WO2018032611A1 - 神经保护剂及用于治疗脑部疾病或病症的方法 - Google Patents

神经保护剂及用于治疗脑部疾病或病症的方法 Download PDF

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WO2018032611A1
WO2018032611A1 PCT/CN2016/103782 CN2016103782W WO2018032611A1 WO 2018032611 A1 WO2018032611 A1 WO 2018032611A1 CN 2016103782 W CN2016103782 W CN 2016103782W WO 2018032611 A1 WO2018032611 A1 WO 2018032611A1
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neuroprotective agent
brain
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侯圣陶
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Southern University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of medicine, and in particular to a neuroprotective agent and a method for treating a brain disease or condition.
  • Stroke has become one of the three major diseases that seriously endanger human health.
  • the clinical treatment of stroke drugs has no other effective neuroprotective agents other than the very limited thrombolytic drugs used in the clinical time window, and there are no therapeutic drugs for promoting the recovery of nerve function.
  • stroke-induced endogenous neuroprotective molecules and mechanisms remains limited (Molecular mechanisms of cerebral ischemia-induced neuronal death. Hou ST, MacManus JP (2002), Int Rev Cytol 221: 93-148.; The science of stroke: mechanisms in search of treatments. Moskowitz MA, Lo EH, Iadecola C (2010). Neuron 67: 181-198).
  • Pharyloic acid is a catabolic product of bismuth compounds of Abscisic Acid (ABA).
  • ABA produces safflower stucic acid under the catalysis of cytochrome P-450 enzyme ((+)-Abscisic Acid 8'-hydroxylase is a cytochrome P450 monooxygenase.
  • Krochko JE, Abrams GD, Loewen MK, Abrams SR, Cutler AJ (1998). Plant Physiol 118: 849-860.).
  • ABA plays an important physiological role in the growth and development of higher plants, including improving stress resistance (drought resistance, cold resistance, salt resistance, etc.), inhibiting growth and germination, promoting shedding and dormancy, accelerating aging and promoting stomatal closure.
  • the level of endogenous ABA in plants is controlled by changes in the balance between biosynthesis and metabolism.
  • ABA acts in plants by inducing the production of second messenger cADP-Ribose to regulate the release of intracellular calcium ions (Guard cellABA and CO 2 signaling network updates and Ca 2+ sensor priming hypothesis.sraelsson M, Siegel RS, Young J, Hashimoto M, Iba K, Schroeder JI (2006).
  • (-)-PA is similar to ABA and is also a plant hormone that inhibits photosynthesis and promotes shedding (ABA biosynthesis and degradation contributing to ABAhomeostasis during barley seed development under control and terminal drought-stressconditions. Seiler C, Harshavardhan VT, Rajesh K, Reddy PS, Strickert M, Rolletschek H, Scholz U, Wobus U, Sreenivasulu N (2011). J Exp Bot 62: 2615-2632.).
  • a neuroprotective agent comprising a compound having the formula:
  • a method for treating a brain disease or condition comprising administering to a subject in need of treatment an effective amount of the above neuroprotective agent.
  • the high level endogenous (-)-PA of the above structural formula exists in the ischemic brain, which is different from other ABA derivatives, and the high content of (-)-PA is present in the semi-hidden region around the ischemic lesion. Reversible inhibition of glutamate receptors, reduction of cellular calcium influx, can protect cortical nerve cells from glutamate toxicity, making the above structural formula (-)-PA an effective neuroprotective agent.
  • Figure 1a is a chromatogram of (-)-PA and (+)-PA of a PA synthesis standard of an embodiment
  • Figure 1b is a (-)-PA chromatogram of a brain test sample of a mouse of an example
  • Figure 1c is a chromatogram of (-)-PA in a blood test sample of a mouse of an example
  • FIG. 2 is a bar graph showing the contents of (-)-PA and its related metabolites in brain test samples and blood test samples of a mouse according to an embodiment
  • Figure 3 is a bar graph of the content of (-)-PA and its related metabolites in the feed of an embodiment
  • cortical neuron supplemented with only 100 ⁇ M of NMDA, cortical neurons supplemented with 100 ⁇ M of NMDA and ( ⁇ )-PA added with 10 ⁇ M, NMRA supplemented with 100 ⁇ M, and cortex added with 100 ⁇ M of (-)-PA.
  • Fluorescence ratio map of calcium influx of neurons 1000 ⁇ M NMDA and 10 ⁇ M (-)-PA plus cortical neurons, and mouse cortical neurons supplemented with only 45 mM potassium chloride;
  • Figure 5 is a graph showing potential changes of cortical neurons added with 50 ⁇ M NMDA and 100 ⁇ M (-)-PA over time in an embodiment
  • Figure 6 is a graph showing potential changes of cortical neurons added with 50 ⁇ M NMDA and 1000 ⁇ M (-)-PA over time in an embodiment
  • Figure 7 is a graph showing the relationship between the concentration of (-)-PA and the potential in cortical neurons of an embodiment
  • Figure 8 is a bar graph of the survival of untreated cortical neurons, 100 ⁇ M NMDA treated cortical neurons, different concentrations of (-)-PA treated cortical neurons, and DMSO treated cortical neurons;
  • Figure 9 is an example of untreated cortical neurons, ALLN-treated cortical neurons, 10 ⁇ M of (-)-PA and 100 ⁇ M of NMDA-treated cortical neurons, 100 ⁇ M of (-)-PA and 100 ⁇ M of NMDA treatment.
  • Figure 10 is a bar graph of (-)-PA content of cerebral ischemic and contralateral samples at 2 hours, 6 hours, and 24 hours after MCAO surgery in experimental and control mice of one example;
  • Figure 11 is a photograph of a mouse brain sample of an embodiment
  • Figure 12 is a bar graph of the content of (-)-PA in the three regions of Figure 11;
  • Figure 13 is an example of perfusion of (-)-PA and saline in mice at 0.5 hours, 6 hours and Survival rate map after 24 hours;
  • Figure 14 is a graph showing the behavioral scores of mice in mice perfused with (-)-PA and saline at 0.5 hours, 6 hours, and 24 hours;
  • Figure 15 is a diagram showing the infarct size of the brain of mice perfused with (-)-PA and saline;
  • Figure 16 is a graph showing the results of mouse behavioral scores of mice perfused with (-)-PA antibody and physiological saline at 0.5 hours, 6 hours, and 24 hours;
  • Figure 17 is a graph showing the results of grasping reflex grip force scores of forefoot of 0.5-hour, 6-hour, and 24-hour mice infused with (-)-PA antibody and physiological saline according to an embodiment
  • Figure 18 is a graph showing the infarct size of the brain of mice perfused with (-)-PA antibody and physiological saline according to an example.
  • a neuroprotective agent comprising a compound having the formula:
  • the neuroprotective agent also includes a pharmaceutically acceptable carrier.
  • the protein molecule is selected from the group consisting of a bovine serum albumin molecule, an ovalbumin molecule, a keyhole limpet hemocyanin molecule, a fibrin molecule, and a human serum protein molecule.
  • polypeptide is selected from the group consisting of polylysine, polyglutamic acid and bis-lipoyl acid.
  • the neuroprotective agent also includes a pharmaceutically acceptable solvent.
  • the solvent is physiological saline or aqueous glucose solution.
  • the high level endogenous (-)-PA of the above structural formula exists in the ischemic brain, which is different from other ABA derivatives, and the high content of (-)-PA is present in the semi-hidden region around the ischemic lesion. Reversible inhibition of glutamate receptors, reduction of cellular calcium influx, can protect cortical nerve cells from glutamate toxicity, making the above structural formula (-)-PA an effective neuroprotective agent.
  • a method for treating a brain disease or condition of an embodiment comprising administering an effective amount of the above neuroprotective agent to a subject in need of treatment.
  • the administration is oral administration, injection administration, respiratory administration, dermal administration, mucosal administration or intraluminal administration.
  • the disease or condition includes senile degenerative encephalopathy, stroke, cerebrovascular disease, intracranial space-occupying lesion, epilepsy, cerebral thrombosis, lacunar infarction or cerebral embolism.
  • the aged degenerative encephalopathy is Alzheimer's disease, Parkinson's disease, and the like.
  • mice 10 C57B/6 mice, each weighing 20 to 37 g, were male.
  • Mouse feed Market purchased feed (LabDiet, Land O'Lakes Inc, Ontario, Canada).
  • mice Collection of brain tissue and blood in mice: The mice were anesthetized with isoflurane, and the mice were anesthetized and blood was collected from the facial veins of the mice to obtain blood samples of the mice; the mice were sacrificed, and the brain tissues were collected to obtain mice. Brain samples, brain samples and blood samples were snap frozen in liquid nitrogen and stored at -80 °C.
  • Mouse brain tissue samples and blood sample pretreatment Mouse brain tissue samples and blood samples were homogenized using a multi-tube ball mill (Mini-BeadBeater-96, Biospec Products, Bartlesville, Oklahoma , USA), weigh about 50 mg of each sample and place them in multiple Falcon tubes.
  • a multi-tube ball mill Mini-BeadBeater-96, Biospec Products, Bartlesville, Oklahoma , USA
  • isotopic internal standards include: d3-DPA, d5-ABA-GE, d3-PA, d4-7'-OHABA, d3-neoPA, d4-ABA, and d4-trans-ABA
  • Synthetic preparation has been reported in the literature Abrams et al., 2013, each substance concentration of 0.2 ng / ⁇ L, dissolved in a mixture of water and acetonitrile, and the volume ratio of water to acetonitrile in a mixture of water and acetonitrile is 1: 1) etc.
  • UPLC/ESI-MS/MS analysis and quantification of PA synthesis standards Chiral separation of PA using the above UPLC/MS equipment, using chiral column for Regis (R, R) Whelko 5/100 Kromasil (4.6 ⁇ 150 mm, 5 ⁇ m, REGIS Technologies)).
  • Mobile phase A was chromatographic grade water containing 0.1% glacial acetic acid and mobile phase B was chromatographic grade methanol containing 0.1% glacial acetic acid.
  • the injection volume is 15 ⁇ L, the flow rate is 0.40 mL/min, and the elution gradient is: 0 min to 1 min 55% mobile phase B; 1 min to 18 min 55% to 90% mobile phase B; 18 min to 19 min 90% to 100% mobile phase B; 19min ⁇ 20min 100% mobile phase B; 20min ⁇ 21min 100% ⁇ 55% mobile phase B; 21min ⁇ 25min 55% mobile phase B balance column, waiting for the next injection.
  • a chromatogram of (-)-PA and (+)-PA of the PA synthesis standard was isolated as shown in Figure 1a.
  • UPLC/ESI-MS/MS analysis and quantification of mouse brain test samples and blood test samples Brain test samples and blood tests of mice obtained by pretreatment of brain tissue samples and blood samples of the above (2) mice The sample was dissolved in an aqueous solution (v/v) containing 40% by volume of methanol, 0.5% acetic acid and 0.1 ng/ ⁇ L of the recovered isotope standard (d6-ABA and d2-ABA-GE). UPLC/ESI-MS/MS analysis and quantification.
  • the injection volume is 10 ⁇ L
  • the flow rate is 0.40 mL/min
  • the elution gradient is: 0 min to 0.2 min 2% mobile phase B, 0.2 min to 0.4 min 2% to 15% mobile phase B; 0.4 min to 5.0 min 15% to 50 % mobile phase B; 5.0 min to 5.5 min 50% to 100% mobile phase B; 5.5 min to 6.2 min 100% mobile phase B; 6.2 min to 6.5 min 100% to 2% mobile phase B; 6.5 min to 8.0 min 2% Mobile phase B.
  • MassLynx v4.1 Waters multiple reaction monitoring mode (MRM), negative ion mode, electrospray ionization, conditions are as follows: capillary voltage 1.75 kV, desolvation gas flow rate 1100 L / h, cone flow 150 L / h, ion
  • the source and desolvation temperatures were 120 ° C and 350 ° C, respectively.
  • Offline chromatographic data processing was performed using QuanLynx v4.1 software (Waters).
  • the calibration curve was created using all target compounds and the (-)-PA chromatogram in the mouse brain test sample was obtained, as shown in Figure 1b; Chromatogram of (-)-PA in the blood test sample, as shown in Figure 1c; and the content of (-)-PA and its related metabolites obtained by chromatograms of mouse brain test samples and blood test samples
  • * indicates P ⁇ 0.05, that is, significant
  • ** indicates P ⁇ 0.01, that is, very significant
  • * in the following figures indicates the above meaning.
  • UPLC/ESI-MS/MS analysis and quantification of mouse feed Quantification and analysis of mouse brain test samples and blood test samples using the same UPLC/ESI-MS/MS method for the feed purchased in the above market (LabDiet, Land O'Lakes Inc (Ontario, Canada) detected a histogram of the content of (-)-PA and its related metabolites in the feed from the chromatogram of the feed obtained by the test as shown in Figure 3.
  • PA is present in the brain and blood of mice and is present in the form (-)-PA instead of (+)-PA.
  • (-)-PA has a higher content in the brain of mice, which is 10 times higher in blood (-)-PA, and the content of ABA in the brain is lower than (-)- 30 times the content of PA, indicating that the mouse There is a high level of (-)-PA in the brain.
  • Cortical neuron culture CD-1 mice aged 15 or 16 days were mechanically isolated from the cerebral hemisphere, the meninges were removed, and then digested with 0.025% (w/v) trypsin solution for 25 min, followed by trypsin inhibition. The agent was neutralized with trypsin, and 0.05% (w/v) Dnase (deoxyribonuclease) was added to remove DNA from dead cells.
  • the cortical neurons were isolated in a medium through a series of disruption treatments and a gentle centrifugation step, and the neurons were followed by the article (Collapsin response mediatorprotein 3deacetylates histone H4to mediate nuclear condensation and neuronal death.
  • the culture method used was cultured for 7 to 14 days in a medium supplemented with B-27 supplement and N2 supplement (Invitrogen) (due to neurons in This medium is fully mature in order to respond to glutamate-induced toxicity, so it is necessary to culture the neurons first).
  • the cells were seeded at a density of 1 ⁇ 10 6 cells/well in a slide of a 24-well plate previously treated with poly-D-lysine, and seeded in a 100 mm cell culture dish at a density of 2 ⁇ 10 7 cells / 10 mL.
  • the cells were cultured in a 37 ° C cell culture incubator.
  • the mouse cortical neurons cultured in vitro for 7 days in the above (1) were added to a mixture of 5 ⁇ M of Fura-2-AM (Molecular Probes, Eugene, CA) and 0.02% pluronic (Molecular Probes, Eugene, USA). and incubated at 37 °C 30min, then the PSS Mg 2+ free buffer (2mM pH of the HEPES 7.2, 140mMNaCl, 5mMKCl, 2.3mM CaCl 2 and 10mM glucose) washed and then in PSS buffer in the absence of Mg 2+ Stabilized for 5 min, the intensity of Fura-2 was measured using a Northern Eclipse Digital Ratio Image System (EMPIX, Mississauga, ON) system and an Axiovert 200 camera, where the emission wavelength was 510 nm and the fluorescence ratio of Fura-2 at 340 nm and 380 nm was measured.
  • EMPIX Northern Eclipse Digital Ratio Image System
  • Dual stimulation screening was performed by the DG-5 system (Sutter Instrument Company, Novato, CA) system.
  • the [Ca 2+ ]i concentration is represented by the ratio of the fluorescence intensity of Fura-2 at the two excitation wavelengths of R340/380 after background correction.
  • R340/380 is the average of the ratio of 20 cells in the same field of view on the flap.
  • the [Ca 2+ ]i ground state level was recorded for 20 s, and 100 ⁇ M NMDA was dissolved in the MgS 2+ -free PSS buffer and added to the cortical neurons, followed by different concentrations of (-)-PA (10 ⁇ M, 100 ⁇ M and 1000 ⁇ M), the [Ca 2+ ]i level was recorded for 60-100 s; the neurons were washed with PGS buffer without Mg 2+ for 300 s, and PES buffer containing 45 mM KCl was added for depolarization treatment, and [Ca 2 was recorded.
  • the above-mentioned (1) cultured cortical neurons were subjected to whole-cell patch clamp recording using an Axopatch 700A patch-clamp amplifier (Axon Instruments, Inverurie, Scotland) at room temperature (22 to 25 ° C). Data was acquired by DigiData 1322A, pClamp 9.0 software with a sampling frequency of 10 kHz and the signal was filtered through 5 kHz. The electrodes were pulled and polished with a Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA). The resistance of the recording electrodes was 4 to 6 M ⁇ in different internal fluids. During voltage clamp recording, resistors and capacitors were used in the amplifier to eliminate transients. Voltage.
  • the series resistance is usually ⁇ 15M ⁇ , the series resistance compensation is set to 70% to 90%, and the liquid junction potential is approximately 2mV.
  • the electrode potential is adjusted by the electrode bias adjustment, and the external solution (NaCl 150mM, KCl) is used.
  • Drugs 50 ⁇ M NMDA and different concentrations of (-)-PA were applied to cortical neurons by external pressure using an 8-channel focused perfusion system (ALA Scientific Instruments, Farmingdale, NY), where (-)- The concentration of PA ranges from 100 to 1000 ⁇ M.
  • the cortical neurons were immersed in the external fluid throughout the recording process, and the drug solution was replaced by electronic control.
  • Patch clamp data were processed using Clampfit 9.0 (Axon Instruments) and analyzed using Origin 7.5 (OriginLab, Northampton, MA) and the changes in potential of cortical neurons with 50 ⁇ M NMDA and 100 ⁇ M (-)-PA were obtained over time. As shown in Fig.
  • cortical neurons of the above (1) cultured at 37 ° C for 7 days in vitro were respectively used with 100 ⁇ M NMDA (N-methyl-D-aspartic acid) at concentrations of 10 ⁇ M, 50 ⁇ M, 100 ⁇ M, 250 ⁇ M, 500 ⁇ M, and 1000 ⁇ M, respectively.
  • 2000 ⁇ M (-)-PA and dimethyl sulfoxide (DMSO) were treated for 15 min and further incubated at 37 ° C for 24 hours.
  • the survival rate of cortical neurons was calculated from the stained area to obtain untreated cortical neurons, 100 ⁇ M NMDA-treated cortical neurons, different concentrations of (-)-PA-treated cortical neurons, and DMSO-treated cortical neurons. a histogram of survival, As shown in Figure 8.
  • cortical neurons of the above (1) cultured in vitro at 37 ° C for 7 days were respectively treated with calpain inhibitor (ALLN), 10 ⁇ M of (-)-PA and 100 ⁇ M of NMDA, 100 ⁇ M of (-)-PA and 100 ⁇ M of NMDA, 1000 ⁇ M of (-)-PA and 100 ⁇ M of NMDA were treated for 15 min and incubated at 37 ° C for an additional 24 hours.
  • ALLN calpain inhibitor
  • Neuronal viability was analyzed by cortical neurons using Alamar blue staining, and the survival rate of cortical neurons was calculated according to the stained area to obtain untreated cortical neurons, ALLN-treated cortical neurons, and 10 ⁇ M (-)- PA and 100 ⁇ M NMDA-treated cortical neurons, 100 ⁇ M (-)-PA and 100 ⁇ M NMDA-treated cortical neurons, columnar survival of cortical neurons treated with 1000 ⁇ M (-)-PA and 100 ⁇ M NMDA Figure, as shown in Figure 9.
  • pretreatment of cortical neurons with (-)-PA can improve the neuronal death to NMDA, and the greater the concentration of (-)-PA used, the cell death rate of cortical neurons. The lower, the dose-dependent.
  • mice in the MCAO group underwent short-term anesthesia with isoflurane, and the left brain surgery was performed on the mouse brain by intraluminal reversible MCAO suture method (Collapsin response) Mediatorprotein 3 deacetylates histone H4 to mediate nuclear condensation and neuronal death.
  • Collapsin response Mediatorprotein 3 deacetylates histone H4 to mediate nuclear condensation and neuronal death.
  • Hou ST Jiang SX, Aylsworth A, Cooke M, Zhou L (2013). Sci Rep 3: 1350.
  • the blood flow returned to normal after the wire plug was removed, monitored by a laser Doppler flowmeter, and the wound was sutured.
  • the rectal probe was used to measure the body temperature of the mouse during the operation, and the body temperature was maintained at 37 ° C by heating pad and heating lamp, and blood pressure, blood gas and pH were detected to ensure the consistency of the experimental operation during the operation.
  • mice were euthanized and the left and right hemispheres of the mice were isolated.
  • the frontal lobe portion of the left hemisphere (L1 and anterior sputum 2.96-0.50) mainly containing ischemic lesion tissue was selected to obtain a brain sample of the mouse, and then the brain sample was rapidly frozen in liquid nitrogen and stored at -80 °C.
  • mice 12 mice were described in (1), of which 3 were in the control group and 9 in the experimental group.
  • mice treatment method The same surgical procedure as described in (2) was accepted, 3 of which were sacrificed 2 hours after surgery, 3 were sacrificed 6 hours after surgery, and 3 were sacrificed 24 hours after surgery.
  • Adopted (2) The method collects mouse brain samples.
  • Control group mice treatment method The surgical procedure described in (2) was accepted, but no middle cerebral artery embolization was performed. He was sacrificed 24 hours after surgery. Mouse brain samples were collected using the method described in (2).
  • the obtained cerebral ischemic side and contralateral samples were subjected to quantitative analysis of (-)-PA by UPLC/ESI-MS/MS, and data were obtained as a histogram, as shown in FIG.
  • mice described in (1) 5 mice described in (1).
  • Experimental mouse treatment method The same surgical procedure as described in (2) was accepted and sacrificed 24 hours after surgery. Mouse brain samples were collected using the method described in (2).
  • Laser microdissection of mouse brain samples was performed using Laser Microdissection Leica LMD6 system, which divided brain samples into three regions, the ischemic core region (region 1) and the penumbra region (region 2). And the contralateral (region 3), as shown in Figure 11, each region was cut, using 10 infrared rays and 2 ultraviolet pulses, wherein all cells in the same region were collected in the same LCM collection tube. Immediately after cleavage, the cells were lysed, and UPLC/ESI-MS/MS was used to analyze the (-)-PA content of the three regions, and the content of (-)-PA in three regions was obtained as a histogram, as shown in Fig. 12. Shown.
  • mice 19 mice were described in (1), of which 11 were in the experimental group and 8 in the control group.
  • mice treatment method The same surgical procedure as described in (2) was accepted, and 24 hours after surgery, according to the contents of the literature (Directintraventricular delivery of drugs to the rodent centralnervous system. DeVos SL, Miller TM (2013) ).J Vis Expe50326..), using a micro pump with an injection speed of 1.0 ⁇ L/hr and a water storage tank (Alzet 1003D) with a displacement of more than 3 days (DURECT Corporation, ALZET Osmotic Pumps, Cupertino, CA) Mice were injected with (-)-PA (50 mg/mL) and control mice were injected with 100% saline (placebo).
  • the micropump was first immersed in a sterile physiological saline solution at 37 ° C overnight in order to rapidly inject (-)-PA after implantation. Mice were performed 0.5 hours, 6 hours, and 24 hours after injection Survival statistics, survival rate statistics are plotted as shown in Figure 13.
  • mice in the experimental group and the control group were scored for neurological deficit assessment at 0.5 hour, 6 hours, and 24 hours after the injection.
  • the neurological deficit score was scored on a six-point scale.
  • the evaluation criteria were: 0, normal no nerve injury symptoms; Slight turn behavior, when the tail is picked up with or without unsatisfactory contracture, side rotation 50%; 2, slight persistent contracture, side end curl 50%; 3, strong and immediate duration Contracted, the mouse held the collapsed position for more than 1 to 2 seconds, the mouse nose almost hit the tail; 4, severe contracture caused by the drum shape, loss of walking or correcting reflex; 5, coma or dying.
  • At least 8 mice were used in each group, and the average of the scores was used for statistical analysis. Among them, the results of neurological deficit assessment are shown in Figure 14.
  • the mice were sacrificed 24 hours after the injection, and the infarct size was measured on the brain.
  • the measurement method is as follows: Chromatetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia.
  • the solvent extract obtained by dissolving was mixed in three tubes with a mixed solvent of fresh ethanol and Me 2 SO (where Me represents methyl) at a volume ratio of 1:20, and placed at a ratio of Colored dish.
  • the average of the absorbance values of the dilution at 485 nm was measured with a spectrophotometer.
  • the infarct size measurement results of the control group and the experimental group were obtained according to the above formula, as shown in FIG.
  • mice 10 mice were described in (1), of which 5 were in the experimental group and 5 in the control group.
  • mice treatment method The same surgical procedure as described in (2) was accepted, and 24 hours after surgery, according to the contents of the literature (Directintraventricular delivery of drugs to the rodent centralnervous system. DeVos SL, Miller TM (2013) ).J Vis Expe50326..), using a micro pump with an injection rate of 1.0 ⁇ L/hr and a water storage tank (Alzet 1003D) with a displacement of more than 3 days (DURECT Corporation, ALZET Osmotic Pumps, Cupertino, CA) Mice were injected with (-)-PA antibody (50 mg/mL), and mice of the control group were injected with 100% saline (placebo).
  • mice of the control group and the experimental group were evaluated according to the neurological deficit in (5) at 0.5 hours, 6 hours, and 24 hours after the injection, and the statistical results were plotted as shown in FIG.
  • the forelimb grip test of the mice was performed 0.5 hours, 6 hours, and 24 hours after the injection.
  • the test method was as follows: forefoot grip test using a grip tester-Columbus instrument (MyNeurolab, St. Louis, MO) to measure muscle strength and related neuromuscular integration.
  • the grip of the forefoot reflects the grip.
  • the peak preamplifier automatically stores the peak pull and displays it on the LCD.
  • mice were sacrificed 24 hours after the injection, and the infarct size was measured on the brain according to the method of infarct size in (5).
  • the infarct size measurement results of the mice were obtained as shown in FIG.
  • MCAO caused a significant increase in the content of (-)-PA in the brain of mice with ischemic ischemia at 2 hours after reperfusion of blood after MACO surgery.
  • MCAO treatment induces (-)-PA expression in the ischemic lateral and contralateral brains, indicating that (-)-PA may protect the contralateral brain.
  • mice had better survival than the saline-treated mice, and the infarct size of the mice perfused with (-)-PA was also significantly reduced. In the nervous system Ratings, these mice showed a better improvement in scores.
  • TTC staining showed that the experimental group had a larger ischemic infarct area and perfused (-)-PA antibody compared with the saline-treated control group.
  • the mice exhibited a neurological deficit score and decreased the change in tensile strength of the forepaws, indicating that the decrease in (-)-PA expression in the brain worsens the behavioral outcome of cerebral ischemia.

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Abstract

一种神经保护剂，包括具有如结构式（Ｉ）的化合物。

Description

神经保护剂及用于治疗脑部疾病或病症的方法 技术领域
本发明涉及药物领域，特别涉及一种神经保护剂及用于治疗脑部疾病或病症的方法。
背景技术
脑卒中已经成为严重危害人类健康的三大疾病之一。然而目前，临床治疗脑卒中的药物除了临床时间窗使用的极为有限的通栓药之外，并无其它有效的神经细胞保护剂，也没有具有促进神经功能恢复的治疗药物。尽管经过几十年的努力，对脑卒中诱发的内源性神经保护分子和机理的认识仍然很局限(Molecular mechanisms of cerebral ischemia-inducedneuronal death.Hou ST，MacManus JP(2002)，Int Rev Cytol 221：93-148.；The science of stroke：mechanisms in searchof treatments.Moskowitz MA，Lo EH，Iadecola C(2010).Neuron 67：181-198)。
红花菜豆酸(Phaseic Acid，PA)是脱落酸(Abscisic Acid，ABA)萜类化合物的分解代谢产物，ABA在细胞色素P-450酶的催化作用下生成红花菜豆酸((+)-Abscisic acid 8′-hydroxylase is a cytochrome P450 monooxygenase.Krochko JE，Abrams GD，Loewen MK，Abrams SR，Cutler AJ(1998).Plant Physiol 118：849-860.)。ABA在高等植物的生长和发育调控中起到重要生理功能，包括提高抗逆性(抗旱、抗寒、抗盐等)，抑制生长和萌发，促进脱落和休眠，加速衰老和促进气孔关闭等。植物内源性ABA的含量水平，受生物合成和代谢之间平衡的变化控制。ABA通过诱导第二信使cADP-Ribose的产生调控细胞内钙离子的释放而在植物中发挥作用(Guard cellABA and CO2signaling network updates and Ca2+sensor priming hypothesis.sraelsson M，Siegel RS，Young J，Hashimoto M，Iba K，Schroeder JI(2006).CurrOpin Plant  Biol 9：654-663.)。(-)-PA与ABA作用相类似，也是抑制光合作用和促进脱落的植物激素(ABA biosynthesis and degradation contributing to ABAhomeostasis during barley seed development under control and terminal drought-stressconditions.Seiler C，Harshavardhan VT，Rajesh K，Reddy PS，Strickert M，Rolletschek H，Scholz U，Wobus U，Sreenivasulu N(2011).J Exp Bot62：2615-2632.)。
发明内容
基于此，有必要提供一种神经保护剂，以使红花菜豆酸(-)-PA应用在神经保护剂中。
一种神经保护剂，包括具有如下结构式的化合物：
Figure PCTCN2016103782-appb-000001
一种用于治疗脑部疾病或病症的方法，包括给需要治疗的客体施用有效剂量的上述神经保护剂。
上述结构式的(-)-PA高水平内源性存在于缺血大脑中，区别于其他ABA衍生物，且(-)-PA较高含量地存在于缺血病灶周围的半隐区中，通过可逆性抑制谷氨酸受体，减少细胞钙内流，能保护皮质神经细胞免受谷氨酸毒性，使得上述结构式的(-)-PA成为一种有效的神经保护剂。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他实施例的附图。
图1a为一实施例的PA合成标准品的(-)-PA和(+)-PA的色谱图；
图1b为一实施例的小鼠的大脑试验样品中的(-)-PA色谱图；
图1c为一实施例的小鼠的血液试验样品中的(-)-PA的色谱图；
图2为一实施例的小鼠的大脑试验样品和血液试验样品的(-)-PA及其相关代谢产物的含量的柱状图；
图3为一实施例的饲料中的(-)-PA及其相关代谢产物的含量的柱状图；
图4为一实施例的仅加入100μM的NMDA的皮层神经元、加入100μM的NMDA和加入10μM的(-)-PA的皮层神经元、加入100μM的NMDA和加入100μM的(-)-PA的皮层神经元、加入1000μM的NMDA和加入10μM的(-)-PA的皮层神经元、以及仅加入45mM氯化钾的小鼠皮层神经元的钙离子内流的荧光比率图；
图5为一实施例的添加有50μM的NMDA和100μM的(-)-PA的皮层神经元随时间的电位变化图；
图6为一实施例的添加有50μM的NMDA和1000μM的(-)-PA的皮层神经元随时间的电位变化图；
图7为一实施例的皮层神经元中的(-)-PA的浓度与电位关系曲线图；
图8为一实施例的未处理的皮层神经元、100μM的NMDA处理的皮层神经元、不同浓度的(-)-PA处理的皮层神经元以及DMSO处理的皮层神经元的存活率的柱状图；
图9为一实施例的未处理的皮层神经元、ALLN处理的皮层神经元、10μM的(-)-PA和100μM的NMDA处理的皮层神经元、100μM的(-)-PA和100μM的NMDA处理的皮层神经元、以1000μM的(-)-PA和100μM的NMDA处理的皮层神经元的存活率的柱状图；
图10为一实施例的实验小鼠和对照组小鼠在MCAO手术后2小时、6小时和24小时的大脑缺血侧和对侧样品的(-)-PA含量的柱状图；
图11为一实施例的小鼠大脑样品的照片；
图12为图11的三个区域的(-)-PA的含量的柱状图；
图13为一实施例的灌注(-)-PA和生理盐水的小鼠在0.5小时、6小时和 24小时后的存活率图；
图14为一实施例的灌注(-)-PA和生理盐水的小鼠在0.5小时、6小时和24小时进行的小鼠行为学评分图；
图15为一实施例的灌注(-)-PA和生理盐水的小鼠的大脑的梗死面积图；
图16为一实施例的灌注(-)-PA抗体和生理盐水的小鼠在0.5小时、6小时和24小时进行的小鼠行为学评分结果图；
图17为一实施例的灌注(-)-PA抗体和生理盐水的小鼠在0.5小时、6小时和24小时的前脚掌的抓握反射握力评分结果图；
图18为一实施例的灌注(-)-PA抗体和生理盐水的小鼠的大脑的梗死面积图。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。
一实施方式的神经保护剂，包括具有如下结构式的化合物：
Figure PCTCN2016103782-appb-000002
其中，神经保护剂还包括药物上可接受的载体。
其中，蛋白分子选自牛血清蛋白分子、卵清蛋白分子、钥孔血蓝蛋白分子、纤维蛋白分子及人血清蛋白分子中的一种。
其中，多肽选自多聚赖氨酸、多聚谷氨酸及二软脂酰氨酸中的一种。
其中，神经保护剂还包括药学上可接受的溶剂。
其中，溶剂为生理盐水或葡萄糖水溶液。
上述结构式的(-)-PA高水平内源性存在于缺血大脑中，区别于其他ABA衍生物，且(-)-PA较高含量地存在于缺血病灶周围的半隐区中，通过可逆性抑制谷氨酸受体，减少细胞钙内流，能保护皮质神经细胞免受谷氨酸毒性，使得上述结构式的(-)-PA成为一种有效的神经保护剂。
一实施方式的用于治疗脑部疾病或病症的方法，包括给需要治疗的客体施用有效剂量的上述神经保护剂。
其中，施用为口服给药、注射给药、呼吸道给药、皮肤给药、粘膜给药或腔道给药。
其中，疾病或病症包括老年退行性脑病、脑中风、脑血管疾病、颅内占位性病变、癫痫、脑血栓形成、腔隙性梗死或脑栓塞。具体的，老年退行性脑病为老年痴呆、帕金森病等。
以下为具体实施例部分：
实施例
一、检测(-)-PA在小鼠大脑中高水平内源性的存在
(1)动物实验及饲料：
实验动物：C57B/6小鼠10只，每只的体重20～37g，均为雄性。
小鼠饲料：市场购买的饲料(LabDiet，Land O′Lakes Inc，加拿大安大略省)。
(2)动物样品收集及预处理：
小鼠的大脑组织和血液的收集：使用异氟烷将小鼠麻醉，小鼠麻醉后从小鼠的面部静脉收集血液，得到小鼠的血液样品；将小鼠处死，收集脑组织得到小鼠的大脑样品，将大脑样品和血液样品在液氮中快速冷冻，并在-80℃保存。
小鼠的大脑组织样品和血液样品预处理：将小鼠的大脑组织样品和血液样品分别采用多管球磨机匀浆(Mini-BeadBeater-96，Biospec Products公司，巴特尔斯维尔市，俄克拉荷马州，美国)，称取每份样品约50mg分别置于多个Falcon管中。将包含同位素内标(同位素内标物包括：d3-DPA、d5-ABA-GE、d3-PA、d4-7′-OHABA、d3-neoPA、d4-ABA和d4-trans-ABA，这些物质的合成制备在文献Abrams et al.，2013中有报道，每种物质浓度各0.2ng/μL，溶于水和乙腈的混合液，且水和乙腈的混合液中水和乙腈的体积比为1∶1)的等 分试样加入每份样品中，并使用提取溶剂(异丙醇∶水∶冰醋酸＝80∶19∶1，v/v/v)进行提取，提取物在避光和4℃下震荡24小时，经离心，取上清液，挥干溶剂。将提取物再溶于100μL的甲醇和冰醋酸的混合溶剂(其中，甲醇∶冰醋酸的体积比为99∶1)中，然后依次加入900μL体积百分含量为1％冰醋酸和2mL正己烷，分离得到水层，挥干溶剂，得到试验样品。试验样品于100μL甲醇和冰醋酸的混合溶剂中(甲醇∶冰醋酸的体积比为99∶1)中复溶，然后加入900μL 1％冰醋酸水的溶液，再经上Oasis HLB固相萃取柱(Waters，米西索加市，加拿大)，洗脱溶剂为乙腈、水和冰醋酸的混合液(乙腈∶水∶冰醋酸＝30∶69∶1，v/v/v)，真空离心，然后用浓缩仪(Labconco公司，堪萨斯城市，美国)浓缩，收集并干燥，分别得到小鼠的大脑试验样品和血液试验样品。
(3)UPLC/ESI-MS/MS(超高效液相色谱串联质谱方法)分析和定量
PA合成标准品的UPLC/ESI-MS/MS分析和定量：采用上述UPLC/MS设备手性分离PA，采用的手性柱为Regis(R，R)Whelko5/100Kromasil(4.6×150mm，5μm，REGIS Technologies公司)。流动相A为含有0.1％冰醋酸的色谱级水，流动相B为含有0.1％冰醋酸的色谱级甲醇。进样量为15μL，流速为0.40mL/min，洗脱梯度为：0min～1min 55％流动相B；1min～18min55％～90％流动相B；18min～19min 90％～100％流动相B；19min～20min 100％流动相B；20min～21min 100％～55％流动相B；21min～25min 55％流动相B平衡柱子，等待下次进样。分离得到PA合成标准品的(-)-PA和(+)-PA的色谱图，如图1a所示。
小鼠的大脑试验样品和血液试验样品的UPLC/ESI-MS/MS分析和定量：将上述(2)小鼠的大脑组织样品和血液样品预处理后得到的小鼠的大脑试验样品和血液试验样品分别溶于含有体积百分含量40％的甲醇、0.5％醋酸和0.1ng/μL的加样回收同位素标准品(d6-ABA和d2-ABA-GE)的水溶液(v/v)，再通过UPLC/ESI-MS/MS分析和定量。UPLC/ESI-MS/MS分析和定量时，采用Waters ACQUITY UPLC系统、二元溶剂输送系统、色谱柱和样品管理器通过Z-spray接口与Waters Micromass Quattro Premier XE四级杆串联质谱耦 合，其中，分析使用的色谱柱为ACQUITY
Figure PCTCN2016103782-appb-000003
HSS C18(2.1×100mm，1.8μm)，预柱为ACQUITY HSS C18VanGuard Pre-column(2.1×5mm，1.8μm)，流动相A为含有体积百分含量0.025％的冰醋酸的色谱级水，流动相B为含有0.025％冰醋酸的色谱级乙腈。进样量为10μL，流速为0.40mL/min，洗脱梯度为：0min～0.2min 2％流动相B，0.2min～0.4min2％～15％流动相B；0.4min～5.0min 15％～50％流动相B；5.0min～5.5min50％～100％流动相B；5.5min～6.2min 100％流动相B；6.2min～6.5min 100％～2％流动相B；6.5min～8.0min 2％流动相B。采用MassLynx v4.1(Waters公司)多反应监测模式(MRM)下采集数据，负离子模式，电喷雾电离，条件如下：毛细管电压1.75kV，脱溶剂气体流速1100L/小时，锥气流150L/小时，离子源和脱溶剂温度分别为120℃和350℃。采用QuanLynx v4.1软件(Waters公司)进行离线色谱数据处理，校准曲线采用所有目标化合物创建，并得到小鼠的大脑试验样品中的(-)-PA色谱图，如图1b所示；小鼠的血液试验样品中的(-)-PA的色谱图，如图1c所示；以及通过小鼠的大脑试验样品和血液试验样品的色谱图得到的(-)-PA及其相关代谢产物的含量的柱状图，如图2所示，在图2中，＊表示P＜0.05，即具有显著性；＊＊表示P＜0.01，即非常显著，且以下图中的＊表示均表示上述意义。
鼠饲料的UPLC/ESI-MS/MS分析和定量：采用小鼠的大脑试验样品和血液试验样品的定量和分析相同UPLC/ESI-MS/MS的方法对上述市场购买得到的饲料(LabDiet，Land O′Lakes Inc，加拿大安大略省)检测，从通过检测得到的饲料的色谱图得到饲料中的(-)-PA及其相关代谢产物的含量的柱状图如图3所示。
(4)结果分析
从图1a～图1c中可以看出，小鼠的大脑和血液中存在PA，其存在形式为(-)-PA，而非(+)-PA。
从图2中可以看出，(-)-PA在小鼠的大脑中的具有较高的含量，是血液中(-)-PA的10倍，而大脑中ABA的含量低于(-)-PA的含量的30倍，说明小鼠 大脑中高水平存在(-)-PA。
从图3中可以看出，饲料中(-)-PA含量很低，这说明小鼠大脑中的(-)-PA并不是来源于食物。显然说明，小鼠大脑中高含量的(-)-PA为内源产生，而非来自于食物等外源性因素。
二、(-)-PA保护皮层神经元免遭谷氨酸毒性实验
(1)皮层神经元培养：将怀孕15或16天的CD-1小鼠使用机械分离大脑半球，去除脑膜，再用0.025％(w/v)的胰蛋白酶溶液消化25min，然后加入胰蛋白酶抑制剂中和胰酶作用，再加入0.05％(w/v)Dnase(脱氧核糖核酸酶)，除去来自死细胞的DNA。在培养基中经过一系列破碎处理以及温和的离心步骤分离出皮层神经元，再将神经元按照文章(Collapsin response mediatorprotein 3deacetylates histone H4to mediate nuclear condensation and neuronal death.Hou S T，Jiang SX，Aylsworth A，Cooke M，Zhou L(2013).Sci Rep 3：1350.)所使用的培养方法，在添加了B-27补充剂和N2补充剂(Invitrogen)的培养基中培养7～14天(由于神经元在这种培养基中完全成熟，才能对谷氨酸盐诱导的毒性有所反应，因此，需先将神经元培养成熟)。将细胞以1×106细胞/孔的密度接种于预先用poly-D-lysine处理的24孔板的玻片中，再以2×107细胞/10mL的密度接种于100mm细胞培养皿中。细胞于37℃细胞培养箱中培养。
(2)使用Fura-2(细胞内钙离子的特异性荧光指示剂)的钙离子比率荧光测量方法(Abscisic acid does notevoke calcium influx in murine primary microglia and immortalised murine microglial BV-2 and N9cells.Jiang SX，Benson CL，Zaharia LI，Abrams SR，Hou ST(2010).BiochemBiophys Res Commun 401：435-439.)测量荧光比值。
将在上述(1)中的体外培养7天的小鼠皮层神经元加入5μM的Fura-2-AM(Molecular Probes，Eugene，CA)和0.02％pluronic(Molecular Probes，Eugene，USA)的混合物中，并在37℃孵育30min，再用无Mg2+的PSS缓冲液(2mM pH为7.2的HEPES、140mMNaCl、5mMKCl、2.3mM CaCl2和10mM葡萄糖) 清洗，然后在无Mg2+的PSS缓冲液里稳定5min，使用Northern Eclipse Digital Ratio Image System(EMPIX，Mississauga，ON)系统和Axiovert 200摄影机对Fura-2的强度进行检测，其中，发射波长为510nm，测量Fura-2在340nm和380nm波长的荧光比值。通过DG-5system(Sutter Instrument Company，Novato，CA)系统进行双重刺激筛选。[Ca2+]i浓度由Fura-2在R340/380这两个激发波长的荧光强度经过背景修正后的比值来表示。R340/380为盖破片上同一个视野中20个细胞的比值平均值。检测过程中先记录[Ca2+]i基态水平20s，用100μM的NMDA溶解于无Mg2+的PSS缓冲液，加入皮层神经元中，随后依次加入不同浓度的(-)-PA(10μM、100μM和1000μM)，记录[Ca2+]i水平60～100s；接着用无Mg2+的PSS缓冲液清洗神经元300s，加入含有45mM KCl的PSS缓冲液进行去极化处理，记录[Ca2+]i水平变化60s，以此来表示神经元的活力，所有测量至少重复3次，所有数据使用Microsoft Excel分析，结果为三次独立实验的平均值，从而得到仅加入100μM的NMDA的皮层神经元、加入100μM的NMDA和加入10μM的(-)-PA的皮层神经元、加入100μM的NMDA和加入100μM的(-)-PA的皮层神经元、加入1000μM的NMDA和加入10μM的(-)-PA的皮层神经元、以及仅加入45mM氯化钾的小鼠皮层神经元的钙离子内流的荧光比率图，如图4所示。
(3)全细胞电生理记录
使用Axopatch 700A patch-clamp amplifier(Axon Instruments，Inverurie，Scotland)在室温下(22～25℃)对上述(1)培养的皮层神经元进行全细胞膜片钳记录。数据通过DigiData 1322A，pClamp 9.0软件采集，采样频率为10kHz，信号通过5kHz过滤。电极用Flaming/Brown micropipette puller(Sutter Instruments，Novato，CA)牵拉和抛光，记录电极的电阻在不同内液中为4～6MΩ，电压钳记录过程中，在放大器中使用电阻电容回路消除瞬变电压。在全细胞模式形成之后，串联电阻通常是＜15MΩ，串联电阻补偿设置为70％～90％，液接电位大约为2mV，通过电极电位偏置调整进行自动调节，使用外液(NaCl 150mM、KCl 5mM、CaCl20.2mM、葡萄糖10mM和HEPES 10mM，NaOH调节pH至7.4)和电极内液(KCl140mM、MgCl22.5mM、HEPES10mM、EGTA 11mM和ATP 5mM，KOH调节pH至7.3)记录NMDA/AMPA激活电流，除非特别说明，电压钳记录膜电位钳制在-70mV。使用8-通道聚焦灌注系统(ALA Scientific Instruments，Farmingdale，NY)通过压力将药物(50μM的NMDA和不同浓度的(-)-PA)在外液中加到皮层神经元上，其中，(-)-PA的浓度范围在100～1000μM。整个记录过程皮层神经元一直浸泡在外液中，通过电子控制完成药物溶液更换。膜片钳数据使用Clampfit 9.0(Axon Instruments)处理，使用Origin 7.5(OriginLab，Northampton，MA)分析，并得到添加有50μM的NMDA和100μM的(-)-PA的皮层神经元随时间的电位变化图，如图5所示，以及得到添加有50μM的NMDA和1000μM的(-)-PA的皮层神经元随时间的电位变化图，如图6所示。并通过记录皮层神经元在各个浓度的(-)-PA中的电位，得到皮层神经元中的(-)-PA的浓度与电位关系曲线图，如图7所示，其中，剂量反应曲线用Logistic方程计算：y＝(A1-A2)/(1+(x/x0)p]+A2，y是响应，A1和A2分别是最大和最小响应，x0是指达到最大反应一半时所对应的浓度，x是药物浓度，p是希尔系数。
(4)神经元存活力
将上述(1)的体外37℃培养7天的皮层神经元分别用100μM的NMDA(N-甲基-D-天冬氨酸)、浓度分别为10μM、50μM、100μM、250μM、500μM、1000μM、2000μM(-)-PA和二甲基亚砜(DMSO)处理15min，并37℃再培养24小时。在处理过程结束之后，使用阿尔玛蓝(Invitrogen)对皮层神经元进行神经元存活力分析，将阿尔玛蓝和培养基的体积比为1∶10，将按照阿尔玛蓝加入培养基中，然后将加入有阿尔玛蓝的培养基加入皮层神经元中，然后将1∶10稀释的阿尔玛蓝加到皮层神经元中，于37℃孵育1小时进行染色。将染色的细胞用倒置显微镜(Carl Zeiss，AX10vert 200M)进行观察，拍照，使用Image J软件进行分析。根据染色面积计算皮层神经元的存活率，从而得到未处理的皮层神经元、100μM的NMDA处理的皮层神经元、不同浓度的(-)-PA处理的皮层神经元以及DMSO处理的皮层神经元的存活率的柱状图， 如图8所示。
将上述(1)的体外37℃培养7天的皮层神经元分别用钙蛋白酶抑制剂(ALLN)、10μM的(-)-PA和100μM的NMDA、100μM的(-)-PA和100μM的NMDA、1000μM的(-)-PA和100μM的NMDA处理15min，并37℃再培养24小时。使用阿尔玛蓝染色法对皮层神经元进行神经元存活力分析，根据染色面积计算皮层神经元的存活率，从而得到未处理的皮层神经元、ALLN处理的皮层神经元、10μM的(-)-PA和100μM的NMDA处理的皮层神经元、100μM的(-)-PA和100μM的NMDA处理的皮层神经元、以1000μM的(-)-PA和100μM的NMDA处理的皮层神经元的存活率的柱状图，如图9所示。
(5)结果分析：
从图4中可以看出，100μM的NMDA能够诱发细胞内钙([Ca2+]i)的快速流入，当使用100μM的NMDA和浓度范围从10μM到1000μM的(-)-PA处理皮层神经元时，使[Ca2+]i流入保持在较低水平，并且以剂量依赖形式改善皮层神经元的[Ca2+]i流入，添加氯化钾对皮层神经元进行去极化处理能引起大量的[Ca2+]i流入。从图中发现，(-)-PA能够有效地减少NMDA诱导增加的[Ca2+]i流入。这说明，(-)-PA是一个谷氨酸受体潜在的可逆性抑制剂。
从图5～7中可以看出，50μM的NMDA可在皮层神经元上可诱导出大量的内向电流，加入100μM和1000μM(-)-PA能有效抑制NMDA激活的电流，洗掉NMDA之后，膜电位又可以恢复到起始水平，说明(-)-PA抑制NMDA激活的电流是可逆的。且在100～1000μM范围内，(-)-PA剂量依赖性的抑制了NMDA激活的电流，IC50为175.4±21.5μM，斜率因子为0.96±0.3(n＝10)。再次证明了，(-)-PA可能是一个谷氨酸受体的可逆性抑制剂。
从图8中可以看出，100μM的NMDA处理培养的皮层神经元会导致细胞大量死亡，而任何浓度的(-)-PA处理的皮层神经元对细胞完全没有影响，即(-)-PA本身对培养的皮层神经元没有毒性，不会导致细胞的死亡。
从图9中可以看出，使用(-)-PA对皮层神经元预处理，可以改善神经元对NMDA毒性的死亡，且使用的(-)-PA浓度越大，皮层神经元的细胞死亡率 越低，即呈剂量依赖性。
综上，上述实验确定(-)-PA对皮层神经元的谷氨酸受体有非常重要的影响。且以上实验结果清楚地证明了(-)-PA是一个可逆的谷氨酸受体抑制剂，能在预防缺血性大脑引起的兴奋性毒性中发挥关键作用。
三、脑缺血诱导(-)-PA在大脑缺血半影区生成，(-)-PA对缺血动物模型的大脑具有保护作用
(1)实验动物：雄性C57B/6小鼠，每只20～37g。
(2)动物模型及样品收集方法：
小鼠MCAO(大脑中动脉闭塞)模型制作：将MCAO组的小鼠在异氟烷短时麻醉作用下，采用管腔内可逆性MCAO线栓法分别对小鼠大脑做左侧手术(Collapsin response mediatorprotein 3 deacetylates histone H4 to mediate nuclear condensation and neuronal death.Hou ST，Jiang SX，Aylsworth A，Cooke M，Zhou L(2013).Sci Rep 3：1350.)。脑缺血1小时，将线栓取出后，血流恢复正常，通过激光多普勒血流仪监测，并且伤口缝合。在手术过程中使用直肠探针测小鼠体温，并通过加热垫与加热灯维持体温在37℃，且为了保证在手术过程中实验操作的一致性，血压、血液气体和pH值都进行了检测(Increased Mdm2expression in rat brain after transient middle cerebral artery occlusion.Tu Y，Hou ST，Huang Z，Robertson GS，MacManus JP(1998).J Cereb BloodFlow Metab 18：658-669.)。
小鼠大脑样品的收集：小鼠安乐死，分离小鼠左右半球大脑。选择主要包含缺血病灶组织的左半球的额叶部分(L1与前囟2.96-0.50)，得到小鼠的大脑样品，然后将大脑样品在液氮中快速冷冻并在-80℃保存。
(3)MCAO缺血后血液再灌注(由于MACO手术后，血液会自然流动到缺血处，即为血液灌注)不同时间对小鼠大脑两侧(-)-PA含量的影响
实验动物：(1)中所述小鼠12只，其中对照组3只，实验组9只。
实验组小鼠处理方法：接受了(2)所述相同的手术操作，其中3只在术后2小时处死，3只在术后6小时处死，3只在术后24小时处死。采用(2) 所述的方法收集小鼠大脑样品。
对照组小鼠处理方法：接受了(2)所述的手术操作，但没有进行脑中动脉栓塞。术后24小时处死。采用(2)所述的方法收集小鼠大脑样品。
采用UPLC/ESI-MS/MS对所得到的大脑缺血侧和对侧样品进行(-)-PA定量分析，得到数据做柱状图，如图10所示。
(4)MCAO对小鼠大脑中缺血核心区域、半影区和对侧区域的影响
实验动物：(1)中所述小鼠5只。
实验小鼠处理方法：接受了(2)所述相同的手术操作，术后24小时处死。采用(2)所述的方法收集小鼠大脑样品。
使用Laser Microdissection Leica LMD6 system(徕卡显微系统)分别对小鼠大脑样品进行激光显微切割，将大脑样品均划分成三个区域，缺血核心区(区域1)、半影区(区域2)和对侧(区域3)，如图11所示，将每个区域切下，使用10个红外线和2个紫外脉冲，其中，相同区域的所有细胞收集在同一个LCM收集管中。切割之后的细胞立即裂解，并采用UPLC/ESI-MS/MS进行三个区域的(-)-PA含量测定分析，得到三个区域的(-)-PA的含量数据做柱状图，如图12所示。
(5)(-)-PA注入实验评估小鼠存活率、神经功能缺陷评估和大脑梗死面积测量
实验动物：(1)中所述小鼠19只，其中实验组11只，对照组8只。
实验组和对照组小鼠处理方法：接受了(2)所述相同的手术操作，术后24小时后，根据文献的内容(Directintraventricular delivery of drugs to the rodent centralnervous system.DeVos SL，Miller TM(2013).J Vis Expe50326..)，使用注射速度为1.0μL/hr的微量泵以及大于3天排量的蓄水槽(Alzet 1003D)(DURECT Corporation，ALZET Osmotic Pumps，Cupertino，CA)，对实验组小鼠进行(-)-PA(50mg/mL)注射，对照组小鼠进行100％生理盐水(安慰剂)的注射。提前先将微量泵浸泡在无菌生理盐水溶液中37℃过夜，以便在植入之后迅速将(-)-PA注入。注入之后0.5小时、6小时和24小时进行小鼠 存活率统计，存活率统计结果作图，如图13所示。
注入之后0.5小时、6小时和24小时对实验组和对照组的小鼠进行神经功能缺陷评估评分，神经功能缺陷评分采用六分制评分，评估标准为：0，正常无神经损伤症状；1，轻微转向行为，当尾巴被拎起来的时候伴有或不伴有不持续的蜷缩，侧端旋度50％；2，轻微持续的蜷缩，侧端旋度50％；3，强烈和即刻的持续蜷缩，小鼠持有蜷缩姿势超过1～2秒，小鼠鼻子几乎碰到尾巴；4，严重蜷缩造成滚筒状，丧失步行或翻正反射；5，昏迷或奄奄一息。每组至少采用8只小鼠，评分的平均值用于统计分析。其中，神经功能缺陷评估评分结果见图14。
注入24小时后处死小鼠，对其大脑进行梗死面积测量。测量方法如下：使用2，3，5-氯化三苯基四氮唑(TTC)的染色法(Chlortetracycline and demeclocycline inhibit calpains andprotect mouse neurons against glutamate toxicity and cerebral ischemia.Jiang SX，Lertvorachon J，Hou ST，Konishi Y，Webster J，Mealing G，Brunette E，Tauskela J，Preston E(2005).J BiolChem280：33811-33818.)计算注入(-)-PA的小鼠和注入生理盐水小鼠的大脑的梗死面积，即将小鼠的大脑样品分别切成4个2mm厚的冠状切片，在37℃下用5mL 2％的TTC染色90min。然后，用生理盐水冲洗，并随后暴露于乙醇和二甲亚砜的混合物(乙醇和二甲亚砜的体积比为1∶1)中以溶解甲瓒产物。在黑暗中孵育24小时后，溶解得到的溶剂提取物在三个管中用新鲜乙醇和Me2SO(其中，Me表示甲基)的混合溶剂按照体积比为1∶20稀释，并放置在比色皿。用分光光度计测试稀释物在485nm处的吸光度值的平均值。TTC染色在脑缺血侧与对侧大脑损失率由以下公式计算得到：损失率＝(1-缺血侧脑的吸光度/对侧脑吸光度)×100％。根据上述公式得到对照组和实验组的小鼠梗死面积测量结果作图，如图15所示。
(6)(-)-PA抗体注入实验评估小鼠神经功能缺陷评估、前肢握力测试和大脑梗死面积测量
实验动物：(1)中所述小鼠10只，其中实验组5只，对照组5只。
实验组和对照组小鼠处理方法：接受了(2)所述相同的手术操作，术后24小时后，根据文献的内容(Directintraventricular delivery of drugs to the rodent centralnervous system.DeVos SL，Miller TM(2013).J Vis Expe50326..)，使用注射速度为1.0μL/hr的微量泵以及大于3天排量的蓄水槽(Alzet 1003D)(DURECT Corporation，ALZET Osmotic Pumps，Cupertino，CA)，对实验组的小鼠进行(-)-PA抗体(50mg/mL)注射，对照组的小鼠进行100％生理盐水(安慰剂)的注射。提前先将微量泵浸泡在无菌生理盐水溶液中37℃过夜，以便在植入之后迅速将(-)-PA抗体注入。注入之后0.5小时、6小时和24小时对对照组和实验组的小鼠按照(5)中的神经功能缺陷评估评分，统计结果作图，如图16所示。
注入之后0.5小时、6小时和24小时进行小鼠前肢握力测试，测试方法如下：前肢握力测试用抓力测试器-Columbus仪器(MyNeurolab，St.Louis，MO)测量肌肉的力量和有关神经肌肉整合的前脚掌的抓握反射握力。峰值前置放大器自动存储的峰值拉力并显示在液晶显示屏上。对于每个动物，在特定的时间点进行至少10次测量，计算平均值和标准误差。所得结果作图，如图17所示。
注入24小时后处死小鼠，按照(5)中梗死面积的测量方法对其大脑进行梗死面积测量。得到小鼠梗死面积测量结果作图，如图18所示。
(7)结果分析：
从图10中可以看出，MACO手术后血液再灌注2小时时，MCAO引起(-)-PA在局部性缺血小鼠的脑中含量显著升高。MCAO处理诱导(-)-PA表达在缺血侧脑和对侧脑，表示(-)-PA可能在对侧脑起到保护作用。
从图12中可以看出，(-)-PA水平在半影区比缺血核心显著提高，大脑对侧的(-)-PA表达水平比缺血病灶区更高。这些研究表明，天然存在的(-)-PA可能在缺血性脑中起保护作用。
从图13～15中可以看出，(-)-PA处理小鼠与生理盐水处理的小鼠相比，具有更好的存活率，灌注(-)-PA的小鼠的梗死面积也显著降低，在神经系统 评分，这些小鼠显示分数也有更好的改善。
从图16～18中可以看出，灌注(-)-PA抗体的小鼠与生理盐水处理的对照组相比，TTC染色表明实验组局部缺血梗死区域更大，灌注(-)-PA抗体的小鼠表现神经功能缺损评分和降低前爪拉伸强度变化更显著，这表明在脑中减少(-)-PA表达恶化脑缺血的行为学结果。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种神经保护剂，包括具有如下结构式的化合物：
    Figure PCTCN2016103782-appb-100001
  2. 根据权利要求1所述的神经保护剂，其特征在于，还包括药学上可接受的载体。
  3. 根据权利要求2所述的神经保护剂，其特征在于，所述载体为多肽或蛋白分子。
  4. 根据权利要求3所述的神经保护剂，其特征在于，所述蛋白分子选自牛血清蛋白分子、卵清蛋白分子、钥孔血蓝蛋白分子、纤维蛋白分子及人血清蛋白分子中的一种。
  5. 根据权利要求3所述的神经保护剂，其特征在于，所述多肽选自多聚赖氨酸、多聚谷氨酸及二软脂酰氨酸中的一种。
  6. 根据权利要求1所述的神经保护剂，其特征在于，还包括药学上可接受的溶剂。
  7. 根据权利要求6所述的神经保护剂，其特征在于，所述溶剂为生理盐水或葡萄糖水溶液。
  8. 一种用于治疗脑部疾病或病症的方法，包括给需要治疗的客体施用有效剂量的根据权利要求1～7中的任何一项所述的神经保护剂。
  9. 根据权利要求8所述的方法，其特征在于，所述施用为口服给药、注射给药、呼吸道给药、皮肤给药、粘膜给药或腔道给药。
  10. 根据权利要求8所述的方法，其特征在于，所述疾病或病症包括老年退行性脑病、脑中风、脑血管疾病、颅内占位性病变、癫痫、脑血栓形成、腔隙性梗死或脑栓塞。
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