CN110339197A - A kind of purposes of the phosphodiesterase 9A inhibitor for the treatment of of vascular dementia - Google Patents
A kind of purposes of the phosphodiesterase 9A inhibitor for the treatment of of vascular dementia Download PDFInfo
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- CN110339197A CN110339197A CN201910551371.1A CN201910551371A CN110339197A CN 110339197 A CN110339197 A CN 110339197A CN 201910551371 A CN201910551371 A CN 201910551371A CN 110339197 A CN110339197 A CN 110339197A
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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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
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- 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
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- 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
Abstract
The invention belongs to technical field of pharmaceuticals, and in particular to phosphodiesterase 9A (PDE9A) inhibitor LW33 shown in formula I, the purposes of pharmaceutical composition composed by pharmaceutically acceptable salt and alternative one.Compound according to the present invention can be applied to the drug of preparation prevention or/and treatment vascular dementia, can also be used for preparation improves the drug of artery ischemia model learning memory dysfunction in mouse unilateral side common carotid artery occlusion (UCCAo) model and rat, it can also be used to which preparation reduces IL-1 β in serum, IL-6, MDA and increases the drug of SOD content in serum and prepare the drug for influencing mouse and rat aorta dementia histopathology structure.
Description
Technical field
The present invention relates to technical field of pharmaceuticals, a kind of specifically phosphodiesterase 9A for treatment of vascular dementia
The purposes of inhibitor.
Background technique
Vascular dementia (Vascular Dementia, VaD) is as flowing one kind caused by cerebripetal blood flow reduction
The progressive disease for influencing cognitive ability, accounts for about the 17-20% of all dementia patients, becomes after Alzheimer disease (AD)
The dementia of the second largest principal mode, and it is generally existing in elderly population.Clinical manifestation is cognition dysfunction, neural function
Can lack etc..
The heterogeneity of cranial vascular disease to illustrate neuropathological mechanisms and vascular dementia mechanism is challenging.Brain
The sufficient supply of blood flow is for maintaining the integrality of brain structure and function to have great importance, and cerebrovascular small lesions are all
Long-range irreversible damage may be caused to cognitive function.It is generally acknowledged at present by heart disease or carotid artery stenosis or blocking
Caused cerebral blood perfusion can lead to cognition dysfunction when being lower than some threshold value.Marshall etc. proposes brain blood in article
Stream, which reduces by 40%~50%, can lead to irreversible cerebral function inhibition and cognition dysfunction.And various cell signallings and
Adjustment mechanism, including Apoptosis, autophagy, oxidative stress and inflammation are related to VaD since they participate in cerebral ischemia.Small glue
Cell plastid, astroglia and some neurons and oligodendroglia by neurotrosis are in the quilt after dangerous sexual stimulus
Inflammatory mediator and other markers can be secreted when activation.Some data reports, the proinflammatory cytokines such as TNF-α, IL-1 β and IL-6 because
Sub- plays key effect in vascular dementia process.
So far, specific drug, current treatment means mainly control cognition barrier caused by VaD not yet for VaD treatment
The development process hindered, but all lack significant curative effect.At present clinically apply anticholinesterase such as huperzine, mostly how piperazine
Equal and nmda receptor blocking agent Memantine (Memantine) and some other Brain circlulation promotor Nimodipine, Shi Pu together
The kind equal sings and symptoms that cannot effectively improve or reverse VaD patient.As the state of an illness of VaD patient is aggravated, life is often
It can not take care of oneself, need the nursing of household or full-time healthcare givers for a long time, bring serious emotional distress and warp to family and society
Ji pressure.And FDA there is no to ratify the active drug for treating VaD at present.Therefore effective medicine of novel targets, new mechanism is found
Object becomes urgent need.
LW33 is the potent selective depressant (IC of PDE9A50For 5.0nM, 50 are greater than to the selectivity of other PDE hypotypes
Times), it is the brand new high-titer N- substituted pyrazolecarboxylic that is designed and synthesized based on PDE9A crystal structure simultaneously [3,4-d] Pyrimdinone
Preferred compound.Molecular formula C18H20CIN5O, molecular weight 357.8.Physicochemical characteristic analysis shows that, white to pale yellow powder, it is readily soluble
In methanol, ethyl alcohol, acetone, it is insoluble in the weakly polar organic solvents such as water, chloroform, ethyl acetate；Drug effect and preliminary safety analysis
It has been shown that, IC50=5.0nM, the anxious poison LD of mouse50Close to 1.5g/kg；Pharmacokinetic analysis shows, rat single oral gavage 5mg/kg
Afterwards, Tmax=2.0h, Cmax=372.54ng/L, AUC(0-t)=1640.87 μ g/L*hr, t1/2=1.46h, oral administration biaavailability
F=57%.
The compound and PDE9A complex crystal structure show, has similar mechanism of action with Pfizer compound.Early period
Pharmacological research shows in hyoscine model in animal body that 1.0mg/kg LW33 causes memory deficits in mice to have hyoscine
The effect of conspicuousness memory improvement, drug effect are consistent with positive control huperzine.Aβ1-40In intracerebral injection rat model,
LW33 low (0.35mg/kg), in (0.7mg/kg), high dose (1.4mg/kg) intervene after, the cognition function of rat model can be improved
Can, shorten incubation period, increases by platform number, extends in the target quadrant residence time.Hippocampus cGMP level significantly rises.
In addition, LW33 can also induce formation and early stage LTP and the later period of hippocampal slices early stage LTP and later period LTP
The fusion of LTP, and AChEI donepezil only can induce early stage LTP and be formed, with document report PDE9 inhibitor BAY73-6691's
Result of study is consistent, and PDE9A inhibitor is prompted more effectively enduringly to enhance learning and memory compared with AChEI.Mechanism Study discovery,
LW33 mainly passes through cGMP/PKG/CREB signal path and generates anti-neuroprotection.But cut-off is till now, there is not yet
Application of the LW33 as treatment vascular dementia.
Summary of the invention
The technical problem to be solved by the present invention is providing LW33 in preparation prevention and/or treatment vascular dementia disease medicine
Purposes in object, Astragaloside IV pass through the mRNA level in-site of IL-1 β, IL-6 and TNF-α in the BV2 inflammatory model for lowering LPS induction,
Improve the damage in learning and memory of UCCAo model mice and rat model, shortens incubation period, increase by platform number, prolong
It grows in the target quadrant residence time.MDA content in rat model serum, increased SOD activity can be reduced simultaneously, and are reduced in serum
The neuron of IL-1 β, the content of IL-6 and improvement vascular dementia model mouse and rat cerebral tissue cortical area and hippocampus is bad
It waits indefinitely pathological state, the new application with treatment of vascular dementia.
To achieve the above object, the present invention provides a kind of phosphodiesterase 9A indicated by following compounds (I)
Inhibitor LW33 or its pharmaceutically acceptable salt are being prepared containing their any pharmaceutical compositions for preventing
Or/and the purposes for the treatment of vascular dementia disease drug.
Further, the LW33 alleviates LPS induction in preparation prevention and/or treatment vascular dementia disease drug
The activity of BV2 inflammatory reaction is 0.1~1 μM.
Further, the LW33 is for reducing inflammatory factor TNF-α, IL1- β and IL-6 when treating vascular dementia
MRNA expression concentration be 0.1~1 μM.
Further, the present invention also provides LW33 or its pharmaceutically-acceptable salts or contain their any drugs
Composition influences the purposes in unilateral common carotid artery occlusion (UCCAo) model mice histopathology structure medicament in preparation.
Further, the present invention also provides LW33 or its pharmaceutically-acceptable salts or contain their any drugs
Composition improves the purposes of unilateral common carotid artery occlusion (UCCAo) model mice damage in learning and memory drug in preparation.
Further, the compound or its pharmaceutically-acceptable salts or contain their any pharmaceutical compositions
Dosage is 10~20mg/kg.
Exist the present invention also provides LW33 or its pharmaceutically-acceptable salts or containing their any pharmaceutical compositions
Preparation improves the purposes of vascular dementia rat models damage in learning and memory.
Further, the compound or its pharmaceutically-acceptable salts or contain their any pharmaceutical compositions
Dosage is 1~9mg/kg.
The present invention also provides a kind of LW33 or its pharmaceutically-acceptable salts or contain their any pharmaceutical compositions
Object preparation for reducing MDA content in vascular dementia rat models serum, increase use in serum in the drug of SOD content
On the way.
Exist the present invention also provides LW33 or its pharmaceutically-acceptable salts or containing their any pharmaceutical compositions
Preparation reduces IL-1 β in vascular dementia rat models serum, the purposes in IL-6 drug.
Exist the present invention also provides LW33 or its pharmaceutically-acceptable salts or containing their any pharmaceutical compositions
Preparation influences the purposes in vascular dementia rat models histopathology structure medicament.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is the cytotoxicity that LW33 is proliferated mouse microglia BV2；
Fig. 2 is influence of the LW33 to the LPS BV2 cell Proliferation induced；
Fig. 3 a is the influence of the mRNA level in-site for the inflammatory factor IL1- β that LW33 induces LPS BV2 inflammatory model；
Fig. 3 b is the influence of the mRNA level in-site for the inflammatory factor IL-6 that LW33 induces LPS BV2 inflammatory model；
Fig. 3 c is the influence of the mRNA level in-site for the inflammatory factor TNF-α that LW33 induces LPS BV2 inflammatory model；
Fig. 4 is that LW33 treats the influence (HE dyeing) to UCCAo model mice brain tissue neurons of hippocampus CA 1 in 21 days；
Fig. 5 is that LW33 treats the influence (HE dyeing) to rat model brain tissue neurons of hippocampus CA 1 in 14 days；
Fig. 6 is that LW33 treats the influence (HE dyeing) to rat model brain tissue cortical area neuron in 14 days；
Fig. 7 is the chemical structural formula of LW33.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The influence of embodiment 1, LW33 to BV2 cell viability
(1) experimental material: BV2 (mouse microglia) is purchased from Cell Bank of Chinese Academy of Sciences；DMEM culture medium (the U.S.
Gibco)；Fetal calf serum (U.S. Gibco)；CCK8 (Japanese colleague's chemistry institute)；Bay73-6691 (1- (2- chlorphenyl)-
6- [the fluoro- 2- methyl-propyl of (2R) -3,3,3- three] -1,5- dihydro -4H- pyrazolo [3,4-d] pyrimidin-4-one, Bayer)；PF-
04447943 (6- [(3S, 4S)-4- methyl-1-(pyrimidine -2-base methyl) pyrrolidin-3-yl]-1- (tetrahydro-2H- pyrans-4-
Base) -1,5- dihydro -4H- pyrazolo [3,4-d] pyrimidin-4-one, U.S. MCE).
(2) experimental method: 5.0 × 103BV2 cell be added in the plate hole of 96 orifice plates, culture for 24 hours.Former culture is sucked out
The complete culture solution of the LW33 containing various concentration (0.1 μM, 0.3 μM, 1 μM) is added in liquid, the complete training for being free of LW33 is added
Nutrient solution is as normal control.Continue culture for 24 hours, 10 μ L CCK8 be added into every hole, culture plate is cultivated into 2h in incubator,
Each hole absorbance value is detected at 450nm with microplate reader, detects cell viability.
(3) experimental result: by Fig. 1, the result shows that, LW33 does not change significantly within the scope of 0.1~1 μM, to cell viability
Become, without obvious cytotoxic effect.
The influence of embodiment 2, LW33 to the LPS BV2 cell Proliferation induced
(1) experimental material
Mouse microglia BV2 (Cell Bank of Chinese Academy of Sciences), DMEM culture medium (U.S. Gibco), fetal calf serum (beauty
State Gibco), CCK8 (Japanese colleague), LPS (Sigma)
(2) experimental method
5.0×103BV2 cell be added in the plate hole of 96 orifice plates, cultivate 24 hours.BV2 cell is divided into:
Control group: LPS processing group (1 μ g/mL action time is for 24 hours)；
0.1 μM of+LW33 of LPS processing group (1 μ g/mL action time is for 24 hours)；
0.3 μM of+LW33 of LPS processing group (1 μ g/mL action time is for 24 hours)；
1 μM of+LW33 of LPS processing group (1 μ g/mL action time is for 24 hours)；
3 μM of+Bay73-6691 of LPS processing group (1 μ g/mL action time is for 24 hours)；
3 μM of+PF-04447943 of LPS processing group (1 μ g/mL action time is for 24 hours)；
CCK8 detects cell proliferative conditions after for 24 hours.It is not stimulated using LPS group as control, analyzes each group BV2 cell proliferation rate
Difference.
(3) experimental result: it is detailed in Fig. 2.Compared with LPS does not stimulate group, LPS stimulates BV2 (mouse microglia) for 24 hours
Afterwards, cell viability declines；And LW33 can increase BV2 vigor in 0.1 μM~0.3 μM concentration range, have protective effect.
The influence of embodiment 3, LW33 to the LPS BV2 inflammatory factor mRNA level in-site induced
(1) materials and methods: HLF cell is purchased from Zhongshan University's Experimental Animal Center；
BV2 is purchased from Cell Bank of Chinese Academy of Sciences, DMEM culture medium (U.S. Gibco), fetal calf serum (U.S. Gibco),
0.25% pancreatin (U.S. Gibco), dual anti-(U.S. Gibco), LPS (Sigma), DMSO (MPBIO), IL-1, IL-1 β, TNF-α
Primer (the raw work in Shanghai), Thermo Revert Aid Kit kit (Thermo Fisher Scientific)
(2) test method
By cell inoculation in 60mm ware, cultivate in born of the same parents' incubator.50~60% are fused to when cell is grown, is used respectively
0.1,0.3,1 μM of LW33 and 3 μM of Bay73-6691, PF-04447943 carry out pretreatment 1h to BV2 cell, then add again
The LPS for entering 1 μ g/mL is stimulated, and effect extracts cell total rna afterwards for 24 hours and carries out real-time fluorescence quantitative PCR test.Measure concentration
Afterwards, taking 1 μ g total serum IgE to reverse according to Thermo Revert Aid Kit kit specification is cDNA, Q-PCR step reference
TOYOBO SYBR Premix EX TaqTMKit specification recommends dosage to optimize, by 95 DEG C of for 60s, 95 DEG C of for
10s, 60 DEG C of for 30s carry out amplification 40 follow it is bad.Using β-action as internal reference, according to 2Δ Δ Ct methodCalculate the opposite table of each gene
Up to amount, i.e. the variation multiple of expression quantity.
(3) Fig. 3 a, 3b, 3c experimental result: are detailed in.Within the scope of 0.1~1 μM, LW33 in dose dependent lower IL-1,
The mRNA level in-site of IL-1 β, TNF-α have statistical difference compared with model group.Compared with model group, LW33 is in 0.1~1 μM of model
In enclosing, the inflammatory reaction of LPS induction can be alleviated.
The influence of embodiment 4, LW33 to unilateral common carotid artery occlusion (UCCAo) model mice
Some researches show that permanent occlusion's model of unilateral CCA (UCCAo) shows CBF (cerebral blood flow (CBF)), WM (white matter) disease
Become and the chronic mild of memory disorders delay is reduced.In addition, the horizontal of proinflammatory cytokine increases, and the water of anti-inflammatory cytokines
It is flat to reduce in brain.This mouse model is research inflammatory reaction, and WM damages the associated powerful between memory disorders, this
Potentially contribute to illustrate the pathomechanism of VaD, especially SIVD (ischemic blood vessels are dull-witted under cortex).
(1) experimental material
Experimental animal: SPF grades KM mouse 40,8~9 week old, 28~40g of weight, male, purchased from Zhongshan University's experiment
Animal center (credit number: SYXK (Guangdong) 2016-0112), environment temperature: 20~25 DEG C, humidity 50~70% is freely ingested
Drinking-water.
LW33 (pharmaceutical college, Zhongshan University), donepezil (Pharmaceutical Co., Ltd. of health material) (day sodium carboxymethylcellulose CMCNa
Jin Zhiyuan chemical reagent Co., Ltd)
(2) experimental method
Animal packet: SPF grades KM mouse 40,5 groups are randomly divided by weight, is sham-operation group, model group, LW33 respectively
Low, high dose group (10,20mg/kg), donepezil group, every group 8.
Model preparation: in addition to sham-operation group, other each group mouse apply 0.45% penta after 1 week laundering period, to mouse
Barbital sodium is used for induced anesthesia.By midline cervical incision, right common carotid artery (CCA) is separated from neighbouring vagus nerve, and
With 6-0 silk suture (n=32).Sham-operation group (n=8), to the identical surgical operation of mouse progress without arteria carotis knot
It pricks.After operation, mouse is placed in cage, and give food and water.
Dosage regimen: isodose solvent 0.5%CMCNa, LW33 low (10mg/kg), LW33 high are given in sham-operation group stomach-filling
Dosage group (20mg/kg) and donepezil group (2.1mg/kg) give corresponding test medicine.Next day starts to be administered after modeling, often
It is administered once, and successive administration 21 days.Index of correlation is surveyed again after last dose 1h.
Testing index: it weighs in, is administered respectively before observation each group mouse general state, administration and after administration daily
21st day is experimental endpoints, carries out Morris water maze test after last dose 1h, sham-operation or UCCAO operation after measurement
30 days afterwards, with yellow Jackets deep anaesthesia mouse, and with 0.01MPBS through heart perfusion, then with containing 4% paraformaldehyde
0.1M phosphate buffer (PB, pH7.4) fixative perfusion.HE dyeing is done in brain tissue fixation afterwards for 24 hours.
(3) experimental result
1) mouse general state and weight
Compared with sham-operation group, modeling groups of animals activity is reduced, and hair owes smooth, other appearance signs, fecal character
Etc. having no notable difference.Compared with model control group, LW33 group and donepezil group animal appearance sign, behavioral activity, excrement
Character etc. has no notable difference.Seen from table 1, compared with Normal group, modeling group the weight of animals is not substantially change, and is had no
Statistical difference (P > 0.05).Compared with model group, LW33 group mouse weight is had no statistical difference (P > 0.05), but 0 week
When, donepezil group mouse weight is mitigated, and has statistical difference (P < 0.05).
Influence (g, Means ± SD, n=8) of 1 LW33 of table to UCCAo mouse weight
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
2) influence of the LW33 to the space exploration of UCCAo mouse and ability of learning and memory
Compared with sham-operation group, model group mouse escape latency was significantly increased, at first day of orientation navigation and the 4th
It, has statistical difference (P < 0.05), and Unilateral ligature CCA is prompted to can lead to memory dysfunction.Compared with model group,
LW33 administration group and donepezil group group can reduce mouse escape latency, but without statistical difference (P > 0.05).
Compared with sham-operation group, model group mouse spanning platform number and (P < 0.01) is significantly reduced in the residence time of target quadrant,
Prompt Unilateral ligature CCA modeling can lead to mouse space exploration reduced capability for 3 weeks.Compared with model group, LW33 high dose group and
Donepezil group can improve the space exploration obstacle (P < 0.05) of model mice.It is shown in Table 2, table 3:
Influence (Means ± SD, n=8) of 2 LW33 of table to UCCAo mouse escape latency
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
Influence (Means ± SD, n=8) of 3 LW33 of table to UCCAo mouse space exploration
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
3) influence that LW33 dyes UCCAo Mice brain tissues HE
As shown in the HE coloration result of Fig. 4: sham-operation CA 1 Zone of Hippocampus in Mouse normal neurons quantity is more, and nucleus is in
Circle, kernel is obvious and endochylema uniform coloring, coating are complete.Model group CA 1 Zone of Hippocampus in Mouse normal neurons significantly reduce, carefully
Nuclear pyknosis, loose around neuronal cell, gap becomes larger.
According to the experimental results, it is small can to improve UCCAo model at high dose (20mg/kg) by PDE9A inhibitor LW33
The damage in learning and memory of mouse acts on suitable with positive drug donepezil effect.LW33 high dose group and donepezil group
CA 1 Zone of Hippocampus in Mouse neure damage can be improved, and neuronal quantity increased compared with model group.
The influence of embodiment 5, LW33 to vascular dementia rat models are caused
It potentially contributes to illustrate the lesion type for causing human cognitive obstacle using the rodent model of ishemic stroke.
Arteria cerebri media (MCA) occlusion is considered as the convenient and reliable model of human brain ischemic in rat aorta.The MCA in rat aorta
Sensorimotor and cognition dysfunction can be induced in Occlusion Model, but sensorimotor caused by being occluded by intravascular MCA lacks
Fall into improve at any time, and data shows in rats, which can lead to the progressive process of cognitive disorder.This
It is consistent with the clinical progress of VaD.Up to the present, it is had been proven that in primary ischemic region using the experimental study of the model
The direct interaction of cerebral ischemia and AD type neuropathology.In addition, it is consistent with the decline of the progressive of cognitive function,
It is had confirmed that in MCA Occlusion Model in rat aorta in primary ischemic region, the delay mind of the remote area of hippocampus distal end
Through being denaturalized.Therefore the model can be used for studying VaD.And provide understanding in depth to some pathophysiological mechanisms of VaD.
(1) experimental material
Experimental animal: SPF grades SD rat 72, male, 200~240g of weight, purchased from Zhongshan University experimental animal
The heart (credit number: SCXK (Guangdong) 2016-0029), environment temperature: 20~25 DEG C, humidity 50~70%, drinking-water of freely ingesting.
LW33 (pharmaceutical college, Zhongshan University), nimodipine tablet (GuangDong HuaNan Pharmacy Group Co., Ltd), carboxymethyl cellulose
Plain sodium (Tianjin Zhi Yuan chemical reagent Co., Ltd), paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.), rat SOD reagent
Box (Bioengineering Research Institute is built up in Nanjing), rat MDA kit (Bioengineering Research Institute is built up in Nanjing), rat IL-6 reagent
Box (Wuhan Sino-American Biotechnology Company), rat TNF-α kit (Wuhan Sino-American Biotechnology Company), rat
IL-1 β kit (Wuhan Sino-American Biotechnology Company).
(2) experimental method
Animal packet:, being randomly divided into 6 groups by weight by SPF grades male Sprague Dawley rat 72, is artificial hand respectively
Art group, model group, LW33 low dose group (1mg/kg), LW33 middle dose group (3mg/kg), LW33 high dose group (9mg/kg), Buddhist nun
Not Horizon group (9.3mg/kg).Every group 12.
Model preparation: in addition to sham-operation group, other each group rats make according to the line brush that the methods of Nagasawa is improved
Model.With 2% yellow Jackets intraperitoneal injection (40mg/kg) anesthesia, lies on the back and be fixed on surgical plate.Right neck preserved skin, cleaning,
About 1cm osculum is cut off in longitudinal direction, and simultaneously hanging wire is spare by separation CCA, ICA and ECA, ligatures ECA and CCA, closes ICA telecentricity with artery clamp folder
Behind end, make a kerf in ECA and ICA crotch rapidly, (diameter is at smooth, spherical nylon wire from incision insertion heating one end
0.25mm, away from marking at the 2cm of ball end).After line is inserted into ICA, nylon wire and inlet ICA sections are ligatured slightly in inlet, so
Unclamp folder afterwards and close the artery clamp of ICA, continue into after nylon wire to slightly resistance and slightly withdraw, until line insertion depth be (18.5 ±
0.5) mm or so realizes that middle cerebral artery occlusion leads to cerebral ischemia.Inlet is ligatured again, about 1cm is stayed outside nylon wire, sutures skin
Skin.Stayed the end of a thread is gently lifted after ischemic 1h to there is resistance, realizes arteria cerebri media Reperfu- sion, then modeling is completed.Sham-operation group
Only ligation ECA and ICA.
Dosage regimen: sham-operation group stomach-filling is given isodose solvent (0.5%CMCNa), LW33 low dose group (1mg/kg),
LW33 middle dose group (3mg/kg), LW33 high dose group (9mg/kg), Nimodipine group (9.3mg/kg) give corresponding test drug
Object.Next day starts to be administered after modeling, is administered once daily, and successive administration 14 days.Index of correlation is surveyed again after last dose 1h.
Testing index: observation each group rat general state daily, administration the 14th day is experimental endpoints, after last dose 1h according to
The test of secondary progress passive avoidance (PAT), Morris water maze test, taken after measurement rat abdominal cavity venous blood be placed in containing
In the anticoagulant blood-collecting pipe of EDTA, 4 DEG C of centrifugation 10min of 3000rpm draw supernatant and measure SOD, MDA, IL-1 β, IL-6 content, take
Neck and chest skin is cut off after blood, exposure tracheae dissects rapidly thoracic cavity, exposes the heart, lung, and with 0.01M PBS through heart perfusion, so
It is perfused afterwards with the fixative of the 0.1M phosphate buffer (PB, pH7.4) containing 4% paraformaldehyde.Brain tissue fixation is done afterwards for 24 hours
HE dyeing.
(3) experimental result
1) compared with sham-operation group, modeling groups of animals activity is reduced rat general state, and hair owes smooth, other appearances
Sign, fecal character etc. have no notable difference.Compared with model control group, each administration group group and animal appearance sign, behavior are lived
Dynamic, fecal character etc. has no notable difference.
2) influence of the LW33 to rat model memory consolidation during obtaining passive avoidance response
It is postoperative give LW33 treat 2 weeks after, PAT test the phase, compared with sham-operation, model group rats enter escaping for camera bellows
Keeping away incubation period is obviously shortened (P < 0.01), and errors number increases (P < 0.05), prompts to can induce the acquired memory consolidation of SD rat
Obstacle；Compared with model group rats, LW33 high dose and Nimodipine group can extend rat enter camera bellows escape it is latent
Phase (P < 0.01) and reduction errors number (P < 0.01).
Table 4.LW33 in training period and tests interim preclinical influence (g, Means ± SD, n=12) to rat model
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
3) influence of the LW33 to rat model long-term memory and space exploration ability in Morris water maze test
In acclimatization training, compared with sham-operation group, each group rats'swimming speed does not have notable difference；Sham-operation group is big
Mouse ability of learning and memory is best, and the time (incubation period) for finding platform shortens with the training time；Model group rats study note
Recall that ability is worst, incubation period substantially change there is no the increase with training number of days, and prompting, which can induce rat, generates cognition
Dysfunction；Compared with model group, LW33 high dose group is suitable with positive drug Nimodipine group effect, especially the 5th day with
The incubation period of model group, there are statistical difference (P < 0.05).It is shown in Table 5, table 6:
Influence (g, Means ± SD, n=12) of the table 5.LW33 to rat model escape latency
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
Influence (g, Means ± SD, n=12) of the table 6.LW33 to rat model space exploration ability
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
4) influence of the LW33 to rat model SOD in serum, MDA content
Compared with sham-operation group, model group rats SOD content is remarkably decreased (P < 0.01), illustrates to can lead to rat blood serum
SOD content is decreased obviously.Compared with model group, the raising of LW33 high dose group rat SOD content has statistical difference (P <
0.01)；Compared with sham-operation group, model group rats Content of MDA dramatically increases (P < 0.01), illustrates to can lead to rat serum
Clear MDA is horizontal significantly raised.Compared with model group, LW33 high dose group and Nimodipine group can reduce rat model serum
MDA level (P < 0.01)；It is shown in Table 7
Influence (Means ± SD, n=6) of 7 LW33 of table to rat model SOD in serum, MDA content
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
5) influence of the LW33 to -1 β, IL-6 content of rat model serum IL
Compared with sham-operation group, model group rats IL-1 β, IL-6 content increase (P < 0.01), illustrate to can lead to rat serum
Clear IL-1 β, IL-6 content are significantly raised.Compared with model group, LW33 high dose group rat IL-1 β, IL-6 content are reduced, respectively
There is statistical difference (P < 0.01, P < 0.05)；It is shown in Table 8
Influence (Means ± SD, n=10) of 8 LW33 of table to -1 β, IL-6 content of rat model serum IL
Compared with model control group, * is P < 0.05, and * * is P < 0.01.
6) influence that LW33 dyes rat model brain tissue HE
As shown in the HE coloration result of Fig. 5, Fig. 6: rats in sham-operated group brain tissue slice HE dyes visible hippocampus under light microscopic
The area CA1 and cortical area neuronal cell number are more, marshalling and morphosis is complete.Kernel is obvious and endochylema coloring is equal
Even, coating is complete.And model group rats Hippocampal CA 1 and cortical area normal neuronal cell significantly reduce, neuron centrum
Cell arrangement is sparse at random, and apparent neuronal necrosis and empty balloon-shaped occurs in cortical area.LW33 high dose group and donepezil
Group CA 1 of Hippocampus and cortical area neure damage make moderate progress, and normal neurons quantity increased compared with model group.
Conclusion: in PAT (passive avoidance test) test, the LW33 and Nimodipine (9.3mg/ of high dose (9mg/kg)
Kg the memory consolidation of rat in passive avoidance task) can be improved；In Morris water maze test, with the increasing of training time
Add, the LW33 and Nimodipine of high dose can be improved the incubation period of rat model, increase the number for passing through target quadrant platform
And target quadrant residence time, learning and memory and space exploration obstacle to rat model make moderate progress.Meanwhile high dose
LW33 and Nimodipine can also increase SOD activity in serum and reduce MDA, IL-1 β, IL-6 content, this prompt LW33 may
Has the effects that anti-oxidation stress, anti-inflammatory.
Those skilled in the art of the present technique are appreciated that unless otherwise defined, all terms used herein (including technology art
Language and scientific term) there is meaning identical with the general understanding of those of ordinary skill in fields of the present invention.Should also
Understand, those terms such as defined in the general dictionary, which should be understood that, to be had and the meaning in the context of the prior art
The consistent meaning of justice, and unless defined as here, it will not be explained in an idealized or overly formal meaning.
It should be noted last that: the above embodiments are only used to illustrate and not limit the technical solutions of the present invention, although ginseng
It is described the invention in detail according to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that: it still can be to this
Invention is modified or replaced equivalently, without departing from the spirit and scope of the present invention.
Claims (9)
1. a kind of phosphodiesterase 9A inhibitor LW33 indicated by following compounds (I) or its pharmaceutically acceptable salt or
The purposes for preventing or/and treating vascular dementia disease drug is being prepared containing their any pharmaceutical compositions.
2. purposes according to claim 1, which is characterized in that in preparation prevention and/or treatment vascular dementia disease medicine
When object, the activity that the LW33 alleviates LPS induction BV2 inflammatory reaction is 0.1~1 μM.
3. purposes according to claim 1, which is characterized in that for when treating vascular dementia, the LW33 to drop
Low inflammatory factor TNF-α, the mRNA expression concentration of IL1- β and IL-6 are 0.1~1 μM.
4. LW33 according to claim 1 or its pharmaceutically-acceptable salts contain their any pharmaceutical compositions
The purposes in unilateral common carotid artery occlusion model mice histopathology structure medicament is influenced in preparation.
5. LW33 according to claim 1 or its pharmaceutically-acceptable salts contain their any pharmaceutical compositions
Improve the purposes of unilateral common carotid artery occlusion model mice damage in learning and memory drug in preparation, which is characterized in that described
The dosage of LW33 is 10~20mg/kg.
6. LW33 according to claim 1 or its pharmaceutically-acceptable salts contain their any pharmaceutical compositions
Improve the purposes of vascular dementia rat models damage in learning and memory in preparation, which is characterized in that the use of the LW33
Dosage is 1~9mg/kg.
7. LW33 according to claim 1 or its pharmaceutically-acceptable salts contain their any pharmaceutical compositions
Preparation for reducing MDA content in vascular dementia rat models serum, increase use in serum in the drug of SOD content
On the way.
8. LW33 according to claim 1 or its pharmaceutically-acceptable salts contain their any pharmaceutical compositions
IL-1 β in vascular dementia rat models serum, the purposes in IL-6 drug are reduced in preparation.
9. LW33 according to claim 1 or its pharmaceutically-acceptable salts contain their any pharmaceutical compositions
The purposes in vascular dementia rat models histopathology structure medicament is influenced in preparation.
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