CN110478351A

CN110478351A - The new application of more target action compound X7

Info

Publication number: CN110478351A
Application number: CN201910931160.0A
Authority: CN
Inventors: 缪朝玉; 王朋; 苏定冯; 周延; 刘建国; 张丽荣; 周杰; 周欣
Original assignee: Shenyang Neptune Biotechnology Co Ltd
Current assignee: Shenyang Neptune Biotechnology Co Ltd
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2019-11-22

Abstract

The present invention relates to the new applications of more target action compound X7 a kind of; the X7 is 3- shown in formula (I) (4- (4- (1H- benzotriazole -1- base) butyl) piperazine -1- base) benzisothia triazole hydrochloride, and the new application of more target action compound X7 of the invention includes: that X7 is preparing the application in antihypertensive drug, the drug of target organ protection function (preventing Cardiovascular Hypertrophy, kidney damage etc.), preventing brain stroke generation and the drug of death.The medical instrument has the advantages of not accelerating heart rate.The invention further relates to the pharmaceutical compositions for having above compound.Its advantage is shown: the present invention expands the purposes of more target action compound X7, provides new therapeutic scheme for patients such as hypertensive patient crowd, patients with cerebral apoplexy crowd, Cardiovascular Hypertrophy, kidney damages, has a good application prospect.

Description

The new application of more target action compound X7

Technical field

The present invention relates to medicine and pharmacology technical fields, specifically, being the new application of more target action compound X7.

Background technique

Hypertension is the clinical syndrome increased with systemic arterial pressure mainly to show, is the most common cardiovascular disease Disease.Primary and secondary two major classes can be divided into, wherein the overwhelming majority belongs to essential hypertension, the total hypertensive patient's of Zhan 95% or more.If hypertension effectively cannot be controlled and be treated, cerebrovascular accident (cerebral apoplexy), renal function can be caused to decline It exhausts, heart failure, coronary heart disease, the complication such as eyeground pathological changes.And these complication mostly can be lethal or disable.Deliver recently I State >=40 year old 170,000 crowd, 8 years Follow-up results show that cardiovascular and cerebrovascular diseases have become the first cause of the death in China, and hypertension is first Risk factor.Drug therapy is the main therapy for effectively controlling hypertension at present.It is traditional in anti-hypertension treatment field Antihypertensive drugs includes following several: 1) diuretics: by natriuretic diuretic, reducing the content of sodium and water in body-internal-circulation, holds blood Amount declines and reduces blood pressure.The side effect of diuretics is mainly Diagnostic value, and Diagnostic value can induce severe arrhythmia, can be with Interfere the antihypertensive effect of diuretics.2) beta-blocker: generating antihypertensive effect by a variety of action pathway, can be summarized as with Under several aspects: 1. reduce heart discharge, body generates adaptation reaction, and peripheral vascular resistance reduces, blood pressure decline；2. blocking Maincenter beta receptor reduces the nerve conduction of sympathetic fiber；3. blocking the excitatory receptor of presynaptic membrane β 2, the release of NE is reduced； 4. inhibiting renin release；5. increasing the vasodilator effect of ANP and PGI2；6. feeling of stress receptor is built again.Beta-blocker Adverse reaction: fatigue, limbs are cold, digestive discomfort, eyes flashing and vision blind spot etc..3) 1 receptor blocker of α: pass through selection Property acts on 1 receptor of cynapse α, expands resistance vessel and capacity vessel all, so that arterial pressure be made to decline.1 receptor blocker of α Decompression is significant, and can reduce cholesterolemia, triglyceride, improves insulin resistance, LVH is slightly reversed, because having suppression to prostate Production is used, therefore can obviously improve the dysuria of hypertrophy of the prostate patient.Suitable for having sugar or Anomalous lipid metablism, hypertrophy of the prostate Patient.Such side effects of pharmaceutical drugs has headache, dizziness, palpitaition, inability etc., the disadvantage is that may occur in which peripheral edema and weight Increase, and may occur in which apparent first dose of phenomenon, i.e. postural hypotension when first administration.4) calcium antagonist: in vascular smooth The special horizontally selected property of L-type calcium channel blocks the interior stream of Ca2+ on cell membrane in flesh and cardiac muscular tissue, to play vascular smooth Muscular relaxation, vascular resistence reduce, and the side effect of calcium antagonist is mainly flush (seeing short-acting dihydropyridines especially), head Bitterly, dizziness, palpitaition, constipation and ankle oedema, it is mainly related with its excessive blood vessel dilatation, to subtract easily since low dose of when use Its light adverse reaction.5) renin-angiotensin-aldosterone system (RAS) system, such drug and blood vessel ACEI: are acted on Angiotensin I converting enzyme combines, and Angiotensin II is inhibited to generate, and slows down bradykinin decomposition, leads to vasodilation, under blood pressure Drop.ACEI more typical adverse reaction is mainly dry cough and angioneurotic edema.If being taken simultaneously containing potassium supplement or protecting potassium Potassemia then easily occurs for diuretics.

Therefore, in view of the deficiencies of the prior art, inventor devises the new application of the more target action compound X7 of the present invention, X7 is a kind of benzotriazole base piperazine compounds.More target action compound X7 are used to treat hypertension and its complication, Achieve apparent technical effect.

Summary of the invention

The first purpose of this invention is in view of the deficiencies of the prior art, to provide the new use of more target action compound X7 On the way.

Second object of the present invention is in view of the deficiencies of the prior art, to provide a kind of pharmaceutical composition.

Third object of the present invention is in view of the deficiencies of the prior art, to provide the purposes of aforementioned pharmaceutical compositions.

To realize above-mentioned first purpose, the technical solution adopted by the present invention is that:

More target action compound X7 refer to formula (I) compound or its pharmaceutically acceptable salt；

Formula (I) compound or its pharmaceutically acceptable salt prevent, treat and delay hypertension, hypertension to cause in preparation Target organ damage and hypertension related disease drug in application,

As a preferred embodiment of the invention, the target organ damage be the heart caused by hypertension, brain, kidney or The damage of blood vessel；The hypertension related disease, including atherosclerosis, hyperlipidemia, obesity, coronary heart disease, dissection of aorta With hyperglycemia, impaired glucose tolerance, metabolic syndrome, diabetes.

As a preferred embodiment of the invention, wherein the target organ damage be left ventricular hypertrophy, cerebral apoplexy, Cortex renis atrophy or active phleboedesis, angina pectoris, myocardial infarction, heart failure, renal failure, retinal arteriosclerosis, Hypertensive Fundus lesion.

To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:

A kind of pharmaceutical composition, including pharmaceutically acceptable carrier and formula as described above (I) compound or its pharmacy Upper acceptable salt.

As a preferred embodiment of the invention, in the drug formula (I) compound containing 20mg-1000mg or Its pharmaceutically acceptable salt.

As a preferred embodiment of the invention, the pharmaceutical dosage form of described pharmaceutical composition is oral preparation or injection Agent, the oral preparation include tablet, capsule, granule pill or suspension.

To realize above-mentioned third purpose, the technical solution adopted by the present invention is that:

Target organ damage caused by pharmaceutical composition as described above prevents, treats and delay hypertension, hypertension in preparation And the application in the drug of hypertension related disease.

X7 chemical name: 3- (4- (4- (1H- benzotriazole -1- base) butyl) piperazine -1- base) benzisothia triazole hydrochloride. Molecular formula: C₂₁H₂₄SN₆HCl, molecular weight: 428.98,

The invention has the advantages that:

1, expand the purposes of X7, experimental result is shown: X7 is definite, rapid-action with antihypertensive effect, is depressured with slight Decreased heart rate does not influence conducting system of heart, generates favorable influence, long-term administration to haemodynamics to caused by hypertension Organ damage has the advantages of protective effect.The present invention had not only expanded the purposes of zofenopril calcium X7, but also was hypertension and its complication New therapeutic scheme is provided, chances of survival is increased for such patient, alleviates patient suffering, before there is application well Scape.

2, the compounds of this invention toxic side effect is small, and safety is good, has obviously to a variety of target organ damages caused by hypertension Curative effect.

Detailed description of the invention

Attached drawing 1, which is that rat is awake, move freely blood pressure continuous measurement schematic device.

Attached drawing 2 is the relation of dosage and effect that X7 reduces SHR blood pressure.

Attached drawing 3 is that X7 reduces the dosage of 2K1C dog blood pressure and the relationship of effect.

Attached drawing 4 is cardiac muscle-model group pathology HE colored graph, A:HE × 100, cardiac muscle fibre marshalling, cardiac interstitium water It is swollen, interstitial fibrosis, dilatation and congestion that cardiac interstitium blood vessel is slight, cardiac muscle cell's sheet necrosis, a small amount of monocyte, lymphocyte Infiltration；B:HE × 200, cardiac muscle fibre marshalling, no fracture, cardiac muscle cell broaden, and slight loose, collagenous fibres are thin between cardiac muscle Born of the same parents' hyperplasia, cardiac muscle cell's sheet necrosis, a small amount of monocyte, lymphocytic infiltration.

Attached drawing 5 is cardiac muscle-positive controls pathology HE colored graph, A:HE × 100, cardiac muscle fibre marshalling, between myocardium Matter oedema, interstitial fibrosis, the denaturation of cardiac muscle cell's sheet have no monocyte, lymphocytic infiltration；B:HE × 200, cardiac muscle are fine Marshalling, no fracture are tieed up, cardiac muscle cell gap reduces, and without obvious loose, interstitial fibrosis, collagenous fibres cell increases between cardiac muscle Raw, the denaturation of cardiac muscle cell's sheet has no inflammatory cell infiltration.

Attached drawing 6 is cardiac muscle-X7 treatment group pathology HE colored graph, A:HE × 100, cardiac muscle fibre marshalling, interstitial fibers Change, the accidental sheet denaturation of cardiac muscle cell, necrotic zone have no inflammatory cell infiltration；B:HE × 200, cardiac muscle fibre marshalling, Without fracture, cardiac muscle cell gap reduces, and nothing is obvious loose, interstitial fibrosis, collagenous fibres hyperplasia between cardiac muscle, cardiac muscle cell Accidental sheet denaturation or necrosis, have no inflammatory cell infiltration.

Attached drawing 7 is kidney-model group pathology HE colored graph, and A:HE × 100, mesangial cell proliferation, matrix diffuse Property increase, it is seen that inflammatory cell infiltration；B:HE × 200, glomerulus atrophy, necrosis, arteria renalis fibrosis and hyalinization, lymph Cell, monocyte and fibroblast infiltration.

Attached drawing 8 is kidney-positive controls pathology HE colored graph, A:HE × 100, mesangial cell proliferation, matrix Diffusivity increases, it is seen that a small amount of inflammatory cell infiltration；B:HE × 200, dia-betic rats are normal, there is a small amount of lymphocytic infiltration, Arteria renalis fibrosis and hyalinization are reduced, a small amount of lymphocyte, monocyte and fibroblast infiltration.

Attached drawing 9 is kidney-X7 treatment group pathology HE colored graph, and A:HE × 100, mesangial cell proliferation, matrix are more Unrestrained property increases, and has no inflammatory cell infiltration；B:HE × 200, dia-betic rats are complete, and individual glomerular matrix gaps increase, but Dia-betic rats are still normal, and a small amount of arteria renalis fibrosis has no inflammatory cell infiltration.

Attached drawing 10 is brain-model group pathology HE colored graph, and A:HE × 100, brain tissue overall structure is normal, have no bleeding and Inflammatory cell infiltration, nerve cell and peripheral clearance are without obvious broadening；B:HE × 200, neurotagma is normal, a small amount of nerve Cell cytoplasm region increases, karyopycnosis, and partial nerve cell expands, oedema.

Attached drawing 11 is brain-positive controls pathology HE colored graph, and A:HE × 100, brain tissue overall structure is normal, has no out Blood and inflammatory cell infiltration, nerve cell and peripheral clearance are without obvious broadening；B:HE × 200, neurotagma is normal, on a small quantity Nerve cell cytoplasmic region increases, nuclear hyperchromatism.

Attached drawing 12 is brain-X7 treatment group pathology HE colored graph, and A:HE × 100, brain tissue overall structure is normal, has and goes out individually Blood point has no inflammatory cell infiltration, nerve cell and peripheral clearance without obvious broadening；B:HE × 200, neurotagma is just Often, a small amount of nerve cell cytoplasmic region increases, nuclear hyperchromatism.Individual nerve cells expand oedema.

Attached drawing 13 is aorta-model group pathology HE colored graph, HE × 40.A: wall thickness: 134 μm, internal diameter: 1293 μm, wall Thickness/internal diameter=0.104, aorta form is normal, and tube wall is thicker；B: wall thickness: 134 μm, internal diameter: 1342 μm, wall thickness/internal diameter= 0.1, aorta form is normal, and tube wall is thicker.

Attached drawing 14 is aorta-positive controls pathology HE colored graph, HE × 40.A: wall thickness: 110 μm, internal diameter: 1647 μ M, wall thickness/internal diameter=0.067, aorta form is normal, and tube wall is relatively thin；B: wall thickness: 122 μm, internal diameter: 1500 μm, wall thickness/internal diameter =0.081, aorta form is normal, and tube wall is relatively thin.

Attached drawing 15 is aorta-X7 treatment group pathology HE colored graph, HE × 40.A: wall thickness: 122 μm, internal diameter: 1586 μm, Wall thickness/internal diameter=0.077, aorta form is normal, and tube wall is relatively thin；B: wall thickness: 122 μm, internal diameter: 1562 μm, wall thickness/internal diameter= 0.078, aorta form is normal, and tube wall is relatively thin.

Attached drawing 16 is SHR-SP survivorship curve.

Attached drawing 17 is X7 compounds on adrenal element α_1AThe effect of receptor.

Attached drawing 18 is X7 compounds on adrenal element α_1BThe effect of receptor.

Attached drawing 19 is X7 compounds on adrenal element α_1DThe effect of receptor.

Attached drawing 20 is X7 compound to serotonin 5-HT_2AThe effect of receptor.

Attached drawing 21 is X7 compound on tissue amine H₁The effect of receptor.

Attached drawing 22 is X7 compounds on adrenal element α_2BThe effect of receptor.

Attached drawing 23 is X7 compound to serotonin 5-HT_1AThe effect of receptor.

Specific embodiment

The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

English abbreviation vocabulary as described in the examples see the table below.

English abbreviation vocabulary

Influence of the 1 X7 single-dose of embodiment to SHR blood pressure and heart rate

1.1. experiment purpose

Understanding influences SHR blood pressure and heart rate by the administration of test product X7 single oral gavage, and content includes the dose-effect to reduce blood pressure Relationship, onset time, maximum reducing amplitude, blood pressure reduce hold time, the influence to heart rate and blood pressure and heart rate Relationship.

1.2. experimental material

1.2.1. test medicine

Test drug: code name X7, white in appearance is powdered, and dress hermetic bag puts 4 DEG C of refrigerator preservations.Product batch number: 2013103001.Physiological saline Extemporaneous is used using preceding.

Positive control drug: amlodipine besylate tablets, pfizer inc, authentication code: national drug standard: H10950224, product batch number: L02430.

1.2.2 experimental group and dosage are arranged

Experimental group: the principle being grouped at random according to animal, experiment set vehicle control group, positive controls, by test product X7 A dosage is first set, reduced downwards according to blood pressure result and increases dosage upwards, finding out minimum effective dose and maximum has Imitate dosage.

Dosage setting: all drug physiological saline solutions, administration volume are 5ml/kg, and various dose group is according to isometric(al) Isoconcentration method does not configure, and vehicle control group gives physiological saline 5ml/kg；Positive controls drug Amlodipine Besylate Tablet, agent Measure 5mg/kg；The dosage of X7 has 5 dosage of 1.25mg/kg, 2.5mg/kg, 5mg/kg, 10mg/kg, 15mg/kg from low to high Group, all experimentss share 7 groups, and every group of size of animal is 10.

1.2.3. administration route and administration number of times

Administration route: arresting rat and stress reaction occur and influence experimental result when in order to avoid with gastric perfusion needle stomach-filling, In Arterial cannulation has also done stomach fistulization and has been intubated while operation, outside fistula lead body.Stomach-filling is replaced by stomach fistula administration when experiment Administration.Administration number of times is single-dose.

1.2.4. reagent is used in other experiments

0.9% sodium chloride injection: Shangdong Hualu Pharmaceutical Co., Ltd.'s production, authentication code: national drug standard H37022749, product batch number: A13011703.

Heparin sodium: Sinopharm Chemical Reagent Co., Ltd.'s production, specification: 1g/ bottles, technical conditions meet Q/CYDZ66- 2012, product batch number: 20130617.

Ketamine Hydrochloride Inj.: Fujian Gutian Pharmaceutical Co., Ltd., specification: 2ml:10mg/ branch, national drug standard: H3502148, lot number: 130303.

Diazepam injection: Anyang City Yikang pharmaceutical factory, specification: 2ml:10mg/ branch, national drug standard: H41021491, lot number: 1403031.

1.2.5. experimental animal

Strain: spontaneous hypertensive rat (spontaneously hypertensive rat, SHR).

Rank: regular grade

Gender: male

Quantity: 70

Weight: 250 ± 20 grams

Source: SHR this teaching and research room, system introduces from the U.S. and breeds.With normal rats forage feed, free water, light 12 is small When light and shade automatic conversion, 20-22 DEG C of room temperature.

1.2.6. laboratory apparatus

Rat is awake to move freely blood pressure continuous measurement device, which mainly forms following (Fig. 1):

Pressure transducer, electronic engineering, Fudan University PT14M2 type Physiological Experiment pressure sensor；

Tee tube, French Plastimed company is perfused；

Four-way pipe, French Plastimed company is perfused；

Single channel rotary device, U.S. SpaldingMedicalproducts；

Pressure signal amplifier, MPA-HBBS, the production of Shanghai Alcott Science and Technology Ltd.；

Constant speed injects pump, Zhejiang Medical university instrument plant WZ-50G injector；

Polyethylene pipe, French Biotrol company, PE10 (internal diameter 0.3mm, outer diameter 0.50mm)；PE50 (internal diameter 0.58mm, Outer diameter 0.96mm)；

Bioelectric amplifier: MPA-SBBB, the production of Shanghai Alcott Biotechnology Co., Ltd.

Electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd., model: AL204.

Surgical instrument etc..

1.3. experimental method

1.3.1. the preparation of artery and vein conduit

Using polyethylene (Polyethylene, PE) conduit of two kinds of specifications, i.e. PE10 manages (internal diameter 0.30mm, outer diameter 0.50mm) and PE50 pipe (internal diameter 0.58mm, outer diameter 0.96mm) makes arterial duct, and PE10 pipe is docked with the heating of PE50 pipe, One is formed at docking and expands protuberance, and examining can securely, after clearness of catheter apply at docking, and conduit is full of test tube of hepari when application PVP liquid, and PE50 pipe one end is closed with metal needle, PE10 pipe is inserted into low level abdominal aorta through femoral artery.

1.3.2. arterial cannulation

Rat is with ketalar 50mg/kg and diazepam 5mg/kg intraperitoneal injection of anesthesia, and rat dorsal position is solid after anesthesia Fixed, cropping, disinfection at the groin of left side, row about 2cm long skin incision expose femoral artery sheath, carefully separate femoral artery, insertion is filled The conduit of full heparinized saline (200IU/ml), used in suturing of arterial duct is fixed on pars inguinalis muscle, will with draw pin Percutaneously down toward neck portion, skin is pierced by conduit.Skin incision at continuous suture groin, the arterial duct that neck is drawn are used certainly Saddle device processed is fixed against to grab after rat regains consciousness and sting.

1.3.3. gastric fistula is made

After venous incubation, abdomen hits exactly cropping, disinfection.Muscle layer is cut along hunter's line, finds stomach under lobe of the liver, Purse string suture is done in stomach wall, insertion self-control silica gel stomach tube tightens pocket.With draw pin by stomach tube from a pleurosternal muscle wear to it is subcutaneous and Skin is pierced by after neck is fixed with saddle, sutures muscle layer and skin.

1.3.4. regain consciousness and move freely the blood pressure measurement of rat

Restore 24 hours after rat intubation, be put into homemade plexiglass cylinder, arterial duct and pressure measuring system are connected It connects, rat can move freely in cylinder and free water is ingested.Blood pressure signal of often fighting through pressure transducer is converted to biology Electric signal is recorded often fight blood pressure and cardiac cycle (Fig. 2) by computer in real time.After adaptations in 1-2 hours are stablized, record 1 is small When blood pressure heart rate based on be worth, be then administered through stomach fistula, 24 hours blood pressures and cardiac cycle data after continuous record administration, Any time segment data that can be transferred in the record time after off line is analyzed.

1.4 statistical disposition

Experimental result indicates that data are with Excel2010 and SPSS18.0 software between group with mean ± standard deviation (_ x ± s) Data use ANOVA variance analysis, carry out two comparison among groups with pairing and non-paired t test, are defined as having with P < 0.05 aobvious The statistical difference of work.

1.5. observation index

Minimum and highest effective dose after X7 single-dose.

Onset time after X7 single-dose.

The relationship of X7 single-dose post dose and effect.

Blood pressure reduces the time maintained after X7 single-dose.

To the relationship of the influence of heart rate and changes in heart rate and blood pressure after X7 single-dose.

1.6. experimental result

1.6.1.X7 the Dosage of SHR blood pressure is reduced

The systolic pressure maximum range of decrease of SHR after the dosage gastric infusion since 1.25mg/kg is explored to the hypotensive dose of X7 Only 11mmHg；Increase that shrink drops when dosage arrives 2.5mg/kg still unobvious, for 13mmHg；But when dosage increases to When 10mg/kg, systolic pressure reduces highly significant, and apparent dose-effect relationship is presented between 2.5mg, 5mg and 10mg/kg dosage, I.e. with the increase of dosage, blood pressure is also accordingly reduced.The absolute value that blood pressure reduces is between 30-36mmHg；However, using being higher than The dosage of 10mg/kg, antihypertensive effect are not increase accordingly.

SHR blood pressure systolic pressure is set to be reduced more than the effective standard judgement of 20mmHg with drug, figure it is seen that The gastric infusion dosage of X75mg/kg can make blood pressure Amplitude of Hypotensive reach 30mmHg, can be set as middle dosage；Downward 2.5mg/kg can It can be used as high dose group as low dose group, upward 10mg/kg.

After X7 is administered to SHR rat oral gavage, there is 30min upon administration in maximum reducing effect.

1.6.2.X7 single oral gavage administration reduces the onset time observation of SHR blood pressure

With X7 and each 5mg/kg of Amlodipine Besylate Tablet to after SHR stomach-filling to blood pressure it has been observed that X7 reduced blood pressure Onset time is obviously faster than Amlodipine Besylate Tablet.Compared with before administration, 2.5min blood pressure reduces 12mmHg after X7 gastric infusion, 5min reduces about 20mmHg, occurs statistical significant difference (P < 0.01) compared with 0min, and 10min, which is reduced, reaches 26mmHg； There is 30min upon administration in maximum reducing effect.Slower, the 10min blood after administration in contrast, Amlodipine antihypertensive effect works The low 9mmHg of pressure drop, reaching the time that blood pressure reduces 20mmHg, 45min, maximum reducing effect occur upon administration upon administration 90min。

1.6.3.X7 influence of the single-dose to SHR blood pressure and heart rate

Influence of the X7 to SHR blood pressure:

SHR femoral artery is implanted into pressure catheter, and connection is awake after restoring for 24 hours move freely rat blood pressure measuring device, even 24 hours blood pressures of continuous record, processed off-line data are found after every 2 hours pressure values are averaged:

Vehicle control group rat systolic pressure is stablized between 175mmHg or so, diastolic pressure 116-120mmHg.

Positive controls SHR gives 5mg/kg Amlodipine, and blood pressure significantly reduces after administration, before systolic pressure is by being administered 172mmHg is down to the minimum point 135mmHg after administration, decreases by 37mmHg, with the extension of time after administration, blood pressure is gradually returned It rises but rise speed is slower, but 20h blood pressure is still maintained at the level before substantially less than administration, the variation of diastolic pressure after administration It is consistent with systolic pressure.

Three test drug X7 low dosage (2.5mg/kg), middle dosage (5mg/kg) and high dose (10mg/kg) dosage groups, give Medicine after-contraction pressure and diastolic pressure also have a significant decrease within 8h, and blood pressure reduction and dosage at dependence, i.e. dosage is got over Greatly, blood pressure reduction is more obvious, and the high dose group blood pressure maximum range of decrease is 26mmHg, and blood pressure reduction is held time as 8-10h, 12h with Afterwards, the systolic pressure of SHR and diastolic pressure rise move closer to level before administration.

Influence of the X7 to SHR heart rate:

Less, fluctuating range is smaller for variation before and after the heart rate medication of vehicle control group SHR.

After positive controls SHR is to Amlodipine 5mg/kg, with the reduction of blood pressure, increased heart rate clearly, is being given (minimum is also in the period blood pressure) in medicine 6 hours, increased heart rate has statistical significant difference compared with before administration (P < 0.01), by 351 beats/min before being administered, highest increases to 416 beats/min, and the variation of heart rate and blood pressure are negatively correlated, i.e. blood Pressure drop is low, increased heart rate, blood pressure rises, decreased heart rate.

X7 is low, and middle high three dosage groups administration front and back is compared, and with the reduction of blood pressure, heart rate is not increased not only, is omited instead Have and slows down.0-2h upon administration, decreased heart rate is obvious, arrives 6-8h, with the gradually rise of blood pressure, heart rate is also gradually extensive Multiple, horizontal before blood pressure is restored to administration, heart rate also substantially returns to that preceding state is administered.

1.7 conclusion

1.7.1.X7 antihypertensive effect is definite, and dose-effect relationship is clear, and being suitble to the low middle high hypotensive dose of SHR is respectively 2.5mg/ Kg, 5mg/kg and 10mg/kg.

1.7.2.X7 single oral gavage administration reduces SHR blood pressure and works rapidly, is depressured onset time compared with Amlodipine Besylate Tablet Fastly.

1.7.3.X7 single oral gavage administration can maintain SHR blood pressure to reduce about 6-8 hours.

1.7.4.X7 single oral gavage administration has slight decreased heart rate while reducing blood pressure.

Influence of the 2 X7 single-dose of embodiment to renovascular hypertension dog blood pressure and heart rate

2.1. experiment purpose

Understand and is administered by test product X7 single oral gavage to Two-kidney One-clip (2K1C) renovascular hypertension under waking state The influence of Beagle dog blood pressure, observed content include dose-effect relationship, blood pressure reduce amplitude peak, blood pressure reduction hold time and Influence to heart rate.

2.2. experimental material

2.2.1. test medicine

2.2.2. reagent is used in other experiments

Yellow Jackets: German Merck biotech company production, import packing, specification: 25g/ bottles, product batch number: WS20130112。

2.2.3. dosage is arranged

Dosage setting: vehicle control group: physiological saline 1ml/kg；

Positive controls: Amlodipine Besylate Tablet 1.5mg/kg；X7:0.25mg/kg, 0.5mg/kg, 1mg/kg, 1.5mg/ Kg, 2mg/kg, 4mg/kg, 6mg/kg totally 7 dosage groups.

2.2.4. administration route, administration volume and administration number of times

Test medicine uses physiological saline solution before use, is configured to the isometric solution of respective concentration；

Administration route: gastric infusion；

Volume: 1ml/kg is administered；Administration number of times is single.

2.2.5. experimental animal

Kind: healthy Beagle dog

Source: Shanghai Jia Gan Biotechnology Co., Ltd

Gender: male female dual-purpose, weight: 8~10kg

Quantity: 22, the dog after Cheng Mo is randomly divided into three groups, every group 6, remaining 3 it is spare, every group be administered once after need to stop It can be reused after medicine 7-10 days.

Licensing: SCXK (Shanghai) 2010-0028

Certification of fitness number: 2010002802091

2.2.6. laboratory apparatus

Four-way PowerLab8/30 high-speed record instrument, the place of production: Australia.

Bridge amplifier: model: ML211, the place of production: Australia.

Pressure transducer, model: MLT844, the place of production: Australia.

Animal specific Doppler's non-invasive blood pressure measuring: model: DS-100 type, the place of production: Hefei ,Anhui.

Surgical instrument: scalpel, operating scissors, haemostatic clamp etc..

2.3. experimental method

Dog is injected intravenously with 3% yellow Jackets 30mg/kg and is anaesthetized, and is lain on the back and is fixed on 39 DEG C of operating tables of constant temperature, upper abdomen Preserved skin, 75% alcohol disinfecting spread aseptic operation list, and upper abdomen median incision about 5cm long successively cuts skin, subcutaneous tissue, abdomen Rectus sheath, open peritonaeum enter abdominal cavity, in margo interior hepatis find right side kidney, with wet gauze by separations such as surrounding small intestine, nethike embranes simultaneously Incision of abdominal wall is pulled open with pull hook, sufficiently exposure right side kidney, blunt separation perirenal fascia.Diagonal trend is found below renal vein The arteria renalis and dissociated, the stainless steel wire of diameter about 1mm is placed in parallel with artery, with 4-0 silk thread by steel wire together with right kidney Artery ligatures together, carefully extracts steel wire, incomplete recovery kidney blood supply after ligation out.(8-10kg Beagle dog arteria renalis diameter It is approximately less than 2mm, the decline of arteria renalis flow will can be made after its constriction about 60%).After checking that gauze, instrument are errorless, abdomen is successively closed, is stitched Skin.Wound is covered and is wrapped up with alcohol gauze.400,000 units of postoperative intramuscular injection Benzylpenicillin sodium salt/day, continuous three days to prevent infection.Kidney Blood pressure is measured with Doppler's non-invasive blood pressure measuring within 45 days after the operation of arteriarctia art, it is hypertension model that systolic pressure, which is greater than 160mmHg, The sodium intravenous anesthesia of amobarbital is used in qualification, then random grouping again, and the operation of row femoral arteriography, post-operative recovery is for 24 hours. Online PowerLab record monitoring blood pressure, stablizes 15-30min start recording work after connecting blood pressure recording system under waking state Based on be worth (blood pressure when 0 after administration), be then administered, 0-120min blood pressure after continuous record administration is remembered every 2h later Blood pressure 5min is recorded, continuously for 24 hours.

2.4. statistical disposition

Experimental result is with mean ± standard deviationIndicate, with Excel2010 and SPSS18.0 software to data between group into Row ANOVA variance analysis carries out two comparison among groups with pairing and non-paired t test, is defined as having significant system with P < 0.05 Meter learns difference.

2.5. observation index

Relationship of the X7 to 2K1C dog dosage and pressure reduction effect.

X7 is to the onset time after the administration of 2K1C dog.

2K1C dog blood pressure maximum reduces amplitude and holds time after the X7 administration of various dose.

The relationship of influence and blood pressure and heart rate of the X7 to 2K1C dog heart rate.

2.6. experimental result

2.6.1.X7 the dose-effect relationship of 2K1C Beagle dog is tested

Beagle dog measures blood pressure after side renal artery stenosis is performed the operation 45 days, with Doppler's non-invasive blood pressure measuring, when flat Equal systolic pressure is in 160mmHg or more, and diastolic pressure in 90mmHg or more, be allowed for access experiment by the dog after meeting hypertension standard.It is real Femoral arteriography operation is done before testing, and connects pressure transducer under the conditions of awake after restoring for 24 hours and PowerLab system records blood Pressure.It is worth (0min) based on dog stable state recording 5-10min blood pressure, 120min is continuously recorded after administration, to divide Analyse dose-effect relationship；Then a blood pressure is recorded at interval of 2h, each 5-10min holds time and right to analyze blood pressure reduction The influence of HR.

To the research experiment lowest dose level of dose-effect relationship since 0.25mg/kg, increase upwards dosage have 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 4mg/kg and 6mg/kg, totally 7 dosage.As it can be seen from table 1 the X7 of various dose gives Maximum blood pressure reduces amplitude appearance 30min or so upon administration after medicine；Be lower than 4mg/kg dosage, blood pressure reduce amplitude with The increase of dosage and increase, there are apparent dose-effect relationships between the two.But after dosage is more than 4mg/kg, increase dosage Blood pressure no longer accordingly reduces afterwards.Hence, it can be determined that X7 is used for the suitable dose of 2K1C hypertension model dog decompression are as follows: 1mg/kg (low dosage), 2mg/kg (middle dosage), 4mg/kg (high dose) (table 1, Fig. 3).

Table 1.X7 single-dose tests (unit: mmHg to the dose-effect relationship of 2K1C dog blood pressure；N=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

2.6.2.X7 observation of the single oral gavage administration to 2K1C Beagle dog blood pressure onset time is reduced

Observation drug has used the onset time of 2K1C dog the middle dosage of X7, i.e. 2mg/kg and positive drug benzene sulfonic acid ammonia chlorine Horizon 1.5mg/kg.Observing time is 120min after administration.Systolic pressure analysis is found after single oral gavage, X7 decompression action when Between it is faster than Amlodipine.Compared with 0min blood pressure, 5min after X7 gastric infusion, systolic pressure reduction has the significant meaning of statistics, 10min decompression is greater than 20mmHg.Slower, the 30min blood pressure reduction after administration in contrast, Amlodipine antihypertensive effect works 20mmHg simultaneously has the significant meaning of statistics (table 2).

To the observation of the blood pressure onset time of 2K1C dog after table 2.X7 single-dose

(unit: mmHg；N=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

2.6.2.X7 influence of the single-dose to 2K1C Beagle dog blood pressure

The blood pressure administration front and back of vehicle control group dog is constantly in opposite steady state, and systolic pressure fluctuation for 24 hours is in 165mmHg Left and right；

Positive controls, drug are amlodipine, and dosage 1.5mg/kg, administration after-contraction pressure, diastolic pressure are in 0-2h It bottoms out, reduction amplitude is respectively 31mmHg and 25mmHg, maintains blood pressure steadily to reduce the time longer, after systolic pressure administration For 24 hours, 10h still has the significant meaning of statistics compared with before administration after diastolic pressure administration.

X7 points of low middle high three dosage reduce with the presence of significant dose-effect relationship, maximum drop blood pressure (systolic pressure, diastolic pressure) Pressure amplitude degree equally occurs in after administration 0-2 hours, maintains blood pressure steadily to reduce systolic pressure and is greater than 6h greater than 12h, diastolic pressure.

2.6.3.X7 influence of the single-dose to 2K1C Beagle dog heart rate

As shown in Table 2-4, the heart rate administration front and back variation of vehicle control group dog less, is stablized at 155 beats/min or so.

Amlodipine Besylate Tablet administration after increased heart rate it is obvious, and increased heart rate hold time it is consistent with blood pressure reduction, It is 22-24h that blood pressure, which maintains the time steadily reduced, and increased heart rate also continues in the corresponding time substantially；Increased amplitude is by giving 154 ± 3 beats/min before medicine, up to 168 ± 11 beats/min, statistical difference are significant (P < 0.05).

The performance of heart rate and positive control drug Amlodipine are exactly the opposite after X7 administration, decreased heart rate after X7 administration, heart rate Slow down duration and blood pressure is maintained to reduce time consistency, within 12h.Hereafter, with the rise of blood pressure, heart rate is also gradually It is preceding horizontal to be restored to administration.Influence from X7 dosage analysis to heart rate, it can be found that the degree of decreased heart rate is related with dosage, i.e., With the increase of dosage, decreased heart rate also increases, and 2h is most obvious upon administration for decreased heart rate, but the amplitude of decreased heart rate is not Greatly, by taking X7 high dose group as an example, minimum 131 beats/min of heart rate, it is 156 beats/min before administration, has slowed down 25 beats/min (tables 3).

Table 3.X7 to Beagle dog HR influence (unit: beat/min,N=6)

* P < 0.01 P < 0.05, * * is compared with 0min

2.7. conclusion

2.7.1.X7 single-dose is definite to reduction 2K1C dog antihypertensive effect, and dose-effect relationship is obvious, the decompression of 4mg/kg dosage It can reach 50mmHg or more.The low middle high drug dose for being suitble to dog decompression is respectively 1mg/kg, 2m/kg and 4mg/kg.

2.7.2.X7 single-dose works to the blood pressure for reducing 2K1C dog very fast, and 10min can make blood pressure significant after administration It reduces, onset time is obviously fast compared with Amlodipine Besylate Tablet.

2.7.3.X7 single-dose can maintain the blood pressure of 2K1C dog steadily to reduce 10-12 hours, hence it is evident that compared with benzene sulfonic acid ammonia chlorine The 22-24h of Horizon is short.

2.7.4.X7 having to the heart rate of dog and slightly slow down effect, the variation of heart rate is related with blood pressure, i.e., blood pressure reduces, Decreased heart rate, blood pressure increase, increased heart rate.

Influence of the 3 X7 multiple dosing of embodiment to 2K1C dog and SHR blood pressure and electrocardiogram

3.1. experiment purpose

Understand whether X7 multiple dosing has an impact to 2K1C dog and SHR to conducting system of heart, if had an impact, after drug withdrawal Whether can restore.

3.2. experimental material

3.2.1. test medicine

X7 white in appearance is powdered, and dress hermetic bag puts 4 DEG C of refrigerator preservations.Product batch number: 2013103001.Use preceding use Physiological saline Extemporaneous.

3.2.2 experimental group and dosage are arranged

Experiment is set according to the principle that is grouped at random holds matchmaker's control and by each one group of test product X7, SHR every group 12, Beagle dog Every group 6；The dosage of dog and rat uses middle dosage, i.e. SHR 5mg/kg, Beagle dog 2mg/kg, and 9 points of every morning or so Gastric infusion, 60min detects blood pressure with non -invasivetesting method after administration.

3.2.3. reagent is used in other experiments

3.2.4. experimental animal

SHR:

Strain: spontaneous hypertensive rat (spontaneously hypertensive rat, SHR).

Rank: regular grade；Gender: male；Quantity: 24；Weight: 250 ± 20 grams.

Source: SHR this teaching and research room, system introduces a fine variety breeding from the U.S..With normal rats forage feed, free water, light 12 is small When light and shade automatic conversion, 20-22 DEG C of room temperature.

Beagle dog:

Source: Shanghai Jia Gan Biotechnology Co., Ltd；Gender: male female dual-purpose；Weight: 9~10kg；Quantity: 6；Perhaps It can demonstrate,prove: SCXK (Shanghai) 2010-0028；Certification of fitness number: 2010002802091.

3.2.5. instrument and material

Rat non-invasive blood pressure measuring instrument: Shanghai Alcott Biotechnology Co., Ltd, model: ALC-NIBP.

4 channel PowerLab8/30 high-speed record instrument, model: ML870/P, the place of production: Australia.

Bioelectric amplifier, model: ML132, the place of production: Australia.

Syringe, gastric perfusion needle etc..

3.3. experimental method

Dog and rat non -invasivetesting blood pressure measurement methods, continuous 7 days once a day.7th day rat under narcosis, Dog uses PowerLab record standard augmented limb lead II lead electrocardiogram under waking state, soft with the included analysis of PowerLab It is worth based on the Electrocardiograph indexes such as part automatic measurement QT time, QRS complex wave time；Then gastric infusion, once a day, even It is 7 days continuous.The daily administration time is relatively fixed, and it is primary to measure blood pressure by 60min after administration.Electrocardiogram is measured again after administration in 7th day, The analysis result of electrocardiogram is worth after being used as administration；Then stopping administration, daily measuring blood pressure is primary, and continuous 7 days, the 7th day after drug withdrawal Measure II lead again afterwards.Electrocardiogram after in front of medication three times is compared, understands whether drug influences cardiac electrical conduction And influence recovery after drug withdrawal situation.

3.4. observation index

The blood pressure of dog and rat normal condition, heart rate and Electrocardiograph index (the QT time, the comprehensive wave width of QRS, T wave width, With ST sections of height).

With the blood pressure of dog and rat, heart rate and Electrocardiograph index after X7 continuous use 7 days, (QT time, QRS complex wave are wide Degree, T wave width and ST sections of height).

Whether if These parameters are by drug influence, being discontinued can restore after a week.

3.5. statistical disposition

3.6. experimental result

3.6.1.SHR test result

Blood pressure: to the blood pressure measurement of SHR week age before administration, systolic pressure is average in 175 ± 3mmHg, then gives X75mg/kg, once a day, continuous one week, detection blood pressure was 140 ± 8mmHg, and mean reduction reaches 35mmHg, was discontinued after a week, Blood pressure is restored to level before administration.The variation of diastolic pressure is consistent with systolic pressure.

Heart rate: administration 346 ± 76 beats/min of the last week average heart rate, medication after a week heart rate not with the reduction of blood pressure And accelerate, slightly slow down instead, 326 ± 54 beats/min of average out to, the measurement heart rate after a week that is discontinued is 329 ± 59 beats/min, with base Plinth value compares no significance,statistical meaning.

Electrocardiogram: through to QT time and ST sections of mutation analysis, X7 multiple dosing does not influence (table 4) to SHR electrocardiogram.

Table 4.X7 multiple dosing to SHR blood pressure, heart rate and electrocardiogram influence (N=12)

P < 0.001 * * is compared with basic value

3.6.1.2 K1C Beagle dog test result

Blood pressure: a blood pressure is measured daily to 6 2K1C dogs before administration, continuous 7 days average value is as the blood before administration Basic value is pressed, the systolic pressure of basic value is 170 ± 4mmHg；Then X72mg/kg is given, it is continuous 7 days once a day, every during administration It is primary that it surveys systolic pressure, time of measuring 60min upon administration, before the mean blood pressure during treatment is 140 ± 4mmHg, with administration Comparing systolic pressure reduces 30mmHg；Hereafter in one week to be discontinued daily still measurement blood pressure it is primary, mean blood pressure be 175 ± 4mmHg has been restored to level before administration,

Heart rate: the heart rate basic value of 2K1C dog is 150 ± 9 beats/min, 138 ± 12 beats/min of heart rate when one week treatment end, Heart rate slightly slows down compared with basic value, reduces 12 beats/min, is discontinued latter week, heart rate substantially returns to that preceding state is administered.

Electrocardiogram: extending 0.01s after QT time administration, QRS complex wave spaced time extends 0.01s, the two and Control basis value is compared to no significance,statistical, also without departing from its physiological fluctuation range, and before being restored to administration after being discontinued Horizontal (table 5).

2K1C dog blood pressure before and after table 5.X7 medication, heart rate and ECG Change (N=6)

* P < 0.01 P < 0.05, * * is compared with basic value

3.7. experiment conclusion

3.7.1.X7 multiple dosing has significant decrease effect to the blood pressure of SHR and 2K1C dog, can be restored to after drug withdrawal to It is horizontal before medicine.

3.7.2.X7 after multiple dosing the heart rate of SHR and 2K1C dog have it is different degrees of slow down, be discontinued heart rate after a week Restore horizontal close to before administration.

3.7.2X7 multiple dosing slightly extends the QT interphase of 2K1C dog electrocardiogram and QRS complex wave time, the two and Control basis value is compared to no significance,statistical, and also without departing from its physiological fluctuation range, and drug withdrawal can restore after a week.

Influence of the 4 X7 single-dose of embodiment to SD hemodynamics

4.1. experiment purpose

Understand whether X7 has an impact to hemodynamics index.

4.2. experimental material

4.2.1. test medicine

4.2.2. experimental group and dosage are arranged

Experimental group: experiment sets vehicle control group, positive controls according to random group forming criterion, is set low middle height by test product X7 Three dosage groups, totally five groups, every group of rat quantity is 10.

Dosage setting: vehicle control group, physiological saline 5ml/kg；Positive controls, Amlodipine 5mg/kg；X7 low dosage Group (2.5mg/kg)；Middle dose group (5mg/kg)；High dose group (10mg/kg).

4.2.3. reagent is used in other experiments

Ethylurethanm: Sinopharm Chemical Reagent Co., Ltd.'s production, specification: 500g/ bottles, purity: >=98%, production batch Number: 20130609.25% solution is prepared with distilled water when use.

4.2.4. administration route, administration number of times and administration volume

Test medicine uses physiological saline solution before use, and the isometric solution of respective concentration is made in slightly ultrasound；Administration route: Gastric infusion is replaced by the administration of stomach fistulization, volume: 5ml/kg is administered；Administration number of times is single.

4.2.5. experimental animal

Strain: 60 healthy SD rats.

Rank: SPF grades.

Gender: male.

Animal productiong licensing number: SCXK (Shanghai) 2008-0016.

The Quality of Experimental Animals quality certification: 2008001641813.

Weight: 200 ± 20 grams.

Source: The 2nd Army Medical College (Experimental Animal Center).

Animal in our unit's experimental animal room divides cage adaptive feeding three days after buying back, with normal rats forage feed, from By drinking water, 12 hours light and shade automatic conversions of light, 20-22 DEG C of room temperature.

4.2.6. instrument

MPA2000: the production of Shanghai Alcott Biotechnology Co., Ltd, model: MPA2000.

Balance: double outstanding fraternal (group) Co., Ltds, the U.S., the production of Changzhou Shuan Jie test equipment factory, model: T1000.

4.3. experimental method

With 25% urethane 1.5g/kg intraperitoneal injection of anesthesia after SD rat weight, and it is fixed, after tongue is involved to prevent tongue Pendant causes to suffocate.Operative region shaving, 75% alcohol disinfecting, separation side femoral artery insertion are full of the heparin physiology of 100u/ml The self-control arterial duct of salt water；Skin of neck is cut, right carotid, insertion self-control cardiac catheter are separated.Arterial duct and the heart Conduit connects pressure transducer, and pressure transducer and bioelectric amplifier are connected through MPA2000 with computer, real-time display electrocardio wave Shape and arterial pressure waveform, are slowly advanced cardiac catheter, gently steering nozzle when reaching aorta petal mouth, when arterial pressure wave mode is prominent The intra-ventricle square wave for so becoming standard indicates that conduit comes into left ventricle, fixed catheter.Needle electrode insertion four limbs subcutaneously connect Connect bioelectric amplifier.Make its test tube of hepari by 500U/kg intravenous injection heparin, to prevent catheter blockage.Surgical procedure finishes, and moves Object is stablized record within 15 minutes following indexs based on value (when 0).Then corresponding test medicine is slowly injected by stomach fistula, 5,15,30,60,90 and 120min minutes duplicate measurements observation index after administration.If systolic pressure is lower than in surgical procedure 80mmHg last up to 5 minutes or more (caused by non-drug) then the animal make waste treatment.

4.4. observation index

Heart rate (HR), mean arterial pressure (MBP), left ventricular systolic pressure (LVSP), ventricular end diastolic pressure (LVEDP), The contraction of the appearances such as left ventricle, diastole maximum rate of change (± dp/dtmax).

4.5. statistical disposition

4.6. experimental result

4.6.1.X7 influence of the single-dose to SD rat MBP and HR

Influence to MBP:

The MBP of volume control group SD rat is average in 93mmHg or so, the 120min mono- terminated since experiment from Directly it is in metastable state；

Positive controls Amlodipine, dosage 5mg/kg, 30min starts blood pressure and is gradually reduced after administration, to after administration 120min Amplitude of Hypotensive is maximum, is 31mmHg；

The onset time to reduce blood pressure after by test product X7 administration is faster than positive control drug Amlodipine, and 5min goes out after administration Existing blood pressure significantly reduces, and with the increase of dosage, MBP reduces amplitude and also increase accordingly, and it is bright to illustrate that X7 reduction Mean Arterial is pressed with Aobvious dose-effect relationship exists.The amplitude peak that MBP is reduced after X7 administration is also in 120min, the maximum blood of low middle high three dosage groups Pressure drop width is respectively 24mmHg, 27mmHg and 30mmHg (table 6).

Influence to heart rate:

As can be seen from Table 7, administration front and back is compared, the heart rate stabilization of vehicle control group rat, and the variation of administration front and back is unknown It is aobvious.

There is increased heart rate upon administration in Amlodipine, and the influence with reference to Amlodipine to blood pressure is it can be found that giving After medicine 30min, blood pressure reduces amplitude and starts to increase, and heart rate is also accordingly accelerated, to (120min after administration) when terminating to test, the heart Rate increases to 390 times/min by 363 times/min before being administered, and administration front and back is compared, the increase highly significant of heart rate.

The low middle high three dosage groups administration of X7 does not increase not only with the reduction heart rate of blood pressure, has instead different degrees of Slow down, after 5min is administered, heart rate slows down, and to after being administered after 15min, decreased heart rate has statistics compared with before administration Significant meaning, and be maintained to experiment and terminate.

Influence (unit: mmHg of the table 6.X7 to rat MBP；N=10)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

Table 7.X7 to rat heart rate influence (unit: beat/min；N=10)

* P < 0.01 P < 0.05, * * is compared with 0min

4.6.2.X7 influence of the single-dose to SD rat left ventricle function

Influence of the X7 to LVSP:

It is consistent with the influence to MBP, compare before and after medication, low middle high three dosage of X7 and positive control drug Amlodipine are equal There is the reduction LVSP of highly significant.120min after administration, Amlodipine reduce LVSP and reach 38mmHg, and X7 is low middle three high The LVSP that dosage reduces rat respectively is 28mmHg, 33mmHg and 41mmHg, and the amplitude for not only reducing LVSP is big, but also in low With the presence of dose-effect relationship (table 8) between high dose.

Influence to LVEDP:

As can be seen from Table 9, the LVEDP value of vehicle control group and medication therapy groups is very close to the variation of administration front and back is not Greatly, all in 10mmHg hereinafter, illustrating X7 not influences the LVEDP of anesthetized rat without statistical difference.

The influence of right ± dp/dtmax:

X7 and positive control drug Amlodipine administration front and back compare or in different time points compared with vehicle control groups, data It is close, without statistical difference, illustrate that X7 and Amlodipine do not influence (table 10,11) to ± dp/dtmax.

Influence (unit: mmHg of the table 8.X7 to rat LVSPN=10)

* P < 0.001 * * P < 0.05, * * P < 0.01, * is compared with 0 min

Influence (unit: mmHg of the table 9.X7 to rat LVEDPN=10)

Table 10.X7 shrinks the influence (unit: mmHg of maximum rate (+dp/dtmax) to rat left ventricleN=10)

* P < 0.01 P < 0.05, * * is compared with 0min

Influence (unit: mmHg of the table 11.X7 to rat left ventricle diastole maximum rate (- dp/dtmax)N=10)

4.7. experiment conclusion

4.7.1.X7 single oral gavage administration has apparent influence to hemodynamics, and be mainly shown as reduces significantly Blood pressure and left ventricular systolic pressure.

4.7.2.X7 slightly slow down while reducing blood pressure with heart rate.

4.7.3.X7 rat left ventricle end-diastolic pressure and left ventricle maximum collapse and relaxation rate are not influenced.

Influence of the 5 X7 single-dose of embodiment to Beagle dog haemodynamics

5.1. experiment purpose

X7 single-dose is understood to anesthesia Beagle dog left ventricle hemodynamic index, peripheral blood vessel blood flow and blood vessel The influence of resistance.

5.2. experimental material

5.2.1. test medicine

5.2.2. experimental group and dosage are arranged

Experimental group: experiment sets vehicle control group, positive controls, is set in low by test product X7 according to the principle being grouped at random High three dosage groups, totally five groups, every group of size of animal is 6.

Dosage setting: vehicle control group, physiological saline 5ml/kg；Positive controls, Amlodipine Besylate Tablet 1.5mg/kg； X7 low dose group (1mg/kg), middle dose group (2mg/kg)；High dose group (4mg/kg).

5.2.3. reagent is used in other experiments

Yellow Jackets: German Merck biotech company production, import packing, specification: 25g/ bottles, lot number: WS20130112。

5.2.4. administration route, administration number of times and administration volume

Test medicine uses physiological saline solution before use, and the isometric solution of respective concentration is made in slightly ultrasound；Administration route: Duodenostomy administration；Volume: 1ml/kg is administered；Administration number of times is single.

5.2.5. experimental animal

Kind: healthy Beagle dog.

Source: mountain animal-breeding field is called in Shanghai.

Gender: male female dual-purpose.

Quantity: 31.

Weight: 8~10kg.

Licensing: SCXK (Shanghai) 2010-0027.

Certification of fitness number: 2005000400050.

5.2.6. laboratory apparatus

PowerLab: angstrom moral instrument international trade (Shanghai) Co., Ltd., model: ML830.

Transonic electromagnetic blood flow meter instrument: model: T420, the place of production: the U.S..

Animal respirator: Shanghai Alcott Biotechnology Co., Ltd, model: ALC-V10B.

Electric knife: Suzhou Kang Di Electronics Co., Ltd., model: S900E.

5.3. experimental method

Beagle dog after shank vein injecting anesthetic, is lain on the back with 3% yellow Jackets (30mg/kg) and is fixed on constant temperature On (39 DEG C) operating table, neck, bilateral femoral arterial ditch, left thoracic operation shaving, 75% ethanol disinfection field of operation；Separation right side Femoral artery is simultaneously intubated connection pressure transducer and is connected with PowerLab for detecting blood pressure, separation left femoral artery connection flow spy Head is for measuring arterial flow；Skin of neck exposure tracheae is cut, the word of falling T notch is done, connects ventilator, do mechanical ventilation and (exhale Suction machine setting: 22 times/min of frequency, respiratory quotient 1:2, tidal volume 20ml/min)；Separation right side external carotid artery connects flow probe For measuring common carotid artery flow；Longitudinal cut on the left of breastbone is taken, cutting 3-4 rib cage exposure heart is opened pericardium and done Pericardium hammock, separates coronary left branch initial part and aortic root, and it is defeated for measuring the heart to place probe for daubing coupling agent Output and coronary blood flow；It is inserted into left ventricle from apex with trochar, connection pressure transducer is for detecting left ventricle The hemodynamic indexs such as internal pressure；Needle electrode insertion distal limbs are subcutaneously used to record electrocardiogram；Duodenostomy is just In administration.Surgical procedure terminates to stablize 15min, records value (0min) based on all data, then duodenum fistula is given Medicine, 5 after record administration, 15,30,60,90,120min haemodynamics data and blood flow data.

5.4. observation index

Record index: heart rate (HR), mean arterial pressure (MBP), left ventricular systolic pressure (LVSP), Left ventricular end diastolic pressure (LVEDP), left ventricle maximum collapse and relaxation rate (± dp/dtmax), cardiac output (CO), coronary flow (CBF), Arteria carotis communis flow (CF), femoral artery flow (FAF).

The calculating of index:

Cardiac index (CI)=cardiac output (L/ml)/body surface area (m²)

Coronary flow (CBF；Ml/min/100g cardiac muscle)=(measured value (ml/min)/myocardial Mass Measured (g)) * 100

Coronary resistance (CAR；MmHg/ml/min)=mean arterial pressure/coronary flow

Body circulation total peripheral resistance (TPR；MmHg/ml/min)=mean arterial pressure (mmHg)/cardiac output (ml/min)

5.5. data and statistical analysis

5.6. experimental result

5.6.1.X7 influence of the single-dose to Beagle dog MBP and HR

Influence to MBP:

Normotensive Beagle dog is only fluctuated in a narrow range to MBP before and after solvent physiological saline, wave Dynamic range is between 86-90mmHg.

Treatment group's Amlodipine Besylate Tablet and X7 play the role of significantly reduce Beagle dog blood pressure, after administration 5min to 120min at the end of experiment, blood pressure persistently reduce.Positive control drug Amlodipine Besylate Tablet 1.5mg/kg dosage, makes dog MBP is gradually reduced to 71mmHg when 120min by the 92mmHg before being administered；And low middle high three dosage groups of X7 not only make averagely Angiosthenia equally reduces, and with the presence of dose-effect relationship between low middle high three dosage groups, i.e., with the increase of X7 dosage, The reduction of MBP is consequently increased (table 12).

Influence to HR:

As can be seen from Table 13, less, positive controls are to benzene sulfonic acid ammonia chlorine for the HR administration front and back variation of solvent group dog HR gradually accelerates after flat, by 154 beats/min, is gradually increased to 173 beats/min when 120min, HR increases by 19 beats/min.After X7 administration The performance of HR and Amlodipine Besylate Tablet are exactly the opposite, X7 administration after HR variation be slow down, and HR slow down it is related with dosage, The bigger HR of dosage, which slows down, to be more obvious.

Influence (unit: mmHg of the table 12.X7 to Beagle dog MBPN=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

Table 13.X7 to Beagle dog HR influence (unit: beat/min；N=6)

* P < 0.01 P < 0.05, * * is compared with 0min

5.6.2.X7 influence of the single-dose to Beagle dog left ventricular function

Influence to left ventricular systolic pressure (LVSP):

Influence to LVSP is consistent with the influence to blood pressure, and the LVSP for the treatment of group dog is persistently reduced upon administration, gives X7 dog LVSP reduce related with dosage, i.e., dosage increases, and LVSP reduces obvious, illustrates that the LVSP of X7 reduction dog also has dose-effect relationship to deposit At (table 14).

Influence to Left ventricular end diastolic pressure (LVEDP):

Vehicle control group, positive controls and low middle high three dosage groups of X7 do not influence LVEDP, tested each group LVEDP value is also at normal range (NR) (table 15).

Influence to left ventricle maximum collapse and relaxation rate (± dp/dtmax):

From table 16,17 as can be seen that comparing the+dp/dtmax after 0min and administration with paired-samples T-test, although absolute value ratio It is closer to, but still has statistical significant difference, but the not presence of dose-effect relationship；By ± the dp/dtmax for the treatment of group with Vehicle control group compares with time point, then absolute value is very close, without statistical significant difference.

Influence (unit: mmHg/s of the table 14.X7 to Beagle dog LVSP；N=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

Influence (unit: mmHg of the table 15.X7 to Beagle dog LVEDP；N=6)

Influence (unit: mmHg/s of the table 16.X7 to Beagle dog+dp/dtmax；N=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

Influence (unit: mmHg/s of the table 17.X7 to Beagle dog-dp/dtmax；N=6)

* P < 0.05 is compared with 0min

5.6.3.X7 influence of the single-dose to Beagle dog blood flow

Influence of the X7 to cardiac output (CO):

Experiment discovery, positive control drug Amlodipine Besylate Tablet play the role of increasing CO, average out to before this group of CO is administered 0.95L/min, most of time of measuring point CO value compares CO difference before and after 1.05L/min, medication after administration has statistics aobvious Work property (P < 0.05), this may cause CO to increase related with the increased heart rate after Amlodipine medication.

There is reducing heart rate after tested X7 administration, therefore CO value also declines upon administration, from low middle high three dosage The CO value analysis of group, the reduction of CO is related with dosage, and dosage is bigger, CO lower (table 18).

Influence of the X7 to coronary blood flow (CBF):

Compare before and after medication, although the CBF of vehicle control group has reduction, but reduce limitation, without statistical difference； CBF increases obvious within 30min after positive control administration, from the 84.2ml/min before administration, increases to 60min after medication 91.7ml/min then gradually decreases to approach the preceding level of administration.

CBF variation is consistent with CO after by test product X7 medication, and CBF is reduced and with dosage in relation to (table 19) after medication.

Influence of the X7 to common carotid artery flow (CF) and femoral artery blood flow (FAF):

Influence of low middle high three dosage groups of vehicle control group, positive controls, X7 to CF is similar to the influence to CO, sun Property comparison medicine plays the role of slightly increasing CF；CF is gradually decreased after X7 administration, and high dose group reduction is most obvious, before medication 84.5ml/min is reduced to 63.3 when 120min after medication, and the range of decrease is obvious (table 20).Compared with CF flow, shadow of the X7 to FAF Sound is smaller, and as can be seen from Table 21, each period, FAF data were opposite compared with 0min after vehicle control group and treatment group's administration Stablize.

Influence (unit: L/min of the table 18.X7 to Beagle dog CO；N=6)

* P < 0.05, * * P < 0.01, P < 0.001 is compared with 0min

Influence (unit: ml/min/100g cardiac muscle of the table 19.X7 to Beagle dog CBF；N=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

Influence (unit: ml/min of the table 20.X7 to Beagle dog CF；N=6)

* P < 0.05, * * P < 0.01, P < 0.001 is compared with 0min

Influence (unit: ml/min of the table 21.X7 to Beagle dog FAF；N=6)

5.6.4.X7 influence of the single-dose to the open chest anesthetized vascular resistence of Beagle dog and cardiac index

Influence to total peripheral resistance (TPR):

The TPR administration front and back of solvent group is highly stable, only in 92-94dynscm^-5Fluctuation in range.Treatment group's ammonia chlorine TPR is gradually decreased after the gentle X7 administration in ground.It is 98.6 before the TPR administration of Amlodipine, is gradually decreased after administration, to after administration 69.4 are reduced to when 120min, reduction difference highly significant (P < 0.0001) is compared in front and back；

Also play the role of reducing TPR after X7 administration, but it is shorter to hold time, only upon administration the reduction of 30minTPR with give Comparing before medicine has the significant meaning of statistics, and hereafter, TPR gradually rises horizontal close to before administration (table 22).

Influence (unit: TPR, dynscm of the table 22.X7 to Beagle dog TPR^-5 N=6)

* P < 0.001 P < 0.05, * * P < 0.01, * * * is compared with 0min

Influence to cardiac index (CI):

CI stablizes 1.78 or so before and after vehicle control group medication；CI increases obvious, administration after the administration of positive control drug Before be 1.87,5min is increased and is maintained 2.0 or more after administration, and is maintained to the 120min after medication；

The performance of CI is just with positive control drug Amlodipine Besylate Tablet on the contrary, CI gradually drops after X7 administration after X7 administration It is low, and the reduction of CI is related with dosage, and dosage is bigger, and CI decline is more obvious (table 23).

Influence (unit: L/min/m of the table 23.X7 to Beagle dog CI² N=6)

* P < 0.01 P < 0.05, * * is compared with 0min

Influence to coronary resistance (CAR):

As shown in table 24, after administration each time point when 0 compared with, the CAR of vehicle control group Beagle dog is slightly increased, but Raising does not reach the significant meaning of statistics.CAR is significantly reduced after treatment group's administration, especially positive controls and X7 high dose The CAR decline of group is most obvious, persistently reduces after administration and is maintained to the 120min at the end of experiment.

Influence (unit: mmHg/ml/min of the table 24.X7 to Beagle dog CAR；N=6)

* P < 0.05, * * P < 0.01, P < 0.001 is compared with 0min

5.7. experiment conclusion

5.7.1.X7 being significantly reduced effect to the mean arterial pressure of anesthetized open-chest dog.

5.7.2.X7 with decreased heart rate while decompression.

5.7.3.X7 the left ventricular systolic pressure to anesthetized open-chest dog can be reduced, but does not influence Left ventricular end diastolic pressure.

5.7.4.X7 play the role of reducing cardiac output and coronary flow, there is reduction to make common carotid artery flow With, but femoral artery blood flow is not influenced.

5.7.5.X7 play the role of reducing coronary resistance and total peripheral resistance, but reduce that total peripheral resistance maintains when Between it is shorter.

5.7.6.X7 there is reduction effect to cardiac index.

6 X7 long term administration of embodiment is to SHR blood pressure, fluctuation of blood pressure, the influence of ABR function and organ protection

6.1. experiment purpose

Understand test drug X7 long term administration to spontaneous hypertensive rat (SHR) blood pressure (BP), fluctuation of blood pressure (BPV), Whether the influence of arterial baroreflex sensibility (BRS) and the organ damage caused by hypertension have protective effect.

6.2. experimental material

6.2.1. test medicine

Test drug: code name X7, white in appearance is powdered, 4 DEG C of closed preservations.Product batch number: 2013103001.X7 experiment In pharmaceutical feed feeding is made.

Positive control drug: Amlodipine Besylate Tablet is produced by Zhejiang Second Pharma Co., Ltd., white in appearance powder Shape, 4 DEG C are sealed, batch number: 07151007.

6.2.2. experiment reagent

0.9% sodium chloride injection: being produced by Shangdong Hualu Pharmaceutical Co., Ltd., specification: 500ml, authentication code: traditional Chinese medicines Quasi- word H37022749, product batch number: E14112114.

Heparin sodium: it is produced by Shanghai Yuan Chuan Biotechnology Co., Ltd, specification: 5g/ bottles, product batch number: Lot73685321。

Ketamine Hydrochloride Inj.: being produced by Fujian Gutian Pharmaceutical Co., Ltd., specification: 2ml:10mg/ branch, and traditional Chinese medicines are quasi- Word: H3502148, lot number: 130303.

Diazepam injection: it is produced by Anyang City Yikang pharmaceutical factory, specification: 2ml:10mg/ branch, national drug standard: H41021491, lot number: 1403031.

Phenylephrine hydrochloride injection: being produced by Shanghai Hefeng Pharmaceutical Co., Ltd., specification: 1ml:10mg, Hu Wei The quasi- word (1995) of medicine the 010045th, batch number: 011001.40ug/ml working solution is configured to when use, i.e. 1ml deoxygenates kidney 250ml normal saline dilution is added in upper parathyrine stoste.

6.2.3. dosage setting and grouping

Experiment is divided into 3 groups, it may be assumed that

Solvent control group: physiological saline；

Positive controls: Amlodipine, 5mg/kg；

Test drug: X7,10mg/kg；

Every group size of animal 20, half male and half female.

6.2.4. administration route

Administration mode: preparation drug containing feed is freely absorbed for rat；

It is repeatedly measured, SHR is averaged every daily inleting appetite for 50g/kg/d, by positive group Amlodipine 5mg+ rat feed powder 50g and test drug X710mg+ rat feed powder 50g, is made two kinds of pharmaceutical feeds, arbitrarily absorbs for SHR after mixing well.

6.2.5. experimental animal

Strain: 4 monthly age spontaneous hypertensive rats (SHR)；

Rank: regular grade；

Gender: half male and half female；

Quantity: 60；

Weight: male SHR weight, 350 ± 20g；Female SHR weight, 200 ± 20g；

6.2.6. laboratory apparatus

Rat tail artery non -invasivetesting device: it is produced by Shanghai Alcott Science and Technology Ltd..

The awake blood pressure continuous measurement device that move freely of rat: Shanghai Alcott Science and Technology Ltd. production.

Pressure transducer: it is produced by Shanghai Alcott Science and Technology Ltd..

Pressure signal amplifier: it is produced by Shanghai Alcott Science and Technology Ltd., model: MPA-HBBS.

Constant speed injects pump: by Zhejiang Medical university, instrument plant is produced, model: WZ-50G injector.

Polyethylene pipe: French Biotrol company, PE10 (internal diameter 0.3mm, outer diameter 0.50mm)；PE50 (internal diameter 0.58mm, Outer diameter 0.96mm).

Bioelectric amplifier: it is produced by Shanghai Alcott Biotechnology Co., Ltd, model: MPA-SBB.

Electronic balance: by Mettler-Toledo Instrument (Shanghai) Co., Ltd., model: AL204.

6.3. experimental method

6.3.1. changes of weight

SHR reach four monthly ages be grouped at random after enter experiment marshalling, number and is weighed with picric acid, later every 30 days weighing Weight.

6.3.2. the SHR blood pressure detecting during long term administration

The preceding method measurement SHR basic blood pressure with arteria caudalis non -invasivetesting of administration, 1 time every other day, continuous 3 times,

It is worth based on the average value of 3 blood pressures.Then starting feeding pharmaceutical feed, (control group is fed with common mouse feed Support), the tail arterial blood pressure of detection in every 3 days after administration, continuous 3 times, monthly detection is primary later, continuous detection 4 months.

6.3.3. after long-term treatment SBPV, HPV and BRS function detection

After administration 4 months, rat ketalar 50mg/kg and diazepam 5mg/kg intraperitoneal injection of anesthesia, dorsal position Fixed, skin is cut in cropping, disinfection at the groin of left side, and exposure femoral artery sheath carefully separates femoral artery and femoral vein.Through stock Artery insertion is full of the arterial duct of heparinized saline, until low level abdominal aorta, then ductus venosus is inserted into through femoral vein. Arterio-venous catheter is subcutaneously gone upward to neck portion skin with draw pin to be pierced by.Skin suture notch, then moved what neck portion was drawn Ductus venosus, which is fixed against to grab after rat regains consciousness with self-control saddle, stings conduit, sends rat back to animal house raising after intubation 24h。

After rat restores for 24 hours, it is put into homemade cylinder, arterial duct is connect with pressure measuring system, rat can be in circle It is move freely in cylinder and drinking-water is ingested.Blood pressure signal of often fighting through pressure transducer is converted to bioelectrical signals, real-time by computer Record often fight blood pressure and cardiac cycle.Adaptation of the rat through 1~2h after online records the blood of 1h after blood pressure heart rate is stablized Pressure and heart rate then while recording blood pressure, inject PHENYLEPHRINE HYDROCHLORIDE through duct of Arantius for calculating SBPV and HPV 5ug/kg makes blood pressure increase 20~50mmHg, restores after blood pressure to basic blood pressure, inject again, continuous 3 times, count after off line Calculate BRS.

6.3.4. hypertension correlation important organ gross specimen is observed

The major organs relevant to hypertension of this experiment detection have heart, brain, kidney and aorta.After BRS detection, mistake Rat is put to death in amount anesthesia, opens thoracic cavity and abdominal cavity, and taking-up heart, aorta pectoralis, bilateral renal and the full brain for intercepting 3cm long claim Weight.Calculate cardiac index (HW/BW), right renal index (RKW/BW), left renal index (LKW/BW), cerebral index (BW/BW) and chest Aortic index (AW/Length).

6.3.5. hypertension correlation important organ HE dyes optics microscopically observation

Every group takes brain, heart, right side kidney and the aorta pectoralis of 1,3, No. 5 animal that pathological section is made, and row HE is dyed, into Row pathological observation and scoring (see pathological photograph and pathological examination results part).

6.4. observation index

Weight and blood pressure during administration.SBPV, HPV between each group.BRS between each group.

Between each group rat whole-heartedly weigh/weight (HW/BW), right kidney weight/weight (RKW/BW), left kidney weight/weight (LKW/BW), Brain weight/weight (BW/BW) and aorta pectoralis weight/length (AW/length).

(GSS) is scored in rat kidney glomerulosclerosis.

It is attached: GSS method: (carrying out GSS using 50 glomerulus as radix at random in kidney cortex)

0 grade: no glomerular mesangium expansion；

1 grade: slight glomerular mesangium expansion (being less than 30%)；

2 grades: moderate glomerular mesangium expands (between 30-60%)；

3 grades: the expansion of severe glomerular mesangium (is more than 60%)；

4 grades: glomerulosclerosis.

GSS score value calculation method=(1 point × 1 grade+3 points of+2 points of Glomerular lesions number × 2 grade Glomerular lesions number × 3 grades of+4 points of Glomerular lesions number × 4 grade glomerulus numbers)/actual measurement Glomerular lesions number × 100%.

Histopathologic examination: brain, heart, right side kidney and Thoracic Aortic Diseases situation.

6.5. statistical disposition

Experimental result is with mean ± standard deviationIt indicates, with Excel 2010 and 18.0 software of SPSS to data between group ANOVA variance analysis is carried out, carries out two comparison among groups with pairing and non-paired t test, is defined as having with P < 0.05 significant Statistical difference.

6.6. experimental result

6.6.1.X7 influence of the long term administration to SHR weight

SHR has been adult there are four the monthly age when due to starting experiment.Therefore, by nursing in four months, weight is more Stablize, does not occur occurring the problems such as weight larger fluctuation is to influence blood pressure (table 25) because of fat or food refusal drug containing feed.

Influence (unit: g of the table 25.X7 long term administration to each period changes of weight of SHR；N=20)

* p < 0.05 is compared with 0 day

6.6.2.X7 during long term administration each group SHR blood pressure and heart rate variation

(1) to the influence of blood pressure

Basic value average value before the SBP administration of solvent control group SHR is after 172 ± 4mmHg, administration with feeding time Extension, blood pressure has slow raising trend, terminates to have raised altogether 111 days to experiment, blood pressure is stable in 170-180mmHg always； Positive controls and test drug X7 treatment group to Amlodipine start blood pressure and solvent control group before treating and approach, and exist 173mmHg starts to treat latter week, and blood pressure is remarkably decreased, and Amlodipine group mean reduction reaches 30mmHg, and X7 treatment group is average The range of decrease is 26mmHg.At the end of experiment, the blood pressure of Liang Ge treatment group reduces amplitude and reaches 20mmHg or more and stablize lower State has significant statistical difference (table 26) compared with solvent control group is with time point.

Influence (unit: mmHg of the table 26.X7 long term administration to SHR SBP；N=20)

P < 0.001 * * is compared with 0 day

(2) to the influence of heart rate

The pretherapy and post-treatment heart rate stabilization of the heart rate of solvent control group SHR is at 400 beats/min or so；Amlodipine positive controls It is slightly increased after the heart rate administration of rat, but increases limited (first 404 beats/min of administration；434 beats/min of peak after administration)；It is tested The heart rate of medicine X7 group rat is more stable before and after treatment, average at 410 beats/min or so；The heart of the positive group and reagent group SHR Rate is compared with solvent control group is with time point without statistical significant difference (table 27,28).

Influence (unit: mmHg of the table 27.X7 long term administration to SHR SBP；N=20)

P < 0.001 * * is compared with 0 day

Table 28.X7 long term administration to SHR HR influence (unit: beat/min；N=20)

6.6.3.X7 influence of the long term administration to SBPV, HPV and BRS function of SHR

(1) to the influence of SBPV and HPV

By long-term treatment in 4 months, it was found that, the HPV of X7 slightly increased with solvent control group, positive controls ammonia chlorine The HPV raising of Horizon is more apparent, but without statistical significant difference；Influence to SBPV is that the SBPV of Liang Ge treatment group is absolute Though value has reduction, limited extent is reduced, statistical significant difference (table 29) is not reached yet.

(2) to the influence of BRS function

Our unit it is previous the study found that hypertension animal has different degrees of BRS to reduce, BRS reduce it is more serious, it is high The target organ damage degree of blood pressure animal is also more serious, if drug can effectively improve BRS, organ damage degree can subtract Gently.This experiment discovery, the BRS testing result of solvent control group SHR are 0.27 ± 0.05, give Amlodipine (F=55.0, P= 0.000) and the treatment group of X7 (F=61.0, P=0.000) is respectively 0.55 ± 0.15 and 0.53 ± 0.14, with solvent control group Compare, BRS function is significantly improved (table 29).

29. long term administration of table to SPPV, HPV and BRS influence (N=20)

* * P < 0.001vs solvent control group

6.6.4.X7 influence of the long term administration to SHR heart, kidney, aorta pectoralis, cerebral index

It was treated by 111 days, Amlodipine and X7 treatment group cardiac index, renal index, cerebral index and aorta pectoralis refer to Number has no significant change (table 30) compared with solvent control group.

Table 30.X7 long term administration to SHR heart, kidney, aorta pectoralis, cerebral index influence (N=20)

6.6.5.X7 the influence that long term administration scores to SHR kidney GSS

It is scored, is tested as standard of the hypertension to kidney damage with the semidefinite quantizating index of glomerulosclerosis degree It was found that the renal lesions of solvent control group rat are more serious, Glomerular lesions based on 2-3 grades, the score value of GSS is 1.75 ± 0.07；Positive drug Amlodipine and the Renal Glomeruli In Rats lesion degree of test drug X7 treatment group are obviously light compared with solvent control group, with Based on 1-2 grades, GSS score value point 1.57 ± 0.08 and 1.51 ± 0.06 is poor with significance,statistical compared with solvent control group It is different, illustrate that X7 and Amlodipine long-term treatment have significant protective effect (table 31) the kidney damage as caused by hypertension.

Influence that table 31.X7 long term administration scores to SHR kidney GSS (N=20)

* P < 0.05, * * P < 0.01VS solvent control group

6.6.6. target organs of patients with essential hypertension damages pathological change

Cardiac muscular tissue (Fig. 4~Fig. 6)

Model group: rat heart muscle fiber alignment is neat, and myocardium gap is broadening, and cardiac muscle cell is slightly loose, cardiac interstitium water It is swollen, interstitial fibrosis, collagen fiber hyperplasia, cardiac muscle cell's sheet necrosis, a small amount of monocyte, lymphocytic infiltration.

Positive controls: cardiac muscle fibre marshalling, cardiac muscle cell have no loose, interstitial fibrosis, collagen fiber hyperplasia, The denaturation of cardiac muscle cell's sheet or necrosis, have no monocyte, lymphocytic infiltration.

X7 treatment group: administration group cardiac muscle fibre marshalling, cardiac muscle cell's form is normal, interstitial fibrosis, collagenous fibres Hyperplasia is reduced, and the denaturation of cardiac muscle cell's sheet or necrosis have no monocyte, lymphocytic infiltration.

Nephridial tissue (Fig. 7~Fig. 9)

Model group: rat kidney can see mesangial cell proliferation, matrix diffusivity increases, glomerulus atrophy, Necrosis, arteria renalis fibrosis and hyalinization, big amount lymphocyte, monocyte and fibroblast infiltration.

Positive controls: dia-betic rats are complete, a small amount of arteria renalis fibrosis and hyalinization, in nephridial tissue and glomerulus Still there are big amount lymphocyte, monocyte and fibroblast infiltration.

X7 treatment group: administration group dia-betic rats are complete, and matrix gap is normal, a small amount of arteria renalis fibrosis, nephridial tissue and Glomerulus endolymph cell, monocyte and fibroblast infiltration mitigate compared with model group.

Brain tissue (Figure 10~Figure 12)

Model group: neurotagma is normal in rat cerebral tissue, and peripheral clearance is without obvious broadening, no inflammation cell leaching Profit, accidental blutpunkte and nerve cell oedema.

Positive controls: neurotagma is normal, and peripheral clearance has no inflammatory cell infiltration without broadening.

X7 treatment group: neurotagma is normal, and peripheral clearance is without obvious broadening, no inflammation cellular infiltration, accidental bleeding Point and nerve cell oedema.

Aorta (Figure 13~Figure 15)

Model group: the vascular wall of rat chest aorta is thicker, wall thickness/internal diameter ratio is larger.

Positive controls and X7 treatment group: the vascular wall of aorta pectoralis is relatively thin, wall thickness/internal diameter ratio is significantly less than model Group.

6.7. experiment conclusion

6.7.1.X7 Long-term Oral approach administration can significantly reduce SHR blood pressure, and pretherapy and post-treatment blood pressure is reduced more than 20mmHg Above and it can maintain to stablize.

6.7.2.X7 long term administration can improve the sensibility of SHR arterial pressure sensitivity reflex function, thus to hypertension Caused by organ damage have protective effect.

6.7.3.X7 long term administration glomerular epithelial cell caused by hypertension has protective effect.

Influence of the 7 X7 long term administration of embodiment to SHR-SP life span

7.1. experiment purpose

Understand whether X7 long term administration can delay the generation of SHR-SP cerebral apoplexy and extend life span.

7.2. experimental material

7.2.1. test medicine

Test drug: code name X7, white in appearance is powdered, 4 DEG C of closed preservations.Product batch number: 2013103001.X7 indissoluble Pharmaceutical feed feeding is made in experiment in Yu Shui.

7.2.2 experiment reagent

Sodium chloride: being produced by Sinopharm Chemical Reagent Co., Ltd., specification: 500g, lot number: F20101203.

7.2.3. dosage setting and grouping

Experiment is divided into 3 groups, it may be assumed that

Solvent control group: physiological saline；

Positive controls: Amlodipine, 5mg/kg；

Test drug: X7,10mg/kg；

Every group size of animal 10.

7.2.4. administration route

It is repeatedly measured, SHR-SP is averaged every daily inleting appetite as 50g/kg/d, raises by positive group Amlodipine 5mg+ rat Feed powder 50g and test drug X710mg+ rat feed powder 50g, is made two kinds of pharmaceutical feeds after mixing well, random for SHR-SP Intake.

7.2.5 experimental animal

Strain: the spontaneous hypertensive rat (SHR-SP) for thering is cerebral apoplexy to be inclined to；

Rank: regular grade；

Gender: male；

Weight: 350g ± 20g；

Quantity: 30；

The strain is this teaching and research room from overseas introduction, in Experimental Animal Center SPF grades of this school animal house captive breeding, June Age transports teaching and research room's experimental animal room back, and normal diet is fed, and drinks 1% salt water to accelerate the generation of cerebral apoplexy, room temperature 20-22 DEG C, 12 hours light and shade automatic conversions of light.

7.3. experimental method

The SHR-SP that every batch of reached for 6 monthly ages enters experiment marshalling after being grouped at random, is numbered and is weighed with picric acid, often The weight of weighing in 30 days.

The basic blood pressure of the preceding method measurement SHR-SP with arteria caudalis non -invasivetesting of administration, 1 time every other day, continuous 3 times, 3 times It is worth based on average blood pressure.Then start feeding pharmaceutical feed (control group is fed with common mouse feed) and change drinking water For 1% salt water, to the blood pressure of survey in initial stage every 3 days of pharmaceutical feed, continuous 3 times, each later moon detects a blood pressure, Continuous 4 months.

Observe and record animal dead situation daily, there were significant differences to stop for the survival rate of observing time to three groups of rats It only tests, counts each group survival of rats rate, draw time-survivor curve.

7.4. observation index

Changes of weight between treatment phase.

Blood pressure (SBP).

Each group SHR survival rate.

7.5 statistical disposition

Experimental result is with mean ± standard deviationIndicate, with Excel2010 and SPSS18.0 software to data between group into Row ANOVA variance analysis carries out two comparison among groups with pairing and non-paired t test, surviving rats percentage Chi-square Test, with P < 0.05 is defined as having significant statistical difference.

7.6. experimental result

7.6.1.X7 influence of the long-term treatment to rat body weight

SHR-SP six months have been adult rats, and therefore, during treatment in four months, weight is relatively stable, without because Occurs the phenomenon that influencing weight (table 32) in mouse grain containing drug influence food ration.

Influence (unit: g of the table 32.X7 long term administration to each period changes of weight of SHR-SP；N=10)

P < 0.01 * is compared with 0 day

7.6.2.X7 to the influence of blood pressure during SHR-SP treatment

Without significant change before and after the blood pressure medication of solvent control group SHR-SP, blood pressure range is in 180mmHg or so, ammonia chlorine It is close with solvent control group before the blood pressure administration of gentle X7 treatment group SHR-SP, and decline highly significant after being administered, blood pressure is average Stablize in 155mmHg or so, mean reduction reaches 20mmHg or more (table 33).

7.6.3.X7 influence of the long-term treatment to SHR-SP life span

Male SHR-SP average life span is last all to die of cerebral apoplexy at 12 months or so.This experiment is at the SHR-SP6 monthly age Into experiment.The sodium chloride that joined 1% in drinking-water is accelerated to the generation of cerebral apoplexy during experiment.Experiment starts rear third Month, solvent control group has 6 death, and Liang Ge treatment group only has 1 death.When by 6 months, the SHR-SP of solvent control group All dead, the SHR-SP The dead quantity of Liang Ge treatment group only has 5.It is shown through Chi-square Test, treatment group SHR-SP Survival rate, with the statistical difference of highly significant, illustrates medicine positive drug Amlodipine and test drug X7 compared with solvent control group To delaying the generation of SHR-SP cerebral apoplexy to have the function of highly significant (table 34, Figure 16).

Influence (unit: mmHg of the table 33.X7 to SHR-SP life span SBP；N=10)

* P < 0.001 * * P < 0.05, * is compared with 0 day

Influence (unit: % of the table 34.X7 to SHR-SP survival rate；N=10)

* P < 0.05, * * P < 0.01vs solvent control group

7.7 experiment conclusion

X7 can delay the time of origin of SHR-SP cerebral apoplexy significantly, extend the life span of SHR-SP.

8 target of embodiment research: 1 μM of results of preliminary screening

Primary dcreening operation experimental method:

In primary dcreening operation experiment, 87 molecular targets are used, to enzymatic activity or radioligand under 1 μM of concentration of detection compound In conjunction with the influence of test, experiment is at least repeated 2 times.As a result suppression percentage or inhibition of enzyme activity are specifically bound with radioligand Percentage indicates that inhibiting effect >=50% has the standard of significant meaning as primary dcreening operation, into secondary screening.

As a result:

As shown in table 35, in 87 molecular targets of primary dcreening operation, 17 molecular targets enter secondary screening.

35 primary dcreening operation experimental result of table

Experimental results

Note: suppression percentage >=50% indicates significant meaning, into secondary screening (totally 17 targets).

* batch: the positive compound of test simultaneously is represented in same analysis.

9 target of embodiment research: secondary screening experimental result

Secondary screening experimental method:

In secondary screening experiment, using 5 concentration (0.1nM, 1nM, 10nM, 0.1 μM, 1 μM) between compound 0.1nM~1 μM The dose-effect relationship acted on target molecule is measured, experiment is at least repeated 2 times.As a result it is indicated with suppression percentage, and calculates IC₅₀ (concentration when 50% inhibition), Ki (inhibition constant), n_H(Hill coefficient).

As a result:

As shown in table 36, in the target for entering secondary screening at 17, the IC of compound₅₀In several nM and molecular target below There are 7, the molecular target of 10 several nM or more indicates 10.Figure 17~Figure 23 also shows the IC of compound₅₀In several nM and The amount effect curve of 7 molecular targets below.

Conclusion: compounds on adrenal element α₁Receptor, serotonin 5-HT_2AReceptor, histamine H₁The molecular targets such as receptor Indicate apparent blocking effect.

36. secondary screening result conclusive table of table

Summary of experimental results

The present invention had not only expanded the purposes of X7, but also provided new therapeutic scheme for hypertension and its complication, and poison is secondary to be made With small, safety is good, has apparent curative effect to a variety of target organ damages caused by hypertension.It is several that existence is increased for such patient Rate alleviates patient suffering, has a good application prospect.

Described is only the preferred embodiment of the present invention, it is noted that for those skilled in the art, In Without departing from the principles of the invention, several improvement and supplement can also be made, these are improved and supplement also should be regarded as this hair Bright protection scope.

Claims

1. formula (I) compound or its pharmaceutically acceptable salt are caused by preparation prevents, treats and delay hypertension, hypertension Application in target organ damage and the drug of hypertension related disease,

2. applying according to claim 1, which is characterized in that the target organ damage is the heart caused by hypertension, brain, kidney Or the damage of blood vessel；The hypertension related disease, including atherosclerosis, hyperlipidemia, obesity, coronary heart disease, aorta clamp Layer and hyperglycemia, impaired glucose tolerance, metabolic syndrome, diabetes.

3. application as claimed in claim 2, which is characterized in that the target organ damage is left ventricular hypertrophy, cerebral apoplexy, kidney Cortical atrophy or active phleboedesis, angina pectoris, myocardial infarction, heart failure, renal failure, retinal arteriosclerosis, height Blood pressure eyeground pathological changes.

4. a kind of pharmaceutical composition, which is characterized in that including pharmaceutically acceptable carrier and formula described in claim 1 (I) Compound or its pharmaceutically acceptable salt.

5. pharmaceutical composition according to claim 4, which is characterized in that the formula (I) in the drug containing 20mg-1000mg Compound or its pharmaceutically acceptable salt.

6. pharmaceutical composition according to claim 5, which is characterized in that the pharmaceutical dosage form of described pharmaceutical composition is oral system Agent or injection, the oral preparation include tablet, capsule, granule pill or suspension.

7. target organ caused by claim 4 described pharmaceutical composition prevents, treats and delay hypertension, hypertension in preparation damages Injure the application in the drug of hypertension related disease.

8. applying according to claim 7, which is characterized in that the target organ damage is the heart caused by hypertension, brain, kidney Or the damage of blood vessel；The hypertension related disease, including atherosclerosis, hyperlipidemia, obesity, coronary heart disease, aorta clamp Layer and hyperglycemia, impaired glucose tolerance, metabolic syndrome, diabetes.

9. application as claimed in claim 8, which is characterized in that the target organ damage is left ventricular hypertrophy, cerebral apoplexy, kidney Cortical atrophy or active phleboedesis, angina pectoris, myocardial infarction, heart failure, renal failure, retinal arteriosclerosis, height Blood pressure eyeground pathological changes.