CN1048158C - Apiolin-A use in preparation of medicine for prevention and treatment of diseases caused by cerebral ischemia - Google Patents
Apiolin-A use in preparation of medicine for prevention and treatment of diseases caused by cerebral ischemia Download PDFInfo
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Abstract
The present invention provides a new purpose of butylphthalide. The compound (butylphthalide) can effectively prevent and treat diseases caused by cerebral ischemia, and particularly has obvious protecting action to ischemic cerebral neuron damage and necrosis.
Description
The present invention relates to the purposes of Butylphthalide, particularly in the disease that preparation prevents and the treatment cerebral ischemia causes, especially to the purposes in the medicine of ischemic brain neuron injury and necrosis.
Be used for the treatment of cerebral ischemia at present and cause that the medicine of disease is a lot, but real responder is few in number.Existing medicine nimodipine has preventive effect to cerebral ischemia, but therapeutical effect is not certainly.EAA antagonists MK801 curative effect certainly, but toxic and side effects is big, can not be used for clinical, duxil, refined companion and piracetam are used for the treatment of apoplexy sequela more, but curative effect is also not ideal enough.
Butylphthalide (3-n-butyl phthalide) (1) is a synthetic natural drug, and Bjeldanes equals report Butylphthalide and Senkyunolide As in 1977 etc. maincenter sedation (Bjeldanes LF:J.Org.Chem.42:2333,1977).Yang Junshan isolated L-Butylphthalide (L-normal-butyl Phthalide) and Senkyunolide A (L-normal-butyl-4,5-dihydro Phthalide) (Yang Junshan, Chen Yuwu from celery seed in 1978; The pharmacy circular, 1984,31:671).The synthetic DL-Butylphthalide of Yang Jing Chinese worker in 1980.
Butylphthalide (3-n-butylphthalide)
Naturally occurring Butylphthalide is a levo form, and synthetic be racemic modification.But no matter be Butylphthalide natural or synthetic, reported all in the document that it has the anti-frightened effect of wide spectrum, to galvanic shock, audiogenic convulsion, the convulsions that rare tetrazole of five first and coriamyrtin cause has antagonism, and can strengthen ability of learning and memory in mice, rat hippocampus CA1 district pathological change due to the convulsions that coriamyrtin is caused has the improvement effect (in timely rain core etc., Acta Pharmaceutica Sinica 1988,19 (8), 566; In timely rain core etc., Acta Pharmaceutica Sinica 1988,23 (9), 656; In timely rain core etc., Acta Pharmacologica Sinica 1988,9:385; In timely rain core etc., Acta Pharmaceutica Sinica 1984,19 (7), 486; In timely rain core etc., pharmacy circular 1985,20,187).But do not see that Butylphthalide has the treatment cerebral ischemia to cause disease, the report of doing as cerebrovascular disease.
Purpose of the present invention just is to provide the new purposes of Butylphthalide, that is, and and the application of Butylphthalide in disease, especially ischemic brain neuron injury that preparation prevention and treatment cerebral ischemia cause and downright bad medicine.The present invention will be described in further detail Butylphthalide as the disease of preventing and the treatment cerebral ischemia causes in conjunction with following result of study, particularly to ischemic brain neuron injury and the downright bad medicine that plays significant protective effect.
For finishing goal of the invention of the present invention, in fact the present invention relates to the application in Butylphthalide reversed or reduced cerebral infarct size that mammal or human local cerebral ischemia cause and neural disappearance as preparation the medicine.
The invention still further relates to Butylphthalide and reduce application in the medicine of the cerebral edema that mammal or human local cerebral ischemia cause as preparation.
The invention still further relates to Butylphthalide as the application in the medicine of preparation prevention mammal or human apoplexy and mortality rate that reduces post-stroke and neural disappearance.
The present invention relates to Butylphthalide as the application that improves in the amnemonic medicine that mammal or human local cerebral ischemia cause.
The present invention also further relates to Butylphthalide as the application in treatment mammal or the human medicine that causes the caused nervous symptoms of cerebral ischemia because of cerebral trauma.
The invention further relates to Butylphthalide as the application in the medicine of the energy metabolism exhaustion of improving mammal or human global brain ischemia.
The present invention also further relates to Butylphthalide as suppressing mammal or human global brain ischemia and heavily irritating application in the medicine that the outer liquid adenosine of phase striatal cell, inosine, hypoxanthine, xanthine content raise.
The invention still further relates to Butylphthalide as suppressing the outer liquid dopamine (DA) and 3 of mammal or human cerebral ischemia phase striatal cell, the application in the medicine of rising of 4-dihydroxyphenyl acetic acid (DOPAC) content and reduction glycine content.
The present invention has opened up a new application to the purposes of already present Butylphthalide in the prior art, and result of study shows that Butylphthalide has following effect:
(1) can reverse or reduce local cerebral ischemia rat cerebral infarction area and neural disappearance, its therapeutic effect is better than nimodipine, and with the instrument medicine, excitatory amino acid receptor antagonists MK801 is suitable, and toxic and side effects is little;
(2) cerebral edema that has obvious reduction local cerebral ischemia to cause;
(3) can reduce the mortality rate and the neural disappearance of apoplexy type spontaneous type Hypertensive Rats post-stroke and have preventive effect;
(4) can improve the dysmnesia that local cerebral ischemia causes;
(5) the cerebral trauma rat is caused cerebral ischemia and the nervous symptoms that causes has the improvement effect;
(6) energy metabolism that can improve the complete cerebral ischemia of mice is exhausted;
(7) can suppress the ischemia of global brain ischemia rat (4 artery ligation) and heavily irritate the outer liquid adenosine of phase striatal cell, inosine, hypoxanthine, xanthine content raises;
(8) content of the outer liquid glycine of the low 4 artery ligation rat cerebral ischemia phase striatal cells of energy battle array and the rising of inhibition DA and DOP AC.
(9) the brain during acute patients with stroke there is the obvious treatment effect.
Acute toxicity test, result of the tests such as teratogenesis, mutagenesis show that the Butylphthalide toxic and side effects is less.
Studies show that Butylphthalide is as the medicine of prevention and the disease that causes of treatment cerebral ischemia, can capsule, dosage form such as injection is combined into compositions with pharmaceutical carrier and is used for the mammal and the mankind, and is wherein, comparatively preferred with capsule.Mice dosage is subcutaneous injection 100～200mg/kg/ days or oral 100～400mg/kg/ days, and rat dosage is oral: 30～240mg/kg/ days or lumbar injection 10～20mg/kg/ days, human dosage was 6～12mg/kg/ days.
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing.In the accompanying drawing:
(A B) is Butylphthalide, MK801 and the nimodipine treatment administration influence to intraluminal middle cerebral artery occlusion in rats ligation (MCAO) back cerebral infarct size and neural disappearance to Fig. 1.
(A B) is the influence of Butylphthalide to intraluminal middle cerebral artery occlusion in rats ligation (MCAO) back cerebral infarct size and neural disappearance to Fig. 2.
(A B) is the influence of Butylphthalide prevention administration to intraluminal middle cerebral artery occlusion in rats ligation (MCAO) back cerebral infarct size and neural disappearance to Fig. 3.
(A B) is the therapeutical effect of Butylphthalide to apoplexy type spontaneous hypertensive rat apoplexy to Fig. 4.
Fig. 5 is the preventive effect of oral Butylphthalide to apoplexy type spontaneous hypertensive rat apoplexy.
Fig. 6 causes that to ligation middle cerebral artery (MCAO) the dysmnesia rat initiatively avoids the influence of number of times for oral Butylphthalide.
Fig. 7 (A, B) causes that to ligation middle cerebral artery (MCAO) the dysmnesia rat avoids preclinical influence for Butylphthalide.
Fig. 8 (A-D) is the influence of Butylphthalide to global brain ischemia rat striatum extracellular fluid purine metabolism thing.
Fig. 9 is the influence of Butylphthalide to global brain ischemia rat striatum extracellular fluid amino acid content.
Butylphthalide is to the influence of treatment administration to intraluminal middle cerebral artery occlusion in rats ligation (MCAO) back cerebral infarct size and neural disappearance
Instrument:
1, SXP-operating microscope (the Shanghai medical optical is according to device factory)
2, high frequency electric knife (62TZ-V type, Beijing medical electronic apparatus factory)
Animal: the male wistar rat is provided by Chinese Academy of Medical Sciences's animal center.Body weight 250～350g
Medicine: the dl-Butylphthalide is provided by the institute of Materia Medica,Chinese Academy of Medical Sciences Beijing XieHe medicine Factory, content 98%, and the pale yellow oily liquid body, oral administration is mixed with desired concn by refined edible oil, and lumbar injection is made into Emulsion with tween 80.
MK 801 purchases order Sigma company
Nimodipine is a street drug
Method:
For simulating clinical cerebral apoplexy patient, adopt intraluminal middle cerebral artery occlusion in rats ligation (MCAO) as the local cerebral ischemia model.
The male wistar rat, 250-350g, lumbar injection chloral hydrate anesthesia (350mg/kg), totally 7 groups, every group 8, press Tamura method (Tamura A, et al.J cereb Blood and Metab 1:53,1981) row right side MCAO, 15min behind MCAO, difference lumbar injection Butylphthalide 10,20mg/kg, oral 40,80mg/kg, subcutaneous injection nimodipine 1mg/kg and lumbar injection MK801 0.5mg/kg, all compare with ischemic control group (give solvent), with Bederson behavior scoring mark (BedersonJ.B Rat middle cerbralArtery occlusion:Evaluation of the model and development of a neurologicexamination, stroke 17 (3): 472-6), concrete grammar is (1) healthy side shoulder inward turning, receives the 0-4 branch in the forelimb; (2) push away healthy side shoulder resistance decline 0-3 branch; (3) muscular strength (snatch) 0-3 branch that descends, totally 10 minutes, 24 hours marks of record postoperative, broken end is got forebrain then, crownly is cut into 5, with TTC (Lundy E.F; J.Pharmacological methods 16:201 1986) dyeing is measured infarct size, and is calculated the percent that it accounts for hemisphere, and measurement result is shown in Fig. 1.The result shows, behind the MCAO 15 minutes with the Butylphthalide treatment, no matter abdominal cavity or oral administration all can obviously reduce cerebral infarct size and neural disappearance.Its effect is better than nimodipine, and suitable with known anti-cerebral ischemia instrument medicine MK 801.Embodiment 2
Butylphthalide is to intraluminal middle cerebral artery occlusion in rats ligation (MCAO) cerebral infarct size and neural influence that lacks after 2 hours.
Instrument, animal and medicine are with embodiment 1.
Method:
Concrete grammar is with embodiment 1,2 hours oral Butylphthalides 80,160 respectively behind the rat MCAO, totally 4 groups (digitized representation animal numbers of elements in the bracket of 240mg/kg and solvent (ischemic control group).Observe 24 hours nerve of postoperative disappearance mark and infarct size, the result shows, 2 hours Butylphthalide oral medications behind the MCAO can obviously reduce cerebral infarct size and improve neural disappearance, result as shown in Figure 2.Embodiment 3
Butylphthalide prevention administration is to the influence of intraluminal middle cerebral artery occlusion in rats ligation (MCAO) back cerebral infarct size and neural disappearance.
Instrument, animal and medicine are with embodiment 1,2.
Method: similar to embodiment 1,2.Administering mode is respectively:
(1) at preceding 1 hour oral Butylphthalide 80 of MCAO or 160mg/kg;
(2) preceding 7 days continuous oral Butylphthalides 40 of MCAO or 80mg/kg/ days;
(3) solvent control (ischemic control),
Totally 5 groups (every group of 7-8 only).Observe 24 hours behavior change of postoperative and infarct size.The result shows that as shown in Figure 3 Butylphthalide prevention administration (oral) has the effect of obvious reduction cerebral infarct size and behavior disappearance.
By embodiment 1-3 as seen, preceding 1 hour of Butylphthalide prevention administration (MCAO), oral Butylphthalide 80 or 160mg/kg or successive administration 7 days, dosage be 40 or 80mg/kg/ days) or treatment administration (15min lumbar injection 10 behind MCAO, 20mg/kg or oral 40,80mg/kg; Or behind the MCAO 2 hours oral 80,160,240mg/kg) all can obviously reverse or reduce the effect of cerebral infarct size and neural disappearance, and tangible dose-effect relationship (Fig. 1,2,3) is arranged.This result shows that Butylphthalide has and reverses or reduce the ischemic neuronal damage, improves neural disappearance effect after the cerebral ischemia, is expected to treat human apoplexy and improves apoplexy sequela.Embodiment 4
Butylphthalide is to the influence of middle cerebral artery ligation rat brain edema
Cerebral hypoxia ischemia energy exhaustion, cell membrane function is impaired, can not keep ion gradient, causes the cerebral tissue edema, and Na piles up, K
+Lowering of concentration, major injury neuronal function or cause neuronal death.
Animal: the male wistar rat, body weight 300～350g is provided by Chinese Academy of Medical Sciences's animal center.
Medicine: the dl-Butylphthalide, provide by institute of Materia Medica,Chinese Academy of Medical Sciences consonance pharmaceutical factory, content 98% is mixed with desired concn with refined edible oil.
Method: this experiment causes cerebral edema with right side middle cerebral artery ligation (MCAO) rat (method is with embodiment 1).The oral dl-Butylphthalide 80,160 of 15min behind MCAO, 240mg/kg, postoperative was put to death in 24 hours, got forebrain, divided the another name weight in wet base with left and right hemisphere.
Bake for 100 ℃ and claim dry weight after 24 hours, the dried wet method calculating of usefulness brain water weight ([(wet tissue-stem organization)/wet tissue] * 100).Then with tissue digestion (with dense sour 0.5ml and the 30%H of disappearing
2O
2(1: 4v/v), transfer pH to 7.2) with HCl 85 ℃ of digestion 4 hours.Be connected (Chemical MicrosensorII., Diamond General Corp.USA) on the oxygen fabric analysis instrument with ion-selective electrode, detect cerebral tissue Na
+And K
+Ion.
The results are shown in table 1 and the table 2 as seen, the d1-Butylphthalide can obviously reduce brain water and Na
+Content increases K
+Content, explanation can alleviate the cerebral edema embodiment 5 that local cerebral ischemia causes
Butylphthalide is to the prevention and the therapeutical effect of apoplexy type spontaneous hypertensive rat apoplexy.
Cause apoplexy for simulating clinical hypertensive patient, this experiment is provided by laboratory animal section of Cardiovascular Disease Inst. Chinese Academy of Medical Sciences with making apoplexy type spontaneous hypertensive rat (SHRSP) 6 weeks, 4～5 animals of every cage.23 ± 1 ℃ of room temperatures, humidity 50 ± 10%.Illumination 12 hours/day is freely drunk water and feeding piece material or soft diet.Experiment divides 5 groups, and 15 every group, experiment begins to apopiecticus insultus certainly, and every animal advances the salt amount and increase to 1.3g gradually by 0.8g every day, uses instead from the apoplexy on diet.
Medicine, the dl-Butylphthalide is provided by the institute of Materia Medica,Chinese Academy of Medical Sciences Beijing XieHe medicine Factory, is the pale yellow oily liquid body, and content is 98%, all is made into desired concn with commercially available refined edible oil.Dosage is 25,50 and 100mg/kg/ days three dosage groups, and other establishes the solvent control group, and the administration volume is 1ml/kg.
Observation index: 1, the natural law of surviving after record apopiecticus insultus time and the apoplexy; 2. post-stroke carries out the nervous symptoms scoring every day.Evaluation criteria is as follows:
1, the standard of cerebral seizure: (1) single or two forelimbs or whole body twitch, hemiplegia or general paralysis appear in (2), and abdominal part can not be stood with pasting, and (3) heavier person trembles for whole body is tiny, and climb volt at last and collapse from physical exhaustion, even death.
Some animal is still with excitation (jump or scurry) or suppress phenomenon.All appearance are assessed as cerebral seizure with the next item up symptom.
2, the standards of grading of nervous symptoms are as follows:
The rank symptom
0 is normal
1 few moving or slight excitation
2 one side forelimbs or head are twitched, and bore the cage angle not
Moving or excitation resists into (jump or scurry)
3 one-sided forward and backward limbs or one-sided forelimb paralysis,
Body is crooked, difficulty in walking.
4 abdominal paries can not be stood with pasting, quadriplegia,
Whole body is tiny to tremble or twitches
Write down 21 days evaluation marks, carry out statistical disposition.
The concrete practice is: 75 male apoplexy type spontaneous hypertensive rats, every group 15, feed 0.8-0.9 gram Sal from the 6th every day in age week, progressively increase to 1.2-1.3 gram Sal/sky later on, every day oral Butylphthalide 25,50 or 100mg/kg/ days, nimodipine 37mg/kg/ days, matched group was oral with the volume solvent control.Take place to end in 3 weeks of back from the 8th beginning in age in week medicine feed to apoplexy.Back meter record neural disappearance mark and death time take place from apoplexy.(A B) shows, having in Butylphthalide 100mg/kg/ days very obviously increases the survival rate behind the cerebral seizure and improve neural disappearance effect, and its effect is similar to nimodipine by Fig. 4.The prompting Butylphthalide causes that to serious hypertension cerebral apoplexy patient has therapeutical effect.
Fig. 5 shows the preventive effect of oral Butylphthalide to apoplexy type spontaneous hypertensive rat apoplexy.Wherein every treated animal number is 15.Embodiment 6
Butylphthalide is to the amnemonic influence of local rats with cerebral ischemia.
Apoplexy causes ischemic brain infarction, the neuron major injury, and function is impaired, and this is to cause the main cause of patients with cerebral apoplexy sequela clinically.Present embodiment is observed the dl-Butylphthalide local cerebral ischemia is caused amnemonic influence.
Instrument:
1, shuttle box: Japanese Taisho Pharmaceutical Co., Ltd makes
2, operating microscope SXP type, the Shanghai medical optical instrument is produced
3, high frequency electric knife: the 62TZ-V type, medical electronic apparatus factory in Beijing produces
Animal:
The Wistar male rat, body weight 250～300g is provided by Chinese Academy of Medical Sciences's Experimental Animal Center.
Medicine:
The dl-Butylphthalide is provided by the institute of Materia Medica,Chinese Academy of Medical Sciences Beijing XieHe medicine Factory, is the pale yellow oily liquid body, and content 98% is mixed with 10,30 and 100mg/ml with refined edible oil, and the gastric infusion volume is 1ml/kg
Idebenone is commercially available
Method:
1, the acquired training of learning and memory
Rat carries out the acquired training of learning and memory earlier.Shuttle box is made up of experimental box and automatic record printing equipment, and the experimental box size is 50 * 16 * 18cm
3The stainless steel rod of case bottom grid for switching on, two Room about central part has the plate washer of a high 1.2cm that the case bottom is separated at the bottom of the case, light source and buzz controller are arranged at the experimental box top, automatically the record printing equipment can write down reaction and the incubation period of animal to electricity irritation or conditional stimulus continuously automatically, and the result printed, during training, rat is put into any Room of case, buzz appears after 10 seconds, continue 10 seconds, the back gave electricity irritation (100V in 10 seconds, 0.2mA, 50Hz, AC), initial animal only responds to electric shock, promptly escape to offside avoiding electric shock, after 10 seconds once more Conditions stimulate and continue impose electricity irritation in animal place side, force animal to skip to another chamber, so shuttle, when buzz is current, rat skips to the offside place of safety immediately to avoid electric shock, promptly think and active avoidance response occurred, every other day train one time, 20 times every time, train 4-5 to return approximately, after last training, active avoidance response reached rat more than 80% except that sham operated rats, all carry out middle cerebral artery ligation (MCAO).
2, MCAO art: (350mg/kg i.p), carries out the MCAO art by the Tamura method with the anesthesia of 12% chloral hydrate.The rat lateral position is fixed on the operating-table, middle point vertical along external auditory meatus and right eye corner of the eyes line is cut skin in line, then under operating microscope, expose zygomatic arch, remove zygomatic arch with rongeur and expose the temporo precoila, go up the microcephalia window that an about 2mm diameter is opened in the place's of the associating boring before zygomatic arch and squamosal bone of little stretching device, coagulate middle cerebral artery, layer-by-layer suture wound then with bipolar electric knife electricity.Sham operated rats is not except that blowing the MCA, and all the other are performed the operation all with above-mentioned step.The postoperative rat is steam again, and room temperature is constant in 24-25 ℃, the experiment of a learning and memory in rats of replication behind the postoperative 24h, and method is identical with the acquired experiment of learning and memory in rats.
3, grouping and drug dose
Fig. 6 shows that oral Butylphthalide causes that to ligation middle cerebral artery (MCAO) the dysmnesia rat initiatively avoids the influence of number of times (mean+SD).
(A B) shows that Butylphthalide causes the influence of dysmnesia rat avoidance incubation period (mean+SD) to ligation middle cerebral artery (MCAO) to Fig. 7.
The result by Fig. 6 and 7 as seen, the active of sham operated rats rat is similar to the numerical value of normal rat with passive avoidance response perioperatively incubation period changing value, though illustrate that animal through surgical injury, does not influence memory function.Otherwise the obvious loss of memory of middle cerebral artery blocking-up back rat is initiatively avoided number of times and is reduced, and incubation period, changing value obviously increased, and illustrated to have produced dysmnesia.To these MCAO rats, behind oral administration 30 or the 100mg/kgdl-Butylphthalide, all can improve dysmnesia, show as 100mg/kg group and initiatively avoid errors number and obviously reduce (promptly initiatively avoiding increased frequency); Active and passive avoidance response changing value incubation period obviously shorten.Above result shows, the dl-Butylphthalide local cerebral ischemia that has clear improvement causes amnemonic effect.Embodiment 7
Butylphthalide is to the influence of rat brain wound
Animal: the wistar rat, body weight 220～245g is provided by Chinese Academy of Medical Sciences's animal center.
Medicine: the dl-Butylphthalide is provided by the institute of Materia Medica,Chinese Academy of Medical Sciences Beijing XieHe medicine Factory, content 98%.The pale yellow oily liquid body.With the preparation of refined edible oil.
Method: freely fall bump rats with left coronal suture rear side parietal bone with heavy 200g cone-shaped metal object from the 30cm eminence.Wound the oral dl-Butylphthalide of back 5min 240mg/kg, marking evaluation behavior changes after 24 hours.The marking standard is as follows:
(1) righting reflex loss person is 3 minutes; (2) put rat on the net, make Mus and net be vertical angle, promptly climb and be hung on the net, do not have that to climb act person be 3.0 minutes (0-3) with forelimb; (3) receive in the strong side forelimb shoulder, flexing person is that 3 (0～3) are divided; (4) the two forelimbs of tractive, unable person 3 (0-3) divides; (5) push away a left side or right shoulder, non-resistance person 3 minutes (0-3).Full marks 15 minutes, normal rat 0 minute.
The result shows, the nervous symptoms that the cerebral trauma rat causes with cerebral ischemia, and after dl-Butylphthalide treatment, nervous symptoms take an evident turn for the better (table 3).Embodiment 8
Butylphthalide causes the influence of global brain ischemia brain energy metabolism to the mice broken end.
Lactic acid obviously raises behind the cerebral hypoxia ischemia, and ATP and PCr descend greatly, cause energy exhaustion, so that has caused that neuronal function is impaired.
Present embodiment male mice in kunming (source is with embodiment 7) 18-22g, every group 10, (medicine is with embodiment 7 for the subcutaneous injection Butylphthalide, subcutaneous injection is made into Emulsion with tween80) 100,150,200mg/kg and solvent (ischemic control), totally 4 groups, after administration, breaked end in 30 minutes to 15 seconds homogenate time as ischemic time, press Folbergrova (FolbergrovaJ.et al., J Neurochem 1972; 19:2497) and Lowry (Lowry OH and Passonneau JV A flexible system of enzymatic analysis New York:Academic Press, 1977) method is carried out centrifugal extraction and is measured lactic acid (ultraviolet spectrophotometer method), ATP and PCr (spectrofluorophotometer) content with the zymetology method.
The result shows that solvent control group brain lactic acid content rises, and ATP and PCr descend; Subcutaneous injection Butylphthalide 200mg/kg, broken end records lactic acid content and obviously descends after 30 minutes, and ATP and PCr content is obviously rising (table 4) then.This results suggest Butylphthalide has the effect that improves the ischemia brain energy metabolism.Embodiment 9
Butylphthalide is to the ischemic stage of global brain ischemia rat and heavily irritate the outer liquid adenosine of phase striatal cell, inosine, the influence of hypoxanthine, xanthine content
The cerebral anoxia ischemia causes brain energy metabolism exhaustion, ATP catabolite such as adenosine (Ade), inosine (Ino), hypoxanthine (Hyp) and xanthine (Xan) are poly-to be increased, and is heavily irritating the phase especially, in the presence of xanthine oxidase, Hyp produces a large amount of oxygen-derived free radicals when changing Xan and uric acid into.In addition, excitatory amino acid (EAA) and DA discharge in a large number, and EAA activates its receptor gated channel and makes Ca
2+, Na
+Interior stream, K
+Go out born of the same parents, make neurocyte swelling, Ca in the cell
2+Over loading causes cell injury or death.For the detection of dynamic cerebral ischemia or heavily irritate phase specific brain regions district extracellular fluid purine metabolism thing, the content of EAA and DA, the global brain ischemia rat model of 4 tremulous pulsies (two survey necks and vertebral artery) ligation is adopted in this research.
Dl-Butylphthalide (source is with embodiment 1) is made into Emulsion with 2% Tween80.
Male Wistar rat (source is with embodiment 1), body weight 280-350g, chloral hydrate anesthesia (350mg/kg ip).Adopt Pulsinelli method (Pulsinelli WA and BrierleyJB, A new model of bilateral hemispheric ischemia in the anesthetized rat stroke 1979; 10:267), go 4-tremulous pulse (bilateral neck and vertebral artery) ligation.By stereotaxic instrument pin is flowed in slight irrigation and insert the right side striatum, use the artificial cerebrospinal fluid perfusion, flow velocity 2.5 μ l/min, collection ischemia (4-artery ligation) 20 minutes and heavily filling (open carotid artery) some parts of perfusate during 2 hours, every part 20 μ l is all through 5%HClO
4Handle.The test condition of HPLC-UV is mobile phase 10mmol/L NH
4H
2PO
4/ CH
3OH (20: 1), pH 3.9-4.0, flow velocity 1.0ml/min, immobile phase: C18-silica gel, 10 μ m UV254nm, sample size are 20 μ l.(510 type Waters, USA), (Japan), UV-detector is formed for SPD-6AV, Shimadzu by integrator (3394A Hewlett-Packard) by constant flow pump for chromatographic system.Dl-Butylphthalide dosage be 20 and 40mg/kg i.p. result as shown in Figure 8, show that Butylphthalide can suppress ischemia and heavily irritate the outer liquid adenosine of phase striatal cell, inosine, hypoxanthine and xanthic rising illustrate that Butylphthalide can improve the generation (it is the substrate that produces oxygen-derived free radicals that ischemia is heavily irritated the phase hypoxanthine) of ATP resynthesis (suppressing adenosine raises) and reduction oxygen-derived free radicals.
Content with dopamine (DA) in the HPLC-EC detection of dynamic ischemic stage perfusate.Test condition is immobile phase YWG-CH
1810 μ m. mobile phases are the 0.1mol/L citric acid, 0.1mol/L acetate buffer solution, pH3.7, the deionized water preparation includes methanol 10%, ion pair B1.2mmol/L, n-butylamine 1.3mmol/L, chromatographic column is 0.46 * 25cm PE, chromatographic condition is flow velocity 1.2ml/mim, running voltage 0.75V, sample size 20 μ l, 20min lumbar injection Butylphthalide 40mg/kg before ischemia, collect ischemia front and back perfusate, the difference of observing DA content between administration group and the matched group.The result shows that Butylphthalide can reduce the rising of the outer liquid DA of ischemic stage striatal cell, illustrates that Butylphthalide has protective effect (seeing Table 5) to cerebral ischemia.
Collect 60 μ l (two sample additions of identical perfusion phase of animal) perfusate; with the L-8500 of Hitachi automatic amino acid analyzer; the pH method is measured excitatory amino acid (EAA) content in the perfusate, and the result shows as shown in Figure 9; Butylphthalide can reduce glycine content; because glycine is the allosteric agonist of nmda receptor, can increase the affinity of EAA and its receptor, so this effect shows; Butylphthalide can reduce the excited toxic action of EAA, and therefore cerebral protection is arranged.
By above-mentioned experimental result as can be known, behind lumbar injection Butylphthalide 20mg/kg or the 40mg/kg, (1) purine compound such as Ade, Ino, Hyp and Xan obviously descend; (2) DA and DOPAC (DA metabolite) obviously descend; (3) glycine concentration is following gust.The result shows: ischemia is heavily irritated phase Hyp in the presence of xanthine oxidase, produce a large amount of toxicity oxygen-derived free radicals when changing Xan and uric acid into, cause neuronal cell film major injury, and the rising effect of this antagonism purine metabolism thing of Butylphthalide, prompting has protective effect to the ischemic neuronal damage; Extracellular dopamine increase is considered to a kind of indication of ischemic brain injury; and Butylphthalide can reduce its rising; point out it that ischemic brain injury is had protective effect; glycine is the allosteric agonist of NMDA subtype acceptor; can increase the affinity of EAA to nmda receptor; increase the frequency of this receptor channel opener, therefore, this effect of Butylphthalide reduction glycine concentration is very favorable to the protection of brain injury.Embodiment 10
Butylphthalide is to the smelting treatment effect of cerebral apoplexy patient
Medicine: the dl-Butylphthalide is provided by the institute of Materia Medica,Chinese Academy of Medical Sciences Beijing XieHe medicine Factory.
The dl-Butylphthalide is made soft capsule preparation, and every ball contains dl-Butylphthalide 100mg and refined edible oil 400mg, and the latter is an excipient.
Case:
19 routine patients with cerebral apoplexy, male's 10 examples, women's 9 examples.45～70 years old age.The course of disease is acute stage (morbidity back 1-4 days).Clinical indexes:
(1) muscle strength test instrument is divided into 5 grades, as the index of function of nervous system;
(2) Chang Gu river formula is checked, as the index of moral function;
(3) clinical laboratory's index: be mainly the CT scan inspection, other is checked as EEG, hemorheology etc.
Medicining mode:
After patient is admitted to hospital certainly and the back of taking medicine was detected These parameters respectively in 2 weeks, and the state of an illness does not have not improvement before and after taking medicine with judgement
Every routine patient takes dl-Butylphthalide 600mg every day after the These parameters inspection, and divide and take for three times, each 2 balls, be 14 days the course of treatment.
The result:
After taking for two weeks, the state of an illness generally alleviates or takes a turn for the better.Judge the 11.2 ± 5.0cm of cerebral infarct volume before by administration by the CT scan result
3Be reduced to 3.2 ± 1.5cm
3, handle P＜0.01 by statistics; Muscular strength generally increases, than increasing by 3～5 times (P＜0.01) before taking medicine.The formula inspection of Chang Gu river also has clear improvement.All be unable to leave the bed during patient admission, after two week treatments, all can walk basically or draw and hold up walking by the people.Show that by above result the dl-Butylphthalide is to the treatment of the cerebral apoplexy patient embodiment 11 that improves significantly
Butylphthalide soft capsule prescription and preparation
(by 1000 calculating that feed intake)
Prescription:
Former, supplementary product consumption
Butylphthalide (source is with embodiment 1) 100 grams
Vegetable oil (commercially available) 350 grams
BHT 1.35 grams
Preparation: BHT is dissolved in (ultrasound wave dissolving) in the vegetable oil,, is sub-packed in the 450mg soft capsule and gets final product again with the Butylphthalide mix homogeneously.
Testing result shows that said preparation has good stability, and stable phase can reach more than 2 years.Meet in Chinese Pharmacopoeia 90 editions (two ones) appendix requirement about capsule.
Acute toxicity test, teratogenesis, mutagenicity test below by Butylphthalide illustrate that this chemical compound is to mammal and human toxicity.Following experiment all is to carry out according to the method for defined in Ministry of Health of the People's Republic of China's " provisions for new drugs approval ".Used Butylphthalide is the dl-Butylphthalide, and the Beijing XieHe medicine Factory provides by institute of Materia Medica,Chinese Academy of Medical Sciences, the pale yellow oily liquid body, and content 98%, oral administration is made into refined edible oil, and lumbar injection is made into Emulsion with 2%Tween80.Experimental example 1
Acute toxicity test in mice is oral:
LD
50=3.19 (2.23～3.85) g/kg lumbar injection:
LD
50=296 (155～354) mg/kg experimental example 2
The acute toxicity test of rat is oral: LD
50=2.79 (2.41～3.45) g/kg lumbar injection DL
50=592.0 (513～680) mg/kg experimental example 3
Teratogenic test
Animal: the Wistar rat is provided by Chinese Academy of Medical Sciences zooscopy center, and female Mus is the nulliparity Mus, body weight 180～280g, male Mus body weight 280～320g.
Gastric infusion, branch 100 and two dosage groups of 400mg/kg.
The result: medicine does not have overt toxicity to female Mus, does not have weight loss during administration, and the miscarriage and the phenomena of mortality are grown obviously influence of nothing to embryo and tire Mus.Do not find outward appearance monster Mus, the skeletal survey no abnormality seen.The mutagenic test of experimental example 4 Butylphthalides
Use the microorganism reverse mutation test, rodent micronucleus test and Chinese hamster pneumonocyte (CHL) chromosomal aberration test show that the dl-Butylphthalide is to the above experiment reaction that all is negative.Experimental example 5
The toxicity test of the oral dl-Butylphthalide of rat (6 months)
Animal: make 160 of Wistar rats 6 weeks, male and female half and half are divided 4 groups, 40 every group
Drug dose divides high, medium and low three groups: 500,250 and 120mg/kg, and administration volume 1.0ml/kg/ days.Administration time is 180 days.
Method: observe all inspection items by provisions for new drugs approval and relevant rules.
The dl-Butylphthalide is given rat continuous irrigation stomach 6 months as a result, the body weight of rarely seen high, middle dosage group rat, and the blood glucose of senior middle school, low dose group has been compared marked difference with the cholesterol of height, middle dosage group rat with matched group; Indivedual organ coefficients of senior middle school dosage group rat compare with control rats that also there were significant differences.When convalescent period finished after drug withdrawal, every index that each dosage group is detected was all in normal range.Show that the dl-Butylphthalide has its safety range, appropriate as clinical medicine dose, safety non-toxic then.Table 1 Butylphthalide is to the influence of middle cerebral artery ligation rat brain water content
Grouping dosage approach brain hemisphere water content (%)
(mg/kg) left side right side sham operated rats--78.41 ± 0.20 78.51 ± 0.30 solvent control--PO 78.44 ± 0.31 80.97 ± 0.79### Butylphthalide, 80 PO 78.44 ± 0.36 80.14 ± 0.56**
160 PO 78.54±0.34 79.98±0.83**
240 PO 78.40 ± 0.18 79.05 ± 0.48*** nimodipine, 5 SC 78.65 ± 0.32 79.99 ± 0.88* mean+SD, every treated animal number is that compare with sham operated rats 8 .###p＜0.001, * p＜0.05, * p＜0.01, compare with the solvent control group * * p＜0.001, the left side is non-ischemia side, and the right side is the ischemia side.Table 2 Butylphthalide is to the influence of middle cerebral artery ligation rat brain cation concn
Grouping dosage approach brain hemisphere cation concn (μ mol/g dry weight)
The sodium ion potassium ion
(mg/kg) left side, right side, left side right side sham-operation group--255.7 ± 7.2 260.2 ± 7.9 484.1 ± 4.1 482.2 ± 4.8 solvent control--PO 259.2 ± plain 80 PO 255.9 of 15.1 349.3 ± 15.7### 484.8 ± 4.3 436.9 ± 11.3### celery first ± 7.8 317.0 ± 27.0** 485.5 ± 4.8 452.2 ± 13.7**
160 PO 263.4±14.8 315.7±37.1** 481.1±7.2 466.3±19.2**
240 PO 253.4 ± 5.3 272.8 ± 18.9*** 485.0 ± 2.2 473.6 ± 7.9*** nimodipine, 5 SC 274.4 ± 16.9 313.9 ± 41.3** 481.8 ± 6.2 453.4 ± 14.5** mean+SD, every treated animal number is that compare with sham operated rats 8 .###p＜0.001, * p＜0.05, * p＜0.01, compare with the solvent control group * * p＜0.001, the left side is non-ischemia side, and the right side is the ischemia side.Table 3. dl-Butylphthalide is to the influence (X ± SD n=10) of head trauma rat
Drug dose approach behavior scoring
(mg/kg) solvent control--po 9.0 ± 2.5dl-Butylphthalide 240 po 3.4 ± 1.0******p＜0.001. table 4 Butylphthalide influences drug dose lactic acid ATP phosphagen to what mice broken end caused the global brain ischemia energy metabolism
(mg/Kg S.C.) (μ mol/g) (μ mol/g) (μ mol/g) ischemic control-14.0 ± 3.6 0.60 ± 0.14 3.90 ± 1.49 Butylphthalide 100 11.0 ± 1.2 0.65 ± 0.14 5.14 ± 1.40
150 10.0±1.2* - 5.50±1.42
200 9.6 ± 3.0*, 0.98 ± 0.30*, 7.64 ± 1.55**n=7, mean+SD: * p＜0.05; * p＜0.01 and the influence of ischemic control group comparison sheet 5 Butylphthalides (40mg/Kg i.p.) to dopamine (DA) in the global brain ischemia rat striatum perfusate and metabolite (DOPAC) concentration thereof
DA(ng/ml) DOPAC(ng/ml)
Ischemia solvent control 4.11 ± 0.45 19.20 ± 3.69** 15.0 ± 2.54 12.75 ± 1.56 before the ischemia ischemia before the ischemia
(n=5) (n=5) (n=7) (n=7) Butylphthalide 5.60 ± 0.89 4.44 ± 3.02## 10.6 ± 0.94 5.60 ± 1.30##
(n=6) (n=6) (n=5) (n=5) mean+SD: before * * p＜0.01 and the ischemia relatively
#p＜0.05, compare with the solvent control group ##p＜0.01
Claims (9)
1, the application of Butylphthalide in the medicine of the disease that preparation prevention and treatment mammal or human cerebral ischemia cause.
2, application as claimed in claim 1 is characterized in that: described disease is mammal or caused cerebral infarct size of human local cerebral ischemia and neural disappearance.
3, application as claimed in claim 1 is characterized in that: described disease is mammal or the caused cerebral edema of human local cerebral ischemia.
4, application as claimed in claim 1 is characterized in that: described disease is the dead and neural disappearance of mammal or human apoplexy and post-stroke.
5, application as claimed in claim 1 is characterized in that: described disease is mammal or the caused dysmnesia of human local cerebral ischemia.
6, application as claimed in claim 1 is characterized in that: described disease is that the energy metabolism of mammal or human global brain ischemia is exhausted.
7, application as claimed in claim 1 is characterized in that: described disease is mammal or human global brain ischemia and irritates the outer liquid adenosine of phase striatal cell, inosine, hypoxanthine, the rising of xanthine content again.
8, application as claimed in claim 1 is characterized in that: described disease is the outer liquid dopamine and 3 of mammal or human cerebral ischemia phase striatal cell, and 4-dihydroxyphenyl acetic acid content raises and glycine content raises.
9, application as claimed in claim 1 is characterized in that: described disease is the nervous symptoms that mammal or human cerebral trauma cause cerebral ischemia to cause.
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| CN100367951C (en) * | 2005-12-16 | 2008-02-13 | 石药集团恩必普药业有限公司 | Butyl benzene phthalein vein emulsion and its application |
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| US20060166931A1 (en) | 2002-08-21 | 2006-07-27 | Niu Zhan-Qi | Clathrates of butylphtualide with cyclodextrin or its derivatives, a process for their preparation and the use thereof |
| CN1565441B (en) * | 2003-06-20 | 2010-05-26 | 中国医学科学院药物研究所 | Use of levobutylphthalide in prevention and cure of dementia |
| CN1605336A (en) * | 2003-10-10 | 2005-04-13 | 中国医学科学院药物研究所 | Application of L-butylphthalide in the process for preparing cerebral infarction preventing and treating medicine |
| CN1647796A (en) * | 2004-01-20 | 2005-08-03 | 中奇制药技术（石家庄）有限公司 | Use of Levo-butylphthalide in preparing medicine for preventing and treating cerebral ischemia diseases |
| CN100435792C (en) * | 2004-04-23 | 2008-11-26 | 石药集团中奇制药技术（石家庄）有限公司 | Application of butylbenzene phthalein homolog in preparation of medicine for treating cerebral ischemia disease |
| EP1757286B1 (en) * | 2004-06-18 | 2017-09-06 | CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. | The application of l-n-butylphthalide in preventing and treating alzheimer's disease |
| CN100361656C (en) * | 2004-08-27 | 2008-01-16 | 石药集团中奇制药技术（石家庄）有限公司 | Butylbenzene phthalein self emulsifying releasing medicine system, preparation method and application |
| TWI419686B (en) * | 2004-10-29 | 2013-12-21 | Inst Materia Medica Cams | The use of therapeutic and preventive action of l-3-n-butylphthalide on dementia, involving alzheimer's disease and vascular dementia |
| CN101289438B (en) * | 2007-04-16 | 2012-04-18 | 山东绿叶天然药物研究开发有限公司 | 3-(3'-hydroxyl)-butyl phthalide ester, and preparation thereof and uses |
| CN102178643B (en) * | 2011-04-29 | 2014-05-14 | 石药集团恩必普药业有限公司 | Microemulsion transdermal gel agent of butylphthalide or derivative thereof, and preparation method thereof |
| CN106963757A (en) * | 2012-04-01 | 2017-07-21 | 石药集团恩必普药业有限公司 | Application of the butylphenyl phthaleine or derivatives thereof in the medicine for preparing therapeutic radiation brain damage |
| CN105130934B (en) * | 2015-08-15 | 2018-11-23 | 石药集团恩必普药业有限公司 | A kind of butylphenyl phthaleine bulk pharmaceutical chemicals product and preparation method thereof |
| CN108947946B (en) * | 2017-05-19 | 2022-10-28 | 泰州华元医药科技有限公司 | Brain injury resistant deuterated compound and medical application thereof |
| CN109419800B (en) * | 2017-08-30 | 2021-04-30 | 石药集团恩必普药业有限公司 | Pharmaceutical composition and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5598169A (en) * | 1979-01-23 | 1980-07-25 | Masayuki Ishikawa | Preparation of 1-phthalazone derivative having aromatic residue substituent at 2-position |
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1993
- 1993-09-09 CN CN93117148A patent/CN1048158C/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5598169A (en) * | 1979-01-23 | 1980-07-25 | Masayuki Ishikawa | Preparation of 1-phthalazone derivative having aromatic residue substituent at 2-position |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100367951C (en) * | 2005-12-16 | 2008-02-13 | 石药集团恩必普药业有限公司 | Butyl benzene phthalein vein emulsion and its application |
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| CN1100097A (en) | 1995-03-15 |
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