CN1784241A - Glutamate receptor antagonists as neuroprotectives - Google Patents

Abstract

The invention relates to the use of an inhibiting agent of t-PA of mediated activation of the glutamate receptor, preferably of the NMDA type, as neuroprotectives.

Description

Glutamate receptor antagonists as neuroprotective

The present invention relates to be used for the treatment of and prevent nerve injury (particularly by the superactivation of methyl-D-asparagic acid type glutamate receptor (hereinafter being called the NMDA-glutamate receptor) indirectly or the damage that directly causes) neuroprotective.The present invention requires the priority of German patent application 103 37 098.6,103 20 336.2 and 103 52 333.2, and the present invention quotes these patent applications with regard to its content.

Within the scope of the invention, term " protective agent " is meant therapeutic agent or the active substance that helps the neuroprotective cell to avoid cell injury and/or opposing neurodegenerative process and neuronal function infringement.

When the exploitation neuroprotective; the effort of essence particularly is to identify and/or further develop glutamate receptor antagonists; this glutamate receptor antagonists prevents that neurocyte from avoiding the influence of the enhanced activity (excitotoxicity) of excitability neurotransmitter receptor, perhaps alleviates superactivation.Most important one group in the described receptor antagonist is the NMDA-antagonist, and it suppresses NMDA type glutamate receptor.At this, be different between the following agonist: (it acts on the glutamic acid binding site to competitive NMDA-antagonist such as 2-amino-5-phosphono valerate for (D)-AP5) and (+/-)-4-(4-phenyl benzoyl)-piperazine-2,3-dicarboxylic acids (PBPD); NMDA-receptor channel blocker such as MK-801, memantine and ketamine, the ion channel of its blocking-up receptor; Act on the NMDA-antagonist such as the GV96771A of glycine binding site point, it suppresses the combination of co-agonists glycine; With polyamines site antagonist such as ifenprodil, it suppresses the combination of the polyamines of costimulatory receptor.

The three class glutamate receptors (AMPA-, Kainat-and NMDA-receptor) that play a role as the ion channel that relies on glutamic acid are passed on the postsynaptic signal of most important excitability neurotransmitter glutamate separately.The NMDA-glutamate receptor is dispersed in the whole brain with different receptor subtype forms, and it mainly is as conclusive excitability neurotransmitter receptor for central nervous system's (ZNS) function.

In recent years, in depth studied the molecular biology of NMDA-glutamate receptor.It shows, these receptors unite one or more NR2-subunits by a NR1-subunit and (more rare) NR3-subunit is formed (Das, S. wait the people: Increased NMDA current and spinedensity in mice lacking the NMDA receptor subunit NR3A.Nature393 (1998), 377-381; Chatterton, people such as J.E.: Excitatoryglycinereceptors containing the NR3 family of NMDA receptor subunits.Nature 415 (2002), 793-798).

Particularly have the NR2-subunit of four kinds of forms of A-D, the selectable splice variant of subunit NR1 has determined the pharmacology of NMDA-glutamate receptor in addition.Express in ZNS on NR1 and NR2A ubiquity ground, and NR2B, NR2C and NR2D are present in the specific region of brain more singularly or only.This of receptor subtype be different forms the therapeutic scheme of the antagonist that makes it possible to adopt selectively acting.

The glutamic acid that presynaptic discharges is as the excitability neurotransmitter, and glycine is all responsible for the complicated activation of NMDA-glutamate receptor as modulability neurotransmitter and co-agonists.The activation of receptor causes the inflow of calcium ion, and combine with complicated cell signal (particularly when the lasting stimulation of long period).This especially causes the phosphorylation of CRE conjugated protein (CREB), and causes multiple gene activation and synaptic plasticity (neural plasticity), and this is the basis of the internal of memory effect and learning process.

Overview about the general pharmacology of NMDA-receptor and NMDA-antagonist can be from Kemp, J.A. and McKernan, R.M.:NMDA Receptor Pathways as Drug Targets, Nature Neuroscience, in November, 2002, the 5th volume supplementary issue obtains in the disclosure of 1039-1042, and its disclosure has relation very widely.

In contrast, the superactivation of receptor and relevant thus Ca ²⁺The increase that flows into causes depending on Ca ²⁺The serious disorder of cellular material metabolism (comprising energy metabolism).Particularly produce the responsible Ca of depending on of cell death that occurs for subsequently ²⁺Activation (Lee, people such as Jin-Mo, " the The changing landscape of ischemic braininjury mechanisms " of catabolic enzymes; Dennis W.Zhol " Glutamat neurotoxicity anddeseases of the nervous system ").This process is called excitotoxicity.

, excitotoxicity causes that this process threatens or the neuron state of damage has the potentiality for the treatment of and/or preventing usually so the antagonist of glutamate receptor is for the ZNS disease or for being subjected to because being the superactivation by glutamate receptor.Cerebral ischemia that occurs under the situation of apoplexy and neurodegenerative disease such as parkinson disease and Huntington Chorea all belong to described " state ", and the wherein main cause of disease is not an excessive glutamic acid but for the enhanced sensitivity of excitotoxicity damage.At last, disease that causes owing to the overactivity of excitability signal pathway such as epilepsy and neuropathy degeneration pain also are the possible applications of the antagonist of glutamate receptor.

Other application examples are as relating to particularly chronic pain of treatment pain status, treatment addictive disorders, treatment mental sickness and stimulation learning and memory efficient.

Antagonist can be competitively or noncompetitive ground suppress receptor.The receptor activation that competitive antagonist opposing is for example caused by natural agonist glutamic acid and glycine, and noncompetitive antaganist with do not rely on agonist exist or the mode of concentration suppresses receptor, for example by the blocking-up ion channel.The for example available 2-amino of the competitive inhibition (antagonism) of NMDA type glutamate receptor-5-phosphono valerate (APV) or 2-amino-5-phosphono enanthate (APH) carry out.On the contrary, the noncompetitive inhibition can be by realizing with the bonded material of phencyclidine one side such as phencyclidine, MK-801, dextrorphan or the ketamine of passage.

Carry out the NMDA-antagonist up to now as protectant clinical research, particularly when apoplexy and traumatic cerebral trauma, still still do not obtained desirable result (Kemp, J.A.; Kew, J.N.C.; Gill, R.:Handbook of ExperimentalPharmakology, the 141st volume (editor Jonas, P.﹠amp; Monyer, H.) 495-527 (Springer, Berlin, 1999); Lees, K.R. wait the people: Glycine antagonist (gavestinel) in neuroprotection (GAIN International) inpatients with acute STROKE:a randomised controlled trial.GAINInternational Investigators.Lancet 355 (2000), 1949-1954; Sacco, people such as R.L.: Glycine antagonist in neuroprotection forpatients with acute stroke:GAIN Americas:a randomizedcontrolled trial.JAMA 285 (2001), 1719-1728).

Except other, so far effect little for the trial of the neuroprotective under the apoplexy situation may owing to, known neuroprotective must make up with thrombolytic agent, so that be deep into the tissue injury position and play a role there.In addition, known NMDA-glutamate receptor antagonists can not be used to obtain desired effect with sufficiently high dosage usually owing to its sizable side effect.When the high dose administration, especially can cause hallucinations and significantly hypertension.The latter is extrahazardous for the paralytic just naturally.

In addition, the known glutamate receptor antagonists of using with the required dosage of neuroprotective usually has the unacceptable stupor property that causes strongly to narcotic effect.Therefore, a large amount of NMDA-antagonist such as phencyclidine and the ketamine purpose that is used for no-feel condition and anesthesia is originally developed.

Therefore, attempting how avoiding using these side effect of receptor-selective NMDA-antagonist.To this, NR2B selective antagonist ifenprodil is verified to be demonstrated favourable effect and has the side effect (Gotti that obviously reduces in the apoplexy animal model, B. wait the people, Ifenprodiland SL 82.0715 as cerebral anti-ischemic agents.Evidence forefficacy in models of focal cerebral ischemia.J.Pharmacoil.Exp.Ther.247 (1988), 1211-1221).Many other antagonist (for example CP-101606, Ro 25-6981 and Ro 63-1908) (Kemp, J.A. of NR2B subunit have been described so far; Kew, J.N.C.; Gill, R.:Handbook of ExperimentalPharmakology, the 141st volume (editor Jonas, P.﹠amp; Monyer, H.) 495-527 (Springer, Berlin, 1999); People such as Gill R.: Pharmacologicalcharacterization of RO63-1908 (1-[2-(4-hydroxyl-phenoxy group)-ethyl]-4-(4-methyl-benzyl)-piperidines-4-01), a novel sinotype-selectiveN-methyl-D-asparatate antagonist.J.Pharmacol.Exp.Ther.302 (2002), 940-948).These antagonisies show neuroprotective fully and have small side effect in animal model.Yet these positive results do not obtain confirming in the clinical research of traumatic cerebral trauma.CP-101606 over and over again inoperative (Pressemitteilung von Pfizer, October calendar year 2001) more precisely.

In addition; in the time of the suitable neuroprotective of the cell injury when searching is used to reduce apoplexy; also have this special problem, promptly neuroprotective must make up with fibrinolytic agent/thrombolytic agent, so that cross thrombosis barrier and the zone interior (on seeing) of going deep into damaged tissues.T-PA uses as thrombolytic agent usually, and it is at present unique thrombolytic agent that allows to be used for the apoplexy treatment.

But, can know t-PA (the Nicole O that in excitotoxicity, plays an important role by people's such as Nicole research; Docagne F Ali C; Margaill I; CarmelietP; MacKenzie ET; Vivien D and Buisson A, 2001:The proteolyticactivity of tissue-plasminogen activator enhances NMDAreceptor-mediated signaling; In:Nat Med 7,59-64).Like this, unpolarized cortical neuron discharges t-PA, and the NR1 subunit of this t-PA and NMDA type glutamate receptor interacts and with its disassociation.Thereby the activation of receptor active appears.So, use the excitotoxicity that t-PA can help damaging cells.The neurotoxic effect that occurs t-PA thus.Therefore; exploitation is used to prevent and the neuroprotective of the cell injury when treating apoplexy is faced with special challenge; this neuroprotective can not only alleviate the cell injury that apoplexy causes, and can consider the damage that these cause or strengthen owing to the therapeutic agent that is used to reopen blood vessel as much as possible.

Therefore, task of the present invention provides a kind of selectable probability that is used for the treatment of and prevents nervous system injury.

This task is accomplished by using the active material as neuroprotective of inhibition t-PA.In this manual, term " inhibition " comprises that all cause the active effect that weakens or reduce of t-PA.At this, this term for example relates to the competitiveness of t-PA or the degraded that noncompetitive suppresses, accelerates, and perhaps also relates to the minimizing that t-PA expresses.Term " inhibitor " correspondingly relates to all and cause the active material that reduces of t-PA in cell.

The t-PA activity is defined as especially the activation of glutamate receptor (preferred NMDA type glutamate receptor).Neuroprotective for the activatory abated effect of glutamate receptor preferably by Ca in the cell that is determined at linked groups under the situation that gives neuroprotective ⁺⁺Influx determine.Be used to describe Ca ⁺⁺Algoscopy be known for the technical staff, and in numbering among the II

Provide among the embodiment 3.

The inhibitor of t-PA is well-known.For example known, the activity of t-PA can be regulated by inhibitors of plasminogen activator inhibitor (PAI).Similarly, t-PA can be connected protein I (PN-1) inhibition by neural serpin of protease or protease.These inhibitor are the serpin that suppresses t-PA in physiology's category.But they are as neuroprotective according to the present invention.

Also may reduce the t-PA activity by the transcription rate that increases physiologic depressor indirectly according to the present invention.For example can use TGF-β (transforming growth factor), hence one can see that, and it has stimulated transcribing of PAI-1.

In particularly preferred embodiment of the present invention, as neuroprotective, it stems from (pathology) state of excitotoxicity especially for treatment with plasminogen activating factors DSPA (desmoteplase).DSPA with isotype is especially at patent document US5,830,849 and US 6,008,019 in be described in detail.The reorganization preparation of DSPA is disclosed in the US patent document 5,731,186.Purpose for the structure, function and the preparation that disclose DSPA is quoted from these patent documentations comprehensively.

The primary structure of the preferred especially desmoteplase isotype that uses is described in (DSPA α 1) among Fig. 1.Yet, can also use the identical DSPA isotype of other functions according to the present invention.These isotypes are disclosed in the above-mentioned US patent equally.These plasminogen activating factors are unified hereinafter to be called DSPA or desmoteplase, and it is not defined as DSPA α 1.

According to the present invention, not only can use natural purified DSPA, and can use the DSPA of reorganization preparation.Can use derivant or the fragment of DSPA equally, as long as it has the neuroprotective of DSPA.Therefore, this DSPA that term " DSPA " should be interpreted as natural or reorganization with and function is identical basically derivant, analog or segmental upperseat concept.

Especially, term DSPA " derivant, analog or fragment " comprises protein or the peptide that all are such, being these protein or peptide have natural DSPA on function distinctive characteristic, mainly is to have the fibrin-specific that increases with respect to natural t-PA.The fibrin-specific with respect to the t-PA increase of DSPA can be known from WO 03/037363.Preferably, the aminoacid sequence of the DSPA of DSPA derivant and analog and Fig. 1 has at least 70%, the preferred homology of 80-90% at least.

Wonderful neuroprotective according to DSPA of the present invention is determined in animal experiment and in vitro tests; show in these trials; DSPA makes it not only not have neurotoxic effect by the neurotoxic effect of opposing t-PA, and has neuroprotective.Wherein known DSPA is as the antagonist of t-PA and play a role.In addition, can also show that when DSPA was not promptly having the outside to give to use under the situation of t-PA separately, the DSPA of high concentration can also cause the minimizing of the neuronal damage that NMDA brings out.

In order to study the neuroprotective of DSPA, handle culture with NMDA, so that with the first cell death of its inducing neural from the isolating cortical neuron of mice embryonic with experiment.Except NMDA, culture medium also contains not commensurability t-PA and/or DSPA.These in vitro testses show, t-PA has the effect of the cell death that reinforcement causes by NMDA, and DSPA does not produce these effects (Fig. 2).If DSPA is used with t-PA, the cell death that brings out of NMDA is compared remarkable minimizing when using t-PA separately so.In addition, this is apparent that DSPA can reduce by the enhanced neural cell injury of t-PA significantly in the mode that depends on concentration.

In addition, under the situation that gives DSPA or t-PA in advance, be used to measure Ca ⁺⁺The research of influx is because the Ca that will be caused by the activation of glutamate receptor ⁺⁺The minimizing that flows into is considered as the indication of the neuroprotective of every kind of test substances.In these trials, the neuroprotective of DSPA has also obtained confirmation, because DSPA does not increase the Ca in the cell ⁺⁺Influx, and the damaging effect (Fig. 4) of opposing t-PA.

The mechanism of the neuroprotective of DSPA is not still thoroughly understood.But, though it may be because DSPA is the same as with t-PA glutamate receptor, these glutamate receptors that do not dissociate, thereby do not activate glutamate receptor.DSPA itself does not contribute for excitotoxicity, and may block the receptor activation that is caused by t-PA.

DSPA is wonderful as the ability of neuroprotective, particularly under this background of neurotoxicity with regard to known t-PA since nineteen ninety-five.Because DSPA and t-PA have the concordance of significant function and structure, thus can estimate in other words DSPA also be have neurovirulent.

DSPA used according to the invention or its derivant or a segmental special advantage are based on following situation; promptly because the neuroprotective of the DSPA that is surprisingly found out that, thereby can use the therapeutic agent that not only has the fibrinolysis characteristic but also have neuroprotective properties.

This advantage particularly plays a role when apoplexy is treated, because the neurotoxic side effects of the t-PA that especially be attributable to self and that use for therapeutic purposes alternatively of the tissue injury relevant with apoplexy.At least can resist these damaging effects of t-PA by the neuroprotective of DSPA.

Therefore, in a particularly advantageous embodiment of the present invention, DSPA can treatment during apoplexy as neuroprotective and with thrombolytic agent for example t-PA unite and use.Therefore, the treatment advantage of t-PA can be utilized, and the neurotoxic side effects of t-PA can be neutralized or weaken at least simultaneously by DSPA as neuroprotective for the patient.

Material according to the present invention as neuroprotective can be used for the treatment of many pathologic state (on seeing).Belong to and treat neurodegenerative disease such as parkinson disease, Alzheimer, Huntington Chorea and diabetes comprising of this application possibility, the treatment pain status, the treatment addictive disorders, treatment neural and mental sickness such as epilepsy, the dyskinesia, depression, frightened state and dysmnesia, the cognitive efficient of general improvement, and treatment amyotrophic lateral sclerosis.In the pathogeny of these diseases or state, the superactivation of NMDA-glutamate receptor respectively plays significant effect.

In I phase clinical research, obtained neuroprotective used according to the invention therapeutic effect for patient's emotional state.In this research, comprised the patient who is in emotional state (depressed and frightened state) with the DSPA treatment.

Term " depression " defines with broad sense at present, and promptly this term comprises that all can not be counted as on the emotion of the appropriate reaction of external condition or spiritual obstacle, and does not rely on the physiology or the Psychology Background of its appearance.Term " depression " especially also comprises frightened state.Therefore, term " antidepressant " comprises the therapeutic agent that is used for the treatment of these obstacles widely.

Execution is used for estimating DSPA α 1 in the clinical efficacy of acute ischemic stroke and the clinical research of safety (Desmoteplase in Acute Ichemic Stroke hereinafter is called DIAS-research).At this, this clinical research is an II phase double-blind study that contrast with placebo, at random, in this research, within 3-9 hour DSPA is used in intravenous mode after the apoplexy symptom beginning to occur.

In the first of this research, there are 46 patients to participate in, whether they still wait to rescue and select according to its brain district that suffers the brain district of apoplexy whether irreversibly to sustain damage or be partially damaged or endangered.Only accept such patient in this research, promptly previous impaired brain district may rescue by infusion again with thrombolytic agent in these patients.This carries out for selecting by MRI (nuclear magnetic resonance, Magnetic ResonanceImaging) of patient.

In 46 patients that study, 16 patients use placebo treatment.Remaining 30 patients have obtained the desmoteplase preparation, wherein 17 patients with the treatment of 25mg active substance and 13 patients with 37.5 or the 50mg active substance treat.

The patient with placebo treatment above 56% reports and depression occurs.Though these depressed quilt treatment doctors separately clearly are diagnosed as depression, can not clearly classify as depressed special representing form owing to lack further information.Thereby this depressive symptom is treated doctors and is categorized as " NOS depression " separately, i.e. " (not otherwisespecified) depression that does not specify in addition ".

As mentioned above, the patient with placebo treatment who surpasses half demonstrates the depression that these do not specify, yet in comparable desmoteplase treatment group, the ratio that relates to depressed patient only is 16.7% altogether.

In the second portion of this research, there are 56 patients to participate in, inter alia, these patients also treat with the DSPA of 62 μ g/kg, 90 μ g/kg, 125 μ g/kg or with placebo.The result of the second portion of this research is not only aspect infusion again but also in the result who has confirmed first aspect the frequent degree reduction of depression.

From these results, for DSPA α 1, greater than 60 and to be used for the treatment of emotionally disturbed less than the dosage, the particularly dosage of 90-125 μ g/kg of 160 μ g/kg be preferred.But, emotionally disturbed to alleviate at higher dosage also be possible.

When using other DSPA isotypes or DSPA derivant, analog or fragment, dosage can depart from above-mentioned scope.In these cases, dosage is advantageously adjusted according to the bioequivalence separately of used material.

This low dosage is favourable during depressed after apoplexy particularly, and in this case, seance is undertaken by the intravenous infusion administering mode.The DSPA of 90 μ g/kg may be just enough so.Under the situation of (Asia) life-time service, much lower dosage also may be enough.This is particularly useful for t-PA inhibitor used according to the invention, the inhibition that this inhibitor causes endogenic t-PA to produce.On the contrary, under the inhibitor situation of the competitive inhibition that causes nmda receptor, above-mentioned dosage is preferred.For the sick type of minority acute illness, subcutaneous, oral or suction-type ingredients is favourable.

T-PA is known for people's the emotional state and the meaning of learning behavior.T-PA plays a part positive (Pawlak, 2002 and 2003) for learning behavior in the human body of health.Similarly, from among the research of t-PA shortage type mice mutant as can be known, the t-PA in the tonsil is a key factor (people such as Pawlak, 2003) of the frightened state of stress-induced and is important (Pawlak, 2003) in the inducing of fear.In contrast, depressed generation is seemingly relevant with so-called neuronal plasticity, is in for example injured health of stress state the neuronal plasticity reaction can take place.According to hitherto known knowledge, this process especially can be regulated by the glutamate receptor activity of t-PA mediation.

From known research, can summarize, by stress or the t-PA of the injured health that discharges itself be a kind of signal for postsynaptic cell, and play a part " the triggering agent " that neuronal plasticity changes.

The basic mechanism of neural plasticity may be so-called long-term reinforcement (long termpotentiation, LTP) and long-term depressed (long term depression, LTD).LTP causes owing to altofrequency ground stimulating neuronal and the enhanced depolarization that causes thus.This long reinforcement is based on the activation of the NMDA-glutamate receptor that relies on glutamic acid, and this activation causes Ca ⁺⁺The increase that discharges.The increase of calcium concentration causes the increase of synapse effect, thereby makes it possible to adapt to situation separately.

LTD can be used as aforementioned LTP's " compensation " or induce generation by low-frequency electrostimulating again.So the same with LTP, its mechanism is based on the activation of NMDA type glutamate receptor.But this causes medium Ca ⁺⁺Influx, thereby cause the just synapse effect of reduction.This causes depression.

Present known t-PA dissociates them in conjunction with NMDA type glutamate receptor, activates them thus.Influence with regard to viewed neuronal plasticities of people such as this Pawlak can be explained by t-PA.According to the quantity of the t-PA that discharges, not only can explain the generation of long-time reinforcement, and can explain the generation of long-time depression.

Therefore, can derive from the described research according to neuroprotective of the present invention that is used for DSPA, DSPA blocks the activation of NMDA-glutamate receptor as antagonist, thereby finally can resist long-time depression.

DSPA and t-PA also can understand (on seeing) by the moving experiment of the calcium current of neuronal cell for the not same-action of LTP and/or LTD thus for synaptic activity.These in vitro testses show, handle the calcium release that neuronal cell causes depending on NMDA with t-PA, and carrying out same processing with DSPA does not then influence (see figure 4) for intracellular calcium concentration.The concentration increase that is caused by t-PA is 30% with respect to matched group.

Embodiment

I. clinical research (DIAS)

Be summarised among Fig. 5 for the result of the DIAS research of the effect of emotionally disturbed (depressed and frightened state) about DSPA.

II. for the in vitro study of murine cortical neuron

Cultivate cortex mammalian nervous unit originally:

The Swiss White mice (the Zentrales Tierhaus of Monash university) of gestation was put to death at gravidic 14-16 days, and take out the uterus.Under aseptic condition, take out the tire Mus, cut down head, the brain that dissociates, and under section microscope (Industrial and ScientificSupply Co.), prepare cerebral neocortex by microscopic section.This slicing processes on ice in containing 3mg/ml bovine serum albumin (BSA) and 1.2mM MgSO ₄Hank ' s balanced salt solution (the HBSS of (pH 7.4); 137mM NaCl, 5.37mM KCl, 4.10mM NaHCO ₃, 44mM KH ₂PO ₄, 0.13mM NaHPO ₄, 10mM HEPES, the 1mM acetone acid, 13mM D (+) glucose and 0.001g/L are phenol red) in carry out.Carefully remove meninges and blood vessel meticulously.To pieces fritter is smashed into tissue in tip with plastic suction pipet, and carry out with the intensity of 1000g at short notice centrifugal, to collect fragment.To segmentally be deposited in warm (37 ℃), contain trypsin 0.2mg/ml) and deoxyribonuclease I (DNase I, HBSS 880U/ml) (has 3mg/ml BSA and 1.2mM MgSO ₄) in suspend again, and in rock type water-bath in 37 ℃ of incubations 5 minutes.Segmental digestion contains trypsin inhibitor (83.2 μ g/ml), DNase I (880U/ml) and MgSO by what add same volume ₄HBSS (1.22mM) (having 3mg/ml BSA) and stopping, and with 1000g centrifugal 5 minutes.Supernatant is removed in suction, and adding contains trypsin inhibitor (0.52mg/ml), DNaseI (880U/ml) and MgSO in precipitation ₄HBSS (2.7mM).Tissue is taken apart (carrying out 15 times with No. 24 pins clashes into) by grinding, and with 1000g centrifugal 5 minutes.Supernatant is removed in suction, cell is being contained 2%B27 fill-in (Invitrogen, USA), NeurobasalTM culture medium (Invitrogen, the USA of 100U/ml penicillin and 100 μ g/ml streptomycins, the hyclone (dFCS) of 0.5mM L-glutaminate and 10% through dialysing; NBM) suspend again in, this culture medium is called complete NBM hereinafter.The cell density of suspension and cultivation yield are determined by carry out multiple cell counting in the blood-counter system cell.Each with cell with 0.3 * 10 ⁶Or 0.12 * 10 ⁶The density of individual cell/sample room is seeded in Nunc ^TMIn the flat board of (Denmark) 24-or 96-sample, this is defined as the external persistent period of 0 day (0div).Realization applies flat board with poly-D-lysine (50 μ g/ml), to promote cell attachment.This coating is to be removed after 37 ℃ of incubations that spend the night.24 hours (1 cell division) replaces complete NBM afterwards with the complete NBM (2.5%B27 fill-in) that does not contain dFCS.After all 3-4 time division, replace half of the complete NBM do not contain serum.Cell remained on 37 ℃ moistening CO ₂In the incubator, and with inverted phase contrast microscope (Olympus IMT-2) studies.In order to prove the cell culture morphology in the cell injury process, and take pictures (Kodacolor Gold 100 ISO films).If do not indicate in addition, all operating procedures are all at room temperature carried out.

1.t-PA and DSPA is for the effect by the inductive neuronal cell death of NMDA

In order to check increase, in the cortical neuron of cultivating according to such scheme, add NMDA (30 μ M or 70 μ M), and culture was cultivated 24 hours again by t-PA and the inductive cell death of the caused NMDA of DSPA.The degree of inductive cell death measure by the lactic acid dehydrogenase (LDH) that measure to discharge, the dissolving fully that wherein will use TX-100 is (Fig. 2) in contrast.Then, under the situation that the concentration of t-PA (5,50,250,500nM) or DSPA (5,50,500nM) increases separately gradually, in culture, add NMDA, and measure cell death in a similar fashion.

The NMDA that adds 30 μ M or 70 μ M all causes significant cell death (see figure 2) under two kinds of NMDA concentration.Under the situation that the concentration of t-PA increases gradually, observe the inductive cell mortality of institute and raise, but (see figure 2) in this highest separately t-PA concentration of 500nM just.In contrast, under the situation of DSPA, do not observe the reinforcement of the inductive cell death of NMDA, particularly when high DSPA concentration, observe the minimizing of the inductive cell death of NMDA even.Under the situation that does not have NMDA, t-PA and DSPA all do not cause the dead (see figure 2) of neuronal cell.

2.DSPA cause the reduction of the inductive cell death of t-PA

In other in vitro tests, find out, add the enhancing whether DSPA can resist the inductive cell death of NMDA of t-PA triggering.For this reason, under the situation of the DSPA concentration that has constant t-PA concentration (500nM) and increase gradually (5,50 and 500nM), each adds the NMDA (Fig. 3) of 70 μ M in the culture originally of cortical neuron.After the subculture 24 hours, the degree of the cell injury that occurs in the culture is determined according to lactic acid dehydrogenase (LDH) quantity that discharges.

In constant t-PA concentration (500nM) with under the situation of the DSPA concentration that increases gradually (5,50,500nM), in the culture originally of cortical neuron, respectively add NMDA (70 μ M), and after 24 hours, measure neuronal cell death according to the LDH quantity that discharges.At this, under the situation of the DSPA concentration that increases gradually, demonstrate suppressed to depend on t-PA for the stimulation of inductive neuronal cell death.

3.Ca ⁺⁺-flowing experiment

Intracellular calcium concentration detects by the algoscopy of using Fluo-3/AM.For this reason, cortical neuron is cultivated 9 days (on seeing), and be loaded into 10 μ M Fluo-3/AM among the buffered saline solution of HEPES.This saline solution contains 135mM NaCl, 5mM KCl, 0.62mM MgSO ₄, 1.8mM CaCl ₂, 10mM HEPES and 6mM glucose, pH 7.4, place 1 hour down at 37 ℃.Add DMSO (1%) and 0.2%Pluronic F-127, to promote dispersing of pigments.

Cell with the above-mentioned HEPES buffer washing that contains the 1mM furosemide, is separated out (described pigment uses subsequently) to prevent pigment in each buffer from buffer.The relative fluorescence unit (RFU) that measures cell after washing to be obtaining baseline thus, and measures in 485/530nm (excitation/emission) during processing period (5 minutes).Use FluoroskanAscent Fluorometer (Labsystems) for this reason.

As shown in Figure 4, handle the remarkable increase (P＜0.05) that after 1 minute, causes calcium concentration with NMDA separately.If cell was used t-PA (30 μ g/ml before adding NMDA; 500nM; P＜0.05) pretreatment is 5 minutes, and calcium concentration still increases by 30% so.

People's (2001) such as these results and Nicole report conforms to, and in people's such as Nicole report, adding t-PA has in advance still increased the calcium that is caused by NMDA and discharge.Handle the variation that does not cause free calcium ion concentration with t-PA separately.In contrast, before adding NMDA, can not exceed the independent degree that NMDA reached of adding with the change for calcium concentration in 5 minutes of DSPA pretreatment neuronal cell.Therefore, independent DSPA is for not influence of intracellular calcium concentration.

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Claims (12)

1.t-PA the activatory inhibitor of glutamate receptor of mediation is as the purposes of neuroprotective, described glutamate receptor is preferably the NMDA type.

2. the purposes of the inhibitor of claim 1, described purposes are to be used for the treatment of or to prevent neuronal damage or state based on the excitotoxicity of glutamate receptor.

3. claim 1 or 2 purposes, described purposes is for being used for the treatment of depression or frightened state.

4. each purposes in the claim 1 or 2 is characterized in that using in the apoplexy treatment.

5. each purposes in the claim 1 or 2 is characterized in that the treatment or the prevention of one of following state: parkinson disease, Alzheimer, Huntington Chorea, diabetes, pain status, epilepsy or dysmnesia.

6. each purposes is characterized in that using the active protease of a kind of inhibition t-PA during aforesaid right required.

7. the purposes of claim 6 is characterized in that the preferred neural serpin of serpin, inhibitors of plasminogen activator inhibitor (PAI) or protease connect protein I (PN-1).

8. each purposes among the claim 1-5 is characterized in that using DSPA or therefrom derivative derivant, analog or fragment on function and/or structure.

9. the purposes of claim 8 is characterized in that, uses the DSPA with aminoacid sequence shown in Figure 1, perhaps has at least 70% with aminoacid sequence shown in Figure 1, DSPA derivant, analog or the fragment of the homology of preferred 80-90%.

10. claim 8 or 9 purposes, it is characterized in that, dosage is greater than 62.5 and less than the DSPA according to Fig. 1 of 160 μ g/kg, the DSPA of preferred 90-125 μ g/kg, the DSPA of preferred especially 90 μ g/kg, perhaps dosage for adjusting according to used derivant, analog or segmental bioequivalence.

11. each purposes is characterized in that among the claim 8-10, DSPA or its derivant, analog or fragment are used as neuroprotective in uniting the apoplexy treatment of using thrombolytic agent.

12. the purposes of claim 11 is characterized in that, t-PA is as thrombolytic agent.

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