL/6 mouse of 8-10 week old be used to carry out TBI modelings.Mouse is fixed on stereotaxic instrument On, scalp is cut off, the line of former posterior fontanels and the intersection point of middle seam and temporalis line are the center of circle, are bored on the skull of left side and open one The hole of 4mm diameters, carefully raises cranium cap (not damage endocranium).Trip rod is set at the vertical angle of brain, if It is 3.0m/s to determine stroke speed, and shock depth is 1.0mm.The mouse of sham-operation group is also removed cranium cap, but does not receive shock and make Mould.During operation and recovery, the central body temperature of mouse maintains 36-37 DEG C.

DHF or control treatment.Before when modeling 1 is small, mouse according to group gives DHF respectively, and (5mg/kg, is dissolved in DMSO) or DMSO intraperitoneal injections are handled.

Tissue treatment.After modeling 24 it is small when, after mouse deep anaesthesia, with 0.9% physiological saline through heart perfusion, then Fixed with 4% paraformaldehyde.Brain tissue is removed, fixed after being placed in 4% paraformaldehyde overnight, is then transferred into 30% Sucrose solution in.48 it is small when after, brain tissue is removed, and carries out frozen section (thickness be 30 μm), and section is saved in -20 degree Refrigerator.Freezing microtome used is Microm HM 500M.

Cortical lesions area measurement.In order to assess the cortex area of remaining, brain tissue takes 1 every 5 (namely per phase Taken every 180 μm a piece of) carry out Nissl's staining (Gao and Chen, 2011).Brain piece is good for side and the profile of damage side is all sketched the contours of Come, carry out three-dimensional reconstruction, microscope used is Olympus BX60, and software used isSystem (Microbrightfield Inc.,Colchester,VT).The degree of cortical lesions is calculated by measuring the cavity of cortex, The percentage of cortex area is accounted for cyst areas to represent.Calculation formula is：(strong side cortex area-Ipsilateral cortex area)/it is strong Side cortex area x100%.

Statistical analysis.All data are represented with mean value ± standard error, followed by the sample size of every group of mouse.Between different groups Data with t examine or One-way ANOVA analyze.

As a result

For sham-operation group although not passing through DHF or DMSO processing, their cortex either Ipsilateral is still good for side all not There is cavity.However, the TBI mouse of DMSO processing, to compared with undamaged strong side, after Ipsilateral damage 24 it is small when cerebral cortex Defect rate is 16.75% ± 1.46%% (Fig. 2 a).The TBI mouse of DHF processing, cortex defect rate is down to 12.43% ± 0.74% (Fig. 2 b), reduces 25.8% compared to DMSO treatment group cortex defect rates.Statistical analysis shows that this difference has significantly Property meaning.Therefore, the results showed that TB models have neuroprotection in vivo for DHF processing.

Moreover, control group and DHF groups and have no that obvious behaviouristics changes, main observation index is revives after a) anaesthetizing Wake up the time；B) motor function；C) pain reaction.These results indicate that DHF has no apparent side effect in vivo.

Embodiment 3：DHF reduces the death of prematurity neuron after TBI, adds prematurity nerve newborn in hippocampus Member

After the effect of hippocampus is proved, DHF is also proved DHF in the protective effect of cortex.In order to detect DHF in skin Effect in terms of layer tissue defect, Nissl's staining hindbrain piece are used for the area for measuring cortex cavity.

Method

Animal：With embodiment 2.

TBI modelings：With embodiment 2.

Animal is through DHF or control treatment：With embodiment 2, modeling previous hour gives DHF or DMSO processing.

Tissue treatment：With embodiment 2, after modeling 24 it is small when, mouse is anesthetized, after heart perfusion with 4% poly first Aldehyde is fixed, and brain tissue is positioned in 4% paraformaldehyde overnight after taking out, and is then transferred into 30% sucrose.48 it is small when after use In frozen section, section is stored in -20 degree.

In order to detect the density of FJB positive cells in hippocampus, every 6 of brain tissue takes a piece of FJB dye markers dead cell Foregoing different subregions are counted (CA1 by (every thickness is 30 μm, is separated by 180 μm)；CA3；Granular cell layer, GCL；Point Sublayer, ML；Door area) (Amaral and Witter, 1989).Counting to FJB positive cells carries out under 40x mirrors, takes double blinding Method of counting, microscope used is Zeiss Axiovert 200M, and software used is AxioVision, v4.0.FJB is positive Density of the cell on unit area is multiplied by brain piece thickness (30 μm) in the hope of bulk density, is described as cell number/mm³。

Immunohistochemistry.Histotomy washs 3 words in PBS, and with confining liquid, (10% donkey serum is dissolved in PBS, includes 0.3% Triton (carried out when) closing 2 is small in 4 DEG C).Then when primary antibody incubation 48 is small, primary antibody used is：Sheep antimouse p-TrkB (phospho Y816),1:20,ab74841,Abcam；Mouse anti-mouse NeuN, 1:100,MAB377,Chemicon；Cavy resists The double cortins (DCX) of mouse, 1:1000,Millipore,ab2253.48 it is small when after, with PBS rinses three times, add anti-sheep secondary antibody (Alexa488IgG,1:1000, A11015, Invitrogen) 4 DEG C be incubated 1 it is small when, then goat anti-mouse (cy3IgG, 1: 400, A10521, Invitrogen) and the anti-cavy of goat (cy5IgG, 1:200, A21450, Invitrogen) secondary antibody in 4 DEG C Be incubated 1 it is small when.Nucleus is redyed 2 minutes with DAPI, with PBS rinses.Finally with being attached to after water rinse on glass slide, sealed after dry Piece.Image is gathered with microscope.

All data of statistical analysis are represented with mean value ± standard error, followed by the sample size of every group of mouse.Between different groups Data with t examine or One-way ANOVA analyze.

As a result

DHF (being dissolved in DMSO, dosage 5mg/kg) or DMSO is injected at when 1 is small before modeling through abdominal cavity and is administered, and mouse exists Draw materials when 24 is small after modeling.Brain piece, which is picked, to be come, and carries out FJB dyeing to assess cell death.Dead cell can be dyed green by FJB Color.In sham-operation group, FJB positive cells (Fig. 3 a) are had no, but in the mouse of TBI modelings, the equal observable in hippocampus To FJB positive cells.Most of FJB positive cells are all located at the inner side 1/3 of hippocampal dentate (Fig. 3 b).Here it is also at the same time Position where prematurity neuron.Research before is it has been shown that most of in FJB positive cells is all prematurity neuron (Gao, 2008), and most cells are all downright bad (Zhou, 2012).Cell counts are shown, in TBI group mouse, tooth The FJB cell densities that shape returns are 25,395 ± 2598/mm³(Fig. 3 d), in DHF treatment groups, the FJB positive cells of dentate fascia are notable Reduce, counting is shown in DHF treatment groups, and FJB positive cell density is down to 19,505 ± 600/mm³(Fig. 3 d), compared with TBI groups Cell mortality has dropped 23.2%.In the control group of DMSO processing, cell death is not decreased obviously, and dead cell is mostly Non- maturation Granule Neurons.

Although most FJB positive cells all in hippocampal dentate, still have a small amount of neuronal death to occur Other regions of hippocampus, such as CA1, CA3 and Men Qu.In sham-operation group, the FJB positive cell density in these three regions is respectively 233±146/mm³(CA1)、3863±1045/mm³(CA3) and 1643 ± 771/mm³(door area), in DHF treatment groups, these three The FJB positive cell density in region is respectively 308 ± 53/mm³(CA1)、3808±504/mm³(CA3) and 1257 ± 226/mm³ (door area), only slight change.

In order to further detect DHF after TBI to the neuroprotection of prematurity neuron, we are to hippocampal dentate Prematurity neuron counted.The Mice brain tissues of mouse and sham-operation group after when modeling 24 is small be used to assess. The brain piece of lesion center is picked to carry out immunostaining, and selected antibody is DCX, is one and is directed to newborn prematurity neuron Specific antibody (Korzhevskii etc., 2006；Mullen etc., 1992).Newborn neuron in dentate fascia is dyed to red (Fig. 4).In sham-operation group, immature newborn neuron number is 187 ± 14 (Fig. 4 a, 4e).In no any processing In the case of, after TBI 24 it is small when, prematurity neuron is reduced to 76 ± 4 (Fig. 4 b, 4e), shows the Leukopenia rate for having 59.4%. However, in DHF treatment groups, the number of prematurity neuron is 142 ± 9 (Fig. 4 c, 4e), and contrast sham-operation group only has 24.1% Slip.There is no the group that DHF is handled 35.3% dead cells more than the group that DHF handle, and it is most to be downright bad.These numbers The non-mature neuron after TBI can be protected according to the single injection for showing DHF.

Claims

1. the 7,8- 5,7-Dihydroxyflavones (DHF) or its pharmaceutically acceptable salt of therapeutically effective amount are being prepared for treating traumatic brain Purposes in the medicine of damage.

2. the purposes of claim 1, wherein the DHF or its pharmaceutically acceptable salt are also applied with least one therapeutic agent With.

3. a effective amount of 7,8- 5,7-Dihydroxyflavones (DHF) or its pharmaceutically acceptable salt are being prepared for before TBI may occur Purposes in the medicine of neuronal necrosis death after prevention or suppression traumatic brain injury (TBI).

4.7,8- 5,7-Dihydroxyflavones (DHF) or its pharmaceutically acceptable salt are being prepared for suppressing that the activity of TBI may occur Purposes in the medicine of period neuronal necrosis death.

5. the purposes of claim 3 or 4, wherein described may be due to participate in movement.

6. the purposes of claim 3 or 4, wherein described may be due to military operation.

7. the purposes of claim 3 or 4, wherein described may be due to fall.

8. the purposes of claim 3 or 4, wherein described may be due to traffic accident.

9. a effective amount of DHF or its pharmaceutically acceptable salt are preparing the composition for treating or preventing traumatic brain injury In purposes.

10. the purposes of claim 9, wherein the composition is bad for the neuron after treating or preventing traumatic brain injury Dead property is dead.