CN101658522A - Application of tacrine short-chain dimer in preparation of medicament for treating neurodegenerative diseases - Google Patents
Application of tacrine short-chain dimer in preparation of medicament for treating neurodegenerative diseases Download PDFInfo
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Abstract
The invention discloses application of a tacrine short-chain dimer compound in preparation of a medicament for treating neurodegenerative diseases, and particularly relates to application of a tacrineshort-chain dimer in preparation of a medicament for treating senile dementia, stroke and Parkinson's disease.
Description
Technical field
The present invention relates to the new medical usage of chemical compound, relate in particular to the application of tacrine short-chain dimer in preparation treatment neurodegenerative diseases medicine.
Background technology
Neurodegenerative diseases comprise senile dementia (Alzheimer ' s disease, AD), parkinson disease (Parkinson ' s disease, PD) and apoplexy (Stroke) etc. be the major disease in the millions of old peoples' of current serious harm quality of life and life-span.Along with the arriving of social senilization, it is more and more serious that problem becomes.Unfortunately at present most of neurodegenerative diseases still there is not effective prophylactic treatment medicine.Although the pathomechanism of neurodegenerative diseases varies, they have a common feature: carrying out property of functional neuron is lost.Programmed cell death (being apoptosis) is lost in the process in carrying out property of functional neuron and is played a significant role.Neuron Apoptosis is reversible within a certain period of time, and therefore, the neuroprotective of prevention Neuron Apoptosis can effectively be controlled the course of disease of neurodegenerative diseases.
Tacrine (tacrine) is that first obtains the cholinesterase inhibitor that the approval of U.S. food and FAD is used for the treatment of senile dementia, its structural formula as shown in Figure 1, it is affirmed the curative effect of senile dementia, but because of it has stronger liver toxicity, especially can cause that transaminase level raises, be close to by clinical and eliminated.According to our research, the homodimer of tacrine can weaken its toxicity by reducing dosage.In addition, simple cholinesterase inhibitor can only improve slight dementia and can not delay neuron degeneration.More and more studies show that: the existing medicine of dimerization is that a class is obtained efficiently effectively, the means of low toxicity, many target drugs.Computer simulation medicine docking system can help to design novel homology or heterodimer.Therefore, we obtain some at comprising acetylcholine esterase by dimerization, N-methyl D-aspartic acid (N-methyl-D-aspartic acid, NMDA) receptor and nitricoxide synthase (Nitric oxide synthase, the newtype drug of many target spots such as NOS).Use the computer docking system of automatization, based on dibit point action principle to acetylcholine esterase, we design and have synthesized a series of dimerization build cholinesterase inhibitor, the tacrine dimer has at acetylcholinesterase (Acetylcholinesterase, AChE) higher selectivity, this innovation result has been applied for United States Patent (USP) (US005783584A).
Research and develop " pathology startability " neuroprotective strategy based on N-methyl D-asparagine acid acceptor:
Though the generation of neurodegenerative diseases and the molecular mechanism of development thereof are not still understood, also lack effective prophylactic treatment medicine, but more and more evidences shows glutamate receptor, and especially the excessive activation of N-methyl D-asparagine acid type glutamate receptor plays a significant role in neurodegenerative diseases.Because energy metabolism impairment, the glutamic acid that synapse discharges can't obtain timely and effective removing, even causes unusual release, and energy battier causes the ion stable state to be destroyed in addition, and lasting depolarization appears in neuron, and neuronic depolarization has weakened magnesium ion (Mg
2+) to the voltage-dependent inhibitory action of N-methyl D-asparagine acid acceptor, cause the excessive activation of N-methyl D-asparagine acid acceptor.This excessive activation causes Ca
2+A large amount of in the generation (comprising nitric oxide and active oxygen) of free radical in stream and the cell, thereby mediate neuronal death.A very long time in past N-methyl D-asparagine acid acceptor is regarded as the drug targets of neuroprotective exploitation always, however the antagonist of numerous N-methyl D-asparagine acid acceptor in clinical experiment, be eliminated, its clinical practice is subjected to very big restriction.This mainly is that N-methyl D-asparagine acid acceptor also mediates important physiological function such as neuronic existence, neural growth, excitatory synapse transmission and synaptic plasticity as the unified important glutamic acid neurotransmitter receptor of nerveous system because glutamic acid is the important biomolecule adjusting molecule in the cell activities.N-methyl D-aspartic acid receptor antagonist has disturbed these normal physiological functions will cause some serious adverse, so clinical tolerance is good, and N-methyl D-aspartic acid receptor blocking agent is the focus that people seek safely and effectively.
It is good that people such as nearest Lipton have proposed an exploitation clinical tolerance; the brand-new strategy of neuroprotective safely and effectively; this tactful marrow acts on the target that pathologic starts with being these drug selectivities; and do not disturb normal physiological function; claimed that this compounds is PAT (Pathologically-activatedtherapy, the treatment of a pathologic actuated type) medicine.For N-methyl D-asparagine acid acceptor, pathologic actuated type drug main will comprise a class uncompetitive, channel blocker (uncompetitive antagonist with fast-off rate with N-methyl D-asparagine acid acceptor of rapid kinetics feature, UFO), this class medicine is expected to reach above-mentioned effect.In addition, the pathogenesis hypothesis of some other senile dementia is also all directly or indirectly relevant with the excitatory toxicity of glutamate induction, and these hypothesis comprise amyloid toxicity theory, cholinergic theory, Protein tau pathology theory, radical damage theory.Therefore, have N-methyl D-aspartic acid receptor antagonist that rapid kinetics and pathologic start feature and be expected to the progress of making a breakthrough property in the treatment of neurodegenerative diseases.
Research and develop " the many targets of a medicine " neuroprotective strategy based on the target that neurodegenerative diseases is relevant:
In recent years, though slight age related disease patient has obtained Drug therapy to a certain degree, but the unable neuron degeneration that delays the patient of these treatment measures changes, and the therapeutic effect of these " medicine one target position " medicines (one-drug-one-target) is very limited.This mainly is because neurodegenerative diseases all is some multi-pathogenesis, the disease of many pathomechanisms, and they need the intervening measure of many target position unit to come at many-sided etiology and pathology.The medicine strategy of this many targets has existed several thousand in traditional medicine (China and other country), for example comes the diffusion of control AIDS at present with HAART.Though a plurality of medicines at different target position of use in conjunction can reach this effect, the medicine with many target position obviously can be avoided the involved bioavailability of the medication combined application of a plurality of lists, the problem that difference caused of pharmacokinetics aspect." the many target position of a medicine " (one-drug-multiple-target) medicine have more clear superiority on clinical therapeutics, for example: (1) better efficacy acts on developing of a plurality of target position antagonism diseases simultaneously; (2) overcome the defective of drug combination, although a plurality of drug combination perhaps can produce similar drug effect, characteristics such as the bioavailability of different pharmaceutical, pharmacokinetics are not quite similar, and make the target difficulty relatively that realizes coupling; (3) greatly improve patient's compliance, dull-witted patient is one of key issue in the disease treatment to the compliance of doctor's advice, single obvious convenient use of medication.More and more evidences shows more or less also to be had the characteristic of " the two targets of a medicine " or " the many targets of a medicine " by U.S. food and FAD approval at four cholinesterase inhibitor of single target treatment senile dementia and the antagonist memantine (memantine) of N-methyl D-asparagine acid acceptor.Therefore, can be subject to people's attention gradually at the single medicine that comprises a plurality of target spots such as amyloid-beta, N-methyl D-asparagine acid acceptor, Protein tau, acetylcholine esterase, metal ion and active oxygen simultaneously.
And research and develop " the many targets of a medicine " neuroprotective and be applied to preparation treatment neurodegenerative diseases based on the relevant target of above-mentioned relevant neurodegenerative diseases; comprise senile dementia (Alzheimer ' s disease; AD); parkinson disease (Parkinson ' s disease; PD) and apoplexy (Stroke), Shang Weijian has report.
Summary of the invention
Technical problem to be solved by this invention provides the new medical usage of tacrine short-chain dimer, i.e. the application of tacrine short-chain dimer in preparation treatment neurodegenerative diseases medicine; Particularly preparation treatment senile dementia (Alzheimer ' s disease, AD), parkinson disease (Parkinson ' s disease, PD) and the application in apoplexy (Stroke) disease medicament.
Tacrine short-chain dimer compounds of the present invention is chemical compound and the pharmaceutically useful salt thereof with following structure:
Wherein, R
1, R
2Be H or (C
1-C
4) alkyl, n=2,3,4.
More preferred example is that the structure of described tacrine short-chain dimer compounds is as follows:
Wherein, n=2,3,4.
Tacrine (tacrine): chemistry tacrinenex by name is an alkaline compound, and commodity are by name
Tacrine short-chain dimer comprises: two (2) tacrine [bis (2)-tacrine], two (3) tacrine [bis (3)-tacrine] and two (4) tacrine three alkaline compounds such as [bis (4)-tacrine] and are white powder behind the hydrochloric acid salify.
Neurodegenerative diseases of the present invention comprises senile dementia, apoplexy and parkinson.
Tacrine short-chain dimer compounds and pharmaceutically useful salt thereof involved in the present invention can be used as raw material, add acceptable accessories and make oral formulations or injection, such as, can add appropriate excipients and make tablet, also can be mixed with aqueous injection or lyophilized injectable powder with its pharmaceutical salts.
The present invention is based on the present pathomechanism theoretical knowledge of the most authoritative up-to-date neurodegenerative diseases; use for reference the lesson of research and development treatment neurodegenerative diseases medicine successful experience and failure; be treatment target, the multidisciplinary novel nerve protective medicine that has the anti-neurodegenerative diseases of pathology startability feature in conjunction with development " the many target position of unimolecule " at the multi-pathogenesis of disease and the too many levels of pathological process.
The present invention overcomes the deficiencies in the prior art, provides a kind of and can have simultaneously the novel dimer medicine of " pathology startability " and " the many targets of a medicine " characteristics.Treatment developing new approaches and new method for neurodegenerative diseases; For the neurodegenerative diseases treatment provides a class brand-new medicine.The object of the present invention is achieved like this:
The application of tacrine short-chain dimer in preparation treatment neurodegenerative diseases medicine; Particularly preparation treatment senile dementia (Alzheimer ' s disease, AD), parkinson disease (Parkinson ' s disease, PD) and the application in apoplexy (Stroke) disease medicament.
The generation of nervous system degenerative disease and the molecular mechanism of development thereof are not still understood, also lack effective prophylactic treatment medicine, so present Therapeutic Method is still based on to the ill.Because these Therapeutic Method are not fundamentally eliminated the cause of disease of nervous system degenerative disease, therefore can not stop course of disease progress, disease still can the deterioration of carrying out property.Along with people to nervous system degenerative disease understanding deeply and the raising that high-quality life is required, the demand of the medicine of antagonism Neuron Apoptosis increases considerably.The medicine of anti-apoptosis prevents the development and the deterioration of nervous system degenerative disease for neuroprotective unit, improves patient's symptom and improves the quality of living, and it is all significant to alleviate burden on society.Along with the world and China's aged tendency of population process are accelerated, the ill colony of nervous system degenerative disease is increasing.Nervous system degenerative disease has become society and heavy family burden, and it is not only a great medical problem, and is a social problem.Aging should not be human helpless wait again, paid close attention to nervous system degenerative disease, and how could let, and a moment is slow! Increasing result of study shows: the pathology startability medicine of " the many target position of unimolecule " is the most promising developing direction of treatment nervous system degenerative disease, and its theory and practice also will help the further pathogeny of understanding nervous system degenerative disease.Therefore, develop many targets treatment nervous system degenerative disease new drugs and have urgent medical demand, and huge science, economic and social benefit.
The present invention provides aspect senile dementia, apoplexy and the parkinson determined curative effect, the lower medication of side effect to select for tacrine short-chain dimer medically treating.
Description of drawings
Fig. 1 is the structural formula of tacrine in the prior art that the present invention relates to.
Fig. 2 is the structural formula of the tacrine short-chain dimer compounds that the present invention relates to.
Fig. 3 is the tacrine short-chain dimer structural formula of one of them embodiment of the present invention.
What Fig. 4 showed is the ideograph of the experimental result of computer molecular docking;
What Fig. 5 showed is the pattern diagram of the experimental result of second computer molecular docking;
Fig. 6 is the cartogram as a result of the glutamate neurotoxicity of 75 μ M and the tetrazolium blue of protection (MTT) colorimetry,
Fig. 7 is the cartogram as a result of the glutamate neurotoxicity of variable concentrations and the tetrazolium blue of protection (MTT) colorimetry;
Fig. 8 is chlorinated triphenyl tetrazole (TTC) coloration result of temporary local cerebral ischemia (MCAO) brain sheet;
Fig. 9 is cartogram-single brain sheet of above-mentioned Fig. 8 coloration result;
Figure 10 is cartogram-total ischemic region of Fig. 8 coloration result;
Figure 11 shows that tacrine short-chain dimer bis (3)-tacrine has the interior neuroprotective of body efficiently.
Figure 12 is the map of current (B3T 0.3 μ M, NMDA30 μ M) of the hippocampal cell of the inductive In vitro culture of NMDA;
Figure 13 is the map of current of the hippocampal cell of the inductive In vitro culture of NMDA;
Figure 14 is receptors ligand competitive binding experiment figure; Show that short chain dimer bis (3)-tacrine is at MK-801 site blocking-up nmda receptor.
Figure 15 is the inductive Hippocampus long time of high frequency stimulation journey enhancing figure; Show that short chain dimer bis (3)-tacrine does not influence nmda receptor mediation long time journey and strengthens (LTP, physiological) effect.
In addition, in description of the present invention, B3T is the abbreviation of tacrine short-chain dimer bis (3)-tacrine.
The specific embodiment
Below by describing the present invention in conjunction with the accompanying drawings and embodiments in detail:
One, the present invention is by means of molecular docking art designs and synthesizing new dimer
We on the basis of the United States Patent (USP) of having applied for (US005783584A), use ICM-Pro 3.4-8a program (Molsoft) thus the research protein-ligand interaction instructs the synthetic of novel N-methyl D-aspartic acid receptor antagonist with rapid kinetics feature uncompetitive.By this method, we have the chemical compound of developing future according to architectural feature and the structure of drug molecule and the function of their interactional characteristics research medicine of receptor protein thereby filter out.Experimental result according to molecular docking, we find two (3) tacrine of short chain dimer to the adhesion of N-methyl D-asparagine acid acceptor than MK-801 a little less than, as Fig. 4 and shown in Figure 5, specifically, Fig. 4: two (3) tacrine of ICM-Pro 3.4-8a program (Molsoft) demonstration and the NR1 of N-methyl D-asparagine acid acceptor and the molecular docking result of NR2B heterodimer; Fig. 5 demonstration: the interaction of tacrine short-chain dimer and N-methyl D-aspartic acid receptor extracellular vestibular area: the M3 of receptor extracellular region and M1 residue are less to dimeric adhesion influence, that is to say, they are very not crucial to dimer and N-methyl D-aspartic acid receptors bind, show dimer [as two (3) tacrine] to the binding ability of N-methyl D-asparagine acid acceptor than MK-801 a little less than.
In addition, the molecular docking sunykatuib analysis of two (3) tacrine and nitricoxide synthase shows that it can be incorporated into the arginine residues in nervous system type nitricoxide synthase or inducible nitric oxide synthase activity district.These results show that tacrine short-chain dimer is the pathology actuated type medicine of potential many target spots.
Two, tacrine short-chain dimer is " pathology startability " and " the many targets of a medicine " pattern of fusion neuroprotective
Acetylcholine esterase, glutamic acid and nitric oxide are that the important biomolecule in the cell activities is regulated molecule, yet, they also play an important role in the pathogeny of senile dementia, such as the acetylcholine esterase of overexpression and the glutamic acid and the nitric oxide energy mediated cell toxic action of concentration abnormality rising.Therefore suppress the N-methyl D-asparagine acid acceptor of excessive activation and nitricoxide synthase and do not influence glutamic acid and nitric oxide production normal physiological function just seems particularly important.We expect that novel dimer can become very promising pathology actuated type medicine by moderate affinity and corresponding target spot effect.In order to confirm this point, we detect the effect of these dimers to corresponding target spot under different physiology and pathological conditions.Simultaneously, a large amount of evidences shows that the medicine of the many target spots of a medicine has the characteristic that is better than single target drug, and such medicine is difficult to obtain by having a mind to design, but can obtain by screening in many ways.Utilize molecular docking simulation, we can study the effect of medicine and acetylcholine esterase or N-methyl D-asparagine acid acceptor, thus design more synthetic have the pathology actuated type dimer of DEVELOPMENT PROSPECT.At first, we find that tacrine short-chain dimer has inside and outside neuroprotective efficiently, as Fig. 6, Fig. 7 and Fig. 8, Fig. 9, Figure 10, shown in Figure 11; Specifically, Fig. 6 shows: rat cerebellum granule neuron (cerebellar granule neurons, CGNs) two (3) tacrine pretreatment of usefulness variable concentrations are two hours, be exposed to the glutamic acid of 75 μ M then, with the 24 hour detection cell survival rates of Si Zuo Blue (MTT) colorimetry in the glutamic acid exposure.Testing result shows: the neurotoxicity of two (3) tacrine concentration dependent ground antagonism glutamate induction; Fig. 7 shows: rat cerebellum granule neuron (CGNs) was with the memantine pretreatment of two (3) tacrine of 3 μ M or 3 μ M two hours, be exposed to the glutamic acid of variable concentrations then, with the 24 hour detection cell survival rates of Si Zuo Blue (MTT) colorimetry in the glutamic acid exposure; The survival rate of cell is expressed as the percentage rate (%) of matched group (untreated fish group).Testing result shows: the protective capability that aminoglutaric acid concentration is got over Gao Shuan (3) tacrine is strong more, obviously is better than the memantine group.
Secondly, by studying the effect of these dimers to acetylcholine esterase and nitricoxide synthase, we find that two (3) tacrine can moderate suppress acetylcholine esterase (IC
50: 0.25 μ M) and neuron pattern nitricoxide synthase (IC
50: 43 μ M).More ironically, patch-clamp and radioligand binding assay result show that two (3) tacrine can suppress N-methyl D-asparagine acid acceptor in moderate uncompetitive ground, as Figure 12, Figure 13 and shown in Figure 14; Simultaneously, our result shows that two (3) tacrine do not influence the receptor-mediated long time journey of N-methyl D-aspartic acid and strengthen phenomenon, as shown in figure 15; Show that this dimer can not disturb the normal physiological function of N-methyl D-asparagine acid acceptor, and he raises along with the concentration of glutamic acid to the protective effect of the neuronal death of glutamate induction and strengthens, as shown in Figure 7; Show that he can resist N-methyl D-asparagine acid acceptor that pathology starts really.These results prove that tacrine short-chain dimer is the chemical compound that a class has pathology actuated type characteristic and many target spots.
Tacrine short-chain dimer comprises: three alkaline compounds such as two (2) tacrine, two (3) tacrine and two (4) tacrine, with be white powder behind the hydrochloric acid salify, its hydrochlorate water soluble (11.3 to 13.7mg/ml), stable in properties, but the powder room temperature preservation is more than 5 years.
According to zoopery (effective dose and pharmacokinetics characteristics), reckoning to the treatment senile dementia (Alzheimer ' s disease, AD) patient's dosage is 5mg/ days, use for convenient, the hydrochlorate of tacrine short-chain dimer and corresponding excipient can be made into 2.5 and the tablet of 5mg, pro ore; Reckoning is that 10mg is each to apoplexy (Stroke) dosage, is that first aid uses, and the hydrochlorate of tacrine short-chain dimer can be made into 5 and the injection of 10mg, uses for vein or intramuscular injection.
Show according to the data that has obtained: at cellular level, the tacrine short-chain dimer of 100 μ M or following concentration is to former neuron free of toxic effects of being commissioned to train foster.In the animal level, the tacrine short-chain dimer of 20mg/kg or following dosage does not have obvious toxic and side effects to muroid.In addition, existing research data proof dimerization tacrine can reduce the original toxic and side effects of tacrine.
The anti-middle cerebral artery occlusion of tacrine short-chain dimer/reperfusion injury analysis (zoopery).The experimental animal model of middle cerebral artery occlusion/filling is again set up and the analysis of tacrine short-chain dimer Chinese People's Anti-Japanese Military and Political College Mus focal cerebral ischemia area, cerebral edema and behavioral function obstacle is carried out according to embodiment 1.
The inductive former foster Neuron Apoptosis (cell experiment) of being commissioned to train of tacrine short-chain dimer antiglutamic acid.The receptor-mediated exitotoxicity of glutamic acid excessive activation N-methyl D-aspartic acid plays a significant role in the pathology process of neurodegenerative diseases.It is significant to the pathomechanism and the screening neuroprotective medicine of research neurodegenerative diseases to set up external glutamic acid mediation exitotoxicity model.Glutamic acid mediation exitotoxicity model is set up and the inductive Neuron Apoptosis effect of tacrine short-chain dimer antiglutamic acid and mechanism analysis are carried out according to embodiment 2 at former neuron of being commissioned to train foster.
Embodiment 1:
Tacrine short-chain dimer blocks the protectiveness intervention effect of focal cerebral ischemia area, cerebral edema and behavioral function obstacle in the ischemia/reperfusion injury experimental animal model to S D intraluminal middle cerebral artery occlusion in rats:
It is the classical model of acute focal cerebral ischemia/reperfusion injury that line bolt method is blocked the middle cerebral artery model, can more reasonably simulate the pathophysiological process of the acute focal cerebral ischemia reperfusion injuries of perioperative such as cervical region, the operation of intracranial trunk.Laboratory animal is selected and grouping: healthy male SD rat, body weight 280-320g.Be divided into 4 groups at random: sham operated rats (S group, n=10 only), normal saline group (NS group, n=10 only), tacrine short-chain dimer processed group [T1 (0.1mg/Kg) and T2 (0.3mg/Kg) group, every group of n=10 only].The preparation of focal cerebral ischemia in rats/re-perfusion model is set up middle cerebral artery occlusion model [Neuroscience Lett.439 (2008): 160-164] with reference to methods such as Longa.Behind 10% chloral hydrate (350mg/kg) intraperitoneal injection of anesthesia, the neck median line incision along rat separates right carotid, external carotid artery, internal carotid artery, and ligation is also cut off the external carotid artery trunk with standby.Bulldog clamp temporary interruption internal carotid artery distal end, common carotid artery proximal part, external carotid artery in nearly crotch inserts the fishing line (the about 0.28mm of diameter) that scribbles silica gel, unclamp internal carotid artery distal end bulldog clamp subsequently, be inserted into when 1.8-2.0mm meets slight resistance and stop.Behind the 120min fishing line is extracted, recovered the middle cerebral artery perfusion.Tighten otch, unclamp bulldog clamp and skin suture.Sham operated rats rat operation technique is identical, and fishing line is only inserted about 1.0cm.Omnidistance laser Doppler flowmetry monitoring in the art before the art; The postoperative rat is kept body temperature at 37.0 ± 0.5 ℃.
Tacrine short-chain dimer reduces cerebral ischemia volume and the cerebral edema that middle cerebral artery occlusion causes: 1,2,3,5-triphenyltetrazolium chloride (TTC) is fat-soluble photaesthesia complex, the synthetic first survival ability that is used for detecting seed in 1894, the ischemia infraction that began to be used for dyeing and to detect mammalian tissues in 1958.It is the proton acceptor of pyridine in the respiratory chain-nucleoside structure enzyme system, take on a red color with dehydrogenase reaction in the normal structure, and dehydrogenase activity descends in the ischemic tissue, can not react, and is not pale so can not change.2, get brain: directly get brain after can anaesthetizing or after the normal saline perfusion, get brain.Because cerebral tissue is not fixed with paraformaldehyde, thus softer, more should be careful when getting brain, keep the integrity of brain.3 ,-20 quick-freezing was convenient to section about 20 minutes in the degree refrigerator.4, section: generally be cut into 7, with special brain groove; Cut a slice every 2mm.First cutter is the utmost point and optic chiasma line midpoint before brain; Second cutter is at the optic chiasma position; The 3rd cutter is at the infundibular stalk position; Four blade is between the infundibular stalk and the posterior lobe tail utmost point.5, section is placed TTC, conventional concentration is 2%, also useful 1% or lower concentration.6, cover with masking foil after, put into 37c incubator 15～30min, stir the brain sheet frequently, make even contact arrive dyeing liquor.7, dyeing back effect: pale district is an infarction under the cortex.8, scanning: calculate Infarction volume and cerebral edema etc. with image analysis software.Pale district is the ischemic injuries district, by formula: V=(S
1+ S
2+ ...+S
nThe ischemic injuries volume is calculated in) * 0.5, and (V is the ischemic injuries volume in the formula, S
1～S
nBe each section ellipse area, 0.5 is constant).The result shows that two (3) tacrine of tacrine short-chain dimer can significantly reduce cerebral ischemia volume and the cerebral edema (Fig. 4) that middle cerebral artery occlusion causes.
Tacrine short-chain dimer is alleviated the behavioral function obstacle of middle cerebral artery occlusion: occur neurological deficit symptom and behavioristics after rat cerebral ischemia/reperfusion injury and change, feed significantly reduces, and body weight obviously alleviates, and activity reduces, bradykinesia.Carry out the nervous symptoms scoring with reference to stagings such as Longa in pouring into corresponding time point again.0: asymptomatic; 1: damage offside forelimb can not stretch when carrying tail; 2: during walking to the damage to sideway swivel; 3: topple over to the damage offside during walking; 4: no spontaneous activity or death.Behind the cerebral ischemia re-pouring 22 hours, each organizes the scoring of rat nervous symptoms disappearance: sham operated rats (S group), n=10 is only) be 0 ± 0.31; Normal saline group (NS group, n=10 only) is 2.3 ± 0.43; Tacrine short-chain dimer processed group [T1 (0.1mg/Kg), n=10 only] be 1.6 ± 0.31 and [T2 (0.3mg/Kg, n=10 are only] be 0.3 ± 0.45.
Embodiment 2: the inductive neuronal death of tacrine short-chain dimer antiglutamic acid
Cerebellar granule neuron is former is commissioned to train to support and carries out (JBC, 280 (2005): 18179-88) by this laboratory method of publishing an article.Take out and gave birth to 7～8 days, the cleaning level SD rat of the about 15～20g of body weight breaks end, opens cranium, gets cerebellum under the aseptic condition, places to dissect liquid removal meninges and blood vessel; Suction removes to dissect liquid, and cerebral tissue is placed on the cutting plate, is cut into 0.5mm with electric slicer then
3The piece of tissue of size; With the cerebral tissue of chopping place dissect liquid resuspended after, centrifugal 3 minutes of 1500rpm is to remove impurity; Get precipitation, place the dissection liquid that contains the 0.25mg/ml pancreatin, place 37 ℃ of water-baths 15 minutes, shook up with digestion promoting even in per 5 minutes with the outer connective tissue of peptic cell; Add the dissection liquid that contains pancreatin inhibitor and DNA enzyme, shake up back 1500rpm and digested and removed the DNA of cell breakage release in centrifugal 3 minutes with termination; Add 5ml and dissect the resuspended precipitation of liquid, and be sub-packed in two 15ml centrifuge tubes, every effective suction pipe piping and druming 20 times dispels into individual cells will assemble agglomerating cell, every then pipe adds dissection liquid mixing and leaves standstill, after 15 minutes, fragment of tissue and the cell mass precipitation that is not dispelled, supernatant is the individual cells suspension; Get the supernatant that contains individual cells after leaving standstill, centrifugal 3 minutes of 1500rpm, precipitation is cell; With 10ml complete medium re-suspended cell precipitation, behind the dilution counting, press 1.5-1.8 * 10
6The density of/ml is inoculated in the culture plate of poly-D-lysine bag quilt, places 5%CO
2And cultivate in 37 ℃ of incubators.Inoculate back 24 hours and add 12 μ mol/L cytosine arabinosides, cultivate the 5th day adding 5mmol/L glucose to replenish cellular metabolism institute energy requirement to suppress the growth and the propagation of glial cell.After 7 days, the neuron differentiation and maturation.The cerebellar granule neuron purity that this method is cultivated is greater than 95%.Cultivate the 8th day, with two (3) tacrine, concentration is at 0.1 to 30 μ M] pretreatment cerebellar granule neuron two hours, hatched altogether 24 hours with 75 μ M glutamic acid respectively then, or, hatched altogether 24 hours with glutamic acid (25 to 500 μ M) respectively then with two (3) tacrine (3 μ M) pretreatment cerebellar granule neuron two hours.Check the survival rate of cell with Si Zuo Blue colorimetry method.The result shows that two (3) tacrine dose dependent ground stop the neuronal death of glutamate induction, as shown in Figure 6.More importantly be that two (3) tacrine manifest stronger protective effect to the high more glutamic acid of concentration, as shown in Figure 7.
Main figure to experimental result of the present invention is further explained below:
As Fig. 4, shown in Figure 5, the experimental result of molecular docking show two (3) tacrine of dimer to the adhesion of N-methyl D-asparagine acid acceptor than MK-801 a little less than.
Specifically, Fig. 4: two (3) tacrine of ICM-Pro 3.4-8a program (Molsoft) demonstration and the NR1 of N-methyl D-asparagine acid acceptor and the molecular docking result of NR2B heterodimer; Fig. 5 demonstration: the interaction of tacrine short-chain dimer and N-methyl D-aspartic acid receptor extracellular vestibular area: the M3 of receptor extracellular region and M1 residue are less to dimeric adhesion influence, that is to say, they are very not crucial to dimer and N-methyl D-aspartic acid receptors bind, show dimer [as two (3) tacrine] to the binding ability of N-methyl D-asparagine acid acceptor than MK-801 a little less than.
Fig. 6, Fig. 7. dimeric two (3) tacrine of short chain have external efficiently neuroprotective.
Specifically, Fig. 6 shows: rat cerebellum granule neuron (cerebellar granule neurons, CGNs) two (3) tacrine pretreatment of usefulness variable concentrations are two hours, be exposed to the glutamic acid of 75 μ M then, with the 24 hour detection cell survival rates of Si Zuo Blue (MTT) colorimetry in the glutamic acid exposure.Testing result shows: the neurotoxicity of two (3) tacrine concentration dependent ground antagonism glutamate induction; Fig. 7 shows: rat cerebellum granule neuron (CGNs) was with the memantine pretreatment of two (3) tacrine of 3 μ M or 3 μ M two hours, be exposed to the glutamic acid of variable concentrations then, with the 24 hour detection cell survival rates of tetrazolium blue (MTT) colorimetry in the glutamic acid exposure; The survival rate of cell is expressed as the percentage rate (%) of matched group (untreated fish group).Testing result shows: the protective capability that aminoglutaric acid concentration is got over Gao Shuan (3) tacrine is strong more, obviously is better than the memantine group.
Fig. 8, Fig. 9, Figure 10, dimeric two (3) tacrine of Figure 11 short chain have the interior neuroprotective of body efficiently.
Specifically, Fig. 8 demonstrates temporary local cerebral ischemia (middle cerebral artery occlusion, MCAO) chlorinated triphenyl tetrazole (TTC) coloration result of brain sheet: left column is a sham operated rats, middle row are solvent control groups, right row are two (3) tacrine (0.3mg/kg) groups, white is the ischemic region of apoplexy rat brain, show among the figure: medicine and normal saline be 15 minutes processing apoplexy rats behind temporary local cerebral ischemia, 24 hours assessment degree of ischemia, with the matched group contrast, the ischemic region of two (3) tacrine (0.3mg/kg) group is littler.Fig. 9 demonstration: behind temporary local cerebral ischemia, handled the apoplexy rat in 15 minutes, 24 hours assessment degree of ischemia, normal saline group and the statistical result of the ischemia volume of seven brains from front to back of two (3) tacrine (0.3mg/kg) group.Figure 10: medicine [two (3) tacrine (0.1,0.3mg/kg), memantine (20mg/kg)] and normal saline 15 minutes processing apoplexy rats behind temporary local cerebral ischemia, 24 hours assessment degree of ischemia, the ischemic region volume statistical result that each organizes total ischemic region shows that two (3) tacrine groups can show the minimizing ischemic region.Figure 11 shows; Medicine [two (3) tacrine (0.1,0.3mg/kg), memantine (20mg/kg)] and normal saline 15 minutes processing apoplexy rats behind temporary local cerebral ischemia, the neurobehavioral function of 22 hours assessment apoplexy rats, the result shows: two (3) tacrine (0.3mg/kg) than memantine (20mg/kg) more apparent improve the behavior disorder that temporary local cerebral ischemia (MCAO) causes (* P＜0.05, * * P＜0.01 and matched group are relatively).
Figure 12, Figure 13. dimeric two (3) tacrine appropriateness uncompetitive blocking-up N-methyl D of short chain-asparagine acid acceptor.
Specifically, Figure 12 shows: two (3) tacrine agonist (N-methyl D-aspartic acid) dependency ground blocking-up N-methyl D-asparagine acid acceptor.Figure 13 shows: two (3) tacrine voltage-dependent blocking-up N-methyl D-asparagine acid acceptor, in the time of cell voltage is from-70 to+50mV, voltage is low more, the ability of two (3) tacrine blocking-up N-methyl D-asparagine acid acceptor strong more (being tacrine appropriateness uncompetitive blocking-up N-methyl D-asparagine acid acceptor).Annotate: the amplitude of electric current is the result according to the current standardization of 30 μ M N-methyl D-aspartic acid group (asterisk person) among the figure.
Dimeric two (3) tacrine of Figure 14 short chain are at MK-801 site blocking-up N-methyl D-asparagine acid acceptor.
Specifically, Figure 14 shows: with the memebrane protein of cortex of cerebellum with [
3H] MK-801 (4nM) and two (3) tacrine of increasing concentration gradually hatched 30 minutes jointly, detected the competitive binding ability with N-methyl D-asparagine acid acceptor to MK-801 of two (3) tacrine.The result shows: two (3) tacrine can competitive be incorporated into the MK-801 site of N-methyl D-asparagine acid acceptor.Two (3) tacrine is to the contention parameter K of MK-801
iBe to utilize formula K
i=IC
50/ (the 1+[part]/K
d) according to corresponding half amount of suppression IC
50, and IC
50Value be utilize at least eight two (3) tacrine that increase concentration gradually with [
3H] MK-801 (4nM) hatch acquisition in 30 minutes (each sample triplicate) jointly.
Dimeric two (3) tacrine of Figure 15 short chain do not influence the receptor-mediated long term potentiation of N-methyl D-aspartic acid.
Preparation of rat hippocampus brain sheet and external electrophysiological technique are pressed document [J Neuroscience 2004,24 (13): 3370-3378] and are carried out.Figure 15 demonstration: at hippocampal slices, the long time journey of two (3) the tacrine processed group of record post-stimulatory matched group of 200Hz and 3 μ M strengthens (Long term potentiation, LTP, physiological) effect; In the time of 60 minutes, the LTP of matched group and two (3) tacrine groups is respectively 149 ± 8% and 152 ± 9%; The result shows: two (3) tacrine does not influence the post-stimulatory long term potentiation of 200Hz.
Claims (3)
1, the purposes of tacrine short-chain dimer compounds in the medicine of preparation treatment neurodegenerative diseases, described tacrine short-chain dimer compounds is chemical compound and the pharmaceutically useful salt thereof with following structure:
Wherein, R
1, R
2Be H or (C
1-C
4) alkyl, n=2,3,4.
2, according to the described purposes of claim 1, it is characterized in that: the structure of described tacrine short-chain dimer compounds is:
Wherein, n=2,3,4.
3, according to the described purposes of claim 1, it is characterized in that: described neurodegenerative diseases comprises senile dementia, apoplexy and parkinson.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104473926A (en) * | 2014-11-27 | 2015-04-01 | 宁波大学 | Medicament for improving synaptic function |
| WO2019130135A1 (en) * | 2017-12-28 | 2019-07-04 | International Business Machines Corporation | Neuron model generation for personalised drug treatment |
| GB2583309A (en) * | 2017-12-28 | 2020-10-21 | Ibm | Neuron model generation for personalised drug treatment |
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| WO2019130135A1 (en) * | 2017-12-28 | 2019-07-04 | International Business Machines Corporation | Neuron model generation for personalised drug treatment |
| GB2583309A (en) * | 2017-12-28 | 2020-10-21 | Ibm | Neuron model generation for personalised drug treatment |
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