WO2006002589A1 - Utilisation medicale de paeoniflorine - Google Patents

Utilisation medicale de paeoniflorine Download PDF

Info

Publication number
WO2006002589A1
WO2006002589A1 PCT/CN2005/000929 CN2005000929W WO2006002589A1 WO 2006002589 A1 WO2006002589 A1 WO 2006002589A1 CN 2005000929 W CN2005000929 W CN 2005000929W WO 2006002589 A1 WO2006002589 A1 WO 2006002589A1
Authority
WO
WIPO (PCT)
Prior art keywords
paeoniflorin
receptor
stroke
parkinson
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2005/000929
Other languages
English (en)
French (fr)
Inventor
Xingzu Zhu
Dazhi Liu
Yang Ye
Huaqing Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institute of Materia Medica of CAS
Original Assignee
Shanghai Institute of Materia Medica of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Institute of Materia Medica of CAS filed Critical Shanghai Institute of Materia Medica of CAS
Priority to US11/631,123 priority Critical patent/US20080275226A1/en
Priority to EP05757963A priority patent/EP1767210A4/en
Priority to JP2007519597A priority patent/JP2008505128A/ja
Publication of WO2006002589A1 publication Critical patent/WO2006002589A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to a medical use of a selective agonist of Adenosine Receptor-1 (AjR), which is specifically for stimulating a receptor, and for treating and preventing a stroke. And the role of neurological diseases such as Parkinson's disease, while eliminating the serious cardiovascular side effects of traditional steroid agonists. Background technique
  • Paeoniflorin is a naturally occurring compound derived from the roots of the white peony roots, red peony, and peony. It is abundant in source plants. Taking the white peony produced in Cao County of Shandong province as an example, the active monomer component is 1.95%; more importantly, the content of paeoniflorin in peony root is as high as 7%. Furthermore, the source plants are widely distributed in regions and are easy to cultivate artificially, and are suitable for large-scale industrial planting.
  • A!R receptor agonists can activate presynaptic A!R receptors, inhibit the release of excitatory amino acids such as glutamate, and alleviate the excitotoxicity and Ca 2+ overload of cells; Acting on postsynaptic receptors, increases the conductance of the K channel, inhibiting neuronal excitation by limiting cell membrane depolarization. Thus, a significant neuroprotective effect is exerted (Heron A, Lekieffre D, Le Peillet E, Lasbennes F, Seylaz J, Plotkine M, Boulu RG., Effects of an A 1 adenosine receptor agonist on the neurochemical, behavioral and histological consequences of ischemia Brain Res.
  • Nervous system diseases such as stroke and Parkinson's disease are among the biggest threats to human health, and the prevalence and disability rate among the elderly is high. In the case of stroke alone, the incidence rate is about 0.35% in the 45-89 age group. About 20% of stroke patients have a survival period of less than 1 month; more than 30% of patients who survive more than 6 months cannot take care of themselves. Individuals, families, and society bear enormous double-psychological and economic burdens.
  • the invention provides a novel medicine for treating and preventing nervous system diseases such as stroke and Parkinson's disease, and has great social and economic benefits. Summary of the invention
  • the object of the present invention is to disclose that paeoniflorin is a non-steroidal A 1 receptor selective agonist.
  • Still another object of the present invention is to provide a use of paeoniflorin for preventing and treating diseases of the nervous system such as stroke and Parkinson's disease.
  • the invention adopts a chemical method to extract and isolate a natural active compound paeoniflorin from the roots of Ranunculaceae, Radix Paeoniae Alba, and Peony. After pharmacological test, the paeoniflorin is regarded as a non-steroidal adenosine receptor-1.
  • Selective agonists can be used in the preparation of drugs for the prevention and treatment of diseases of the nervous system such as stroke and Parkinson's disease.
  • the invention adopts the roots of white peony, red peony, and peony as raw materials, and the preparation process is as follows - the roots of white peony, red peony, or peony are extracted by refluxing with ethanol to obtain a dry extract, and the extract is fully dissolved by water, and is passed through a large hole.
  • the resin was eluted with water to a negative reaction of sugar, and eluted with 2 column volumes of ethanol to obtain an extract, which was separated by silica gel column chromatography to obtain a pure compound paeoniflorin.
  • the related pharmacological efficacy test is carried out by using the above-mentioned method to extract the paeoniflorin.
  • the rat cerebral cortex synaptic membrane was used, and after the rats were decapitated, the cerebral cortex was separated, then homogenized in a 1:15 (w/v) sucrose solution, centrifuged, and the supernatant was centrifuged. After discarding the supernatant, resuspend the pellet in 1:30 (w/v) Tris-Hcl buffer (50 mM, pH 7.4), homogenate and centrifuge, and repeat the washing process 3 times, using 1: 5 ( W/V) Tris-Hcl buffer (50 mM, pH 7.4) resuspend the pellet again, store at 80 °C, and determine the protein concentration by Bradford method (Bradford MM. A rapid and sensitive method for the The quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-254.), the above operations were all carried out at 4 °C.
  • the above model was used to verify the competitive binding of paeoniflorin to the A 2a R receptor mixed agonist [ 3 H]-ethylamide adenosine ([3HJ-NECA) and receptor selective agonist [3H]-CCPA. .
  • the plug line was removed after 1.5 hrs of ischemia, and reperfusion was performed for 22.5 hr.
  • saline and different doses of paeoniflorin were injected subcutaneously;
  • the permanent MCAO model group The suture was pulled out, continuous ischemia for 24 hr, and 15 minutes and 6 hours after ischemia, respectively, saline, paeoniflorin, and DPCPX + paeoniflorin were administered subcutaneously, and DPCPX was pre-administered 15 min before ischemia. .
  • the score of the defect is comprehensively graded (Sydserff SG, Borelli AR, Green AR, Cross AJ., Effect of NXY-059 on infarct volume after transient or permanent middle cerebral artery occlusion in the rat; studies on dose, Plasma concentration and therapeutic time window. Br J Pharmacol. 2002;135(1):103-12.).
  • paeoniflorin significantly improved the neurological symptoms of transient middle-vessel embolism in rats at medium dose (Smg.kg sc), and significantly reduced the damage of cerebral cortex and cerebral cortex caused by ischemia.
  • the dependent spoon reduces the damage of the cerebral cortex and cerebral cortex caused by ischemia, and this effect can be antagonized by the selective receptor antagonist DPCPX.
  • paeoniflorin may exert neuroprotective effects by activating A and R receptors, which can be used for clinical treatment and prevention of stroke.
  • Mouse PD model induced by l-metliyl-4-phenyl-I,2,3,6-tetrahydro-pyridine was used to detect the peony trapping of PD model mice.
  • Male C57B1/6J mice were used in 20-25 g, and the time of climbing by the mice was measured before the start of the experiment, and 5 groups were randomly divided according to the time of climbing, 8 in each group.
  • Low-dose and medium-dose paeoniflorin were administered subcutaneously for 11 consecutive days, and 2 hours on the 8th day of the experiment.
  • a mouse PD model was induced by intraperitoneal injection (ip) of 4 times of N-methyl-4-phenyl-l,2,3,6-tetmhydro-pyridine (MPTP).
  • mice were measured for their ability to exercise by climbing the rod.
  • the head of the mouse was placed down on a vertical rough rod with a length of 55 cm and a diameter of 8 mm.
  • the time from when the rod climbed down from the rod to when all the limbs touched the ground was recorded (Hamaue N).
  • the tail pulse sensor was used to transmit the pressure pulse to the phototransistor, and the corresponding blood pressure and heart rate values were converted. After the rats returned to calm, their blood pressure and heart rate were recorded as the baseline values. Subsequently, the saline vein, different doses of paeoniflorin and respirin (reserpi ne ) were separately injected through the tail vein.
  • FIG 3.1 Effect of paeoniflorin (PF) (2.5 mg-kg 1 , 5 mg-kg sc) on MPTP-induced motor bradykinesia.
  • Figure 3.2 Effect of paeoniflorin (PF) (2.5 mg-kg " 1 , 5 mg-kg sc) on MPTP-induced loss of dopaminergic neurons and reduction of striatal dopaminergic nerve fibers.
  • the compound paeoniflorin provided by the present invention competes for binding to the A!R receptor and has high selectivity. In terms of its chemical structure, it is distinguished from the traditional A!R receptor agonist as a novel non-steroidal A!R receptor agonist. It has been confirmed by related animal experiments that paeoniflorin has the effect of treating and preventing neurological diseases such as stroke and Parkinson's disease; further studies have shown that it has small cardiovascular side effects and has clinical application value, and can be used in nervous system such as stroke and Parkinson's disease. It is used in the treatment and prevention of diseases. detailed description
  • Example 1 Paeoniflorin is a selective agonist of non-steroidal receptors
  • the cerebral cortex was immediately separated and placed in ice-cold saline. Subsequently, the mixture was well homogenized in a 1:15 (W/V) sucrose solution (0.32 mol/L), centrifuged (l, 000 x g, 10 minutes), and the supernatant was centrifuged (30,000 x g, 30 minutes). After discarding the supernatant, the pellet was resuspended in 1:30 (W/V) Tris-Hcl buffer (50 mM, pH 7.4), homogenized, and centrifuged (48,000 x g, 10 minutes).
  • the reaction system is 200 ⁇ , of which the pre-test drug is 5 ⁇ , which is a paeoniflorin, a selective agonist.
  • CPA CBA
  • a 2a R receptor selective agonist CV-1808
  • the final concentration was from 0, 1 ( ⁇ 1 ⁇ to 10_ 4 moI / L; cerebral cortex synaptic suspension 20 ⁇ 25nmol/L [ 3H]-NECA or 0.2 nmol/L of [3H]-CCPA 20 ⁇ , assay buffer (50 mmol/L Tris-HCl, 1 mmol EDTA, 0.5% BSA, pH 7.4) 155 ⁇ . Samples were incubated at 25 ° C 3 After an hour, use a cell harvester to aspirate the reaction solution through a GF/C filter to stop the reaction, then rinse the filter with filtration buffer (50 mmol/L Tiis-HCl, 0.1% BSA, pH 7.4).
  • assay buffer 50 mmol/L Tris-HCl, 1 mmol EDTA, 0.5% BSA, pH 7.4
  • the membrane was dried 3 times, then dried at 40 ° C for 1 hour.
  • the membrane was placed in a sealed bag, 5 mL of MicroScint 20 was added, and the hook was pressed and sealed for radioactivity counting (Micro ⁇ , PerkinElmer). The statistical results were based on the double site.
  • the competition model is fitted.
  • Example 2 Application of paeoniflorin in prevention and treatment of stroke
  • the comprehensive grade score was evaluated according to the degree of forelimb bending, spontaneous rotation, and the absence of ipsilateral stimuli.
  • the brain was immediately removed and stored at 20 ° C for 15 min, and continuous coronal sections (sheet thickness 2 mm).
  • the brain slices were stained with 1% TTC (pH 7.2), incubated at 37 ° C for 10 min, and the staining solution was decanted and fixed in 4% formaldehyde solution.
  • Taking a digital camera to perform a pixel scan of the cortical and subcortical lesions of the brain slice (Adobe ImageReady 7.0), calculate the percentage of the area of the cortical damage area, the subcortical damage area and the total damage area, and further refer to the whole brain volume. , to estimate the volume of the cortical damage zone, the subcortical damage zone and the total damage zone.
  • sputum sputum significantly improved the neurological symptoms of transient MCAO rats, and significantly reduced the damage of the cerebral cortex and cerebral cortex caused by ischemia; ⁇ ⁇ (2.5, 5, 10 m Kg S . C . ) Dosage reduces the damage of the cerebral cortex and cerebral cortex caused by ischemia, and this effect can be antagonized by the selective A!R receptor antagonist DPCPX. It is suggested that paeoniflorin may exert neuroprotective effects by activating receptors and can be used for clinical treatment and prevention in stroke and its sequelae.
  • Example 3 Application of paeoniflorin in the prevention and treatment of Parkinson's disease
  • mice 20-25 g male C57B1/6J mice were randomly divided into 5 groups according to the time of climbing, and 8 rats in each group were measured before the start of the experiment.
  • Paeoniflorin 2.5mg kg- 1 , 5mg kg 1
  • ip intraperitoneal injection
  • 4-phen y ll, 2,3,6-tetrahydro-pyridine, MPTP induced mouse PD model.
  • the dosage regimen is as follows:
  • mice were recorded to climb down from the rod to Time when all limbs touch the ground
  • the heart was perfused with 4% paraformaldehyde for pre-fixation, and then the brain was removed and fixed in 4% formaldehyde solution for one week.
  • Tyrosine hydroxylase TM was used as a specific marker for dopamine neurons.
  • the primary antibody was monoclonal mouse TH antibody (1:1000, Sigma), and the secondary antibody was Alexa fluor 488 fluorescently labeled goat anti-mouse (1:1000, Molecular Probes). A fluorescence microscope (Olympus DP70) was photographed and black TH positive cells were counted.
  • Paeoniflorin (S mgJcg -i s.c.) is a potent anti-MPP-induced loss of dopaminergic neurons and motor dysfunction, suggesting that paeoniflorin can be used for clinical treatment and prevention of Parkinson's disease.
  • the tail pulse sensor was used to transmit the pressure pulse to the phototransistor, and the corresponding blood pressure and heart rate values were converted. After the rats returned to calm, their blood pressure and heart rate were recorded as the baseline values. Subsequently, normal saline, paeoniflorin (10, 40, 160 mg kg"' and reserpine were injected through the tail vein.
  • Paeoniflorin (10, 40, 160mg kg- 1 , iv) does not affect the blood pressure and heart rate of rats, and it is obvious that there is no significant heart in the therapeutic dose range of treatment and prevention of diseases such as wind and Parkinson's disease. Vascular side effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Cardiology (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Psychology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Description

芍药甙的医药用途
技术领域
本发明涉及一种非嘌呤腺苷受体一 1 (Adenosine Receptor- 1, AjR) 的选择性激动剂—— 芍药甙的医药用途， 具体指该化合物可选择性激动 受体， 发挥治疗和预防中风及帕金 森病等神经系统疾病的作用， 同时消除了传统的嘌呤类 激动剂的严重心血管副作用。 背景技术
芍药甙是一种天然活性化合物， 源于毛茛科植物白芍、 赤芍、 及牡丹的根。 其在源植物 中的含量丰富， 以山东曹县产白芍为例， 该活性单体成分达 1.95 % ; 更甚之， 芍药甙在牡丹 根中的含量高达 7%。 再者， 其源植物地域分布广泛， 易于人工栽培， 适于大规模产业化种 植。 药效学研究表明， 其具有镇痛、 镇静、 解痉、 抗炎、 抗溃疡、 扩张冠状动脉、 对抗急性 心肌缺血， 以及抑制血小板凝聚等诸多方面的药效， 且安全范围大。
大量研究发现， A!R受体激动剂一方面可激活突触前 A!R受体， 抑制谷氨酸等兴奋性 氨基酸的释放， 缓解细胞的兴奋毒性及 Ca2+超负荷； 另一方面， 作用于突触后 受体， 使 K通道的电导增加， 通过限制细胞膜去极化抑制神经元的兴奋。 从而， 发挥明显的神经 保护作用 （Heron A, Lekieffre D, Le Peillet E, Lasbennes F, Seylaz J, Plotkine M, Boulu RG.， Effects of an A 1 adenosine receptor agonist on the neurochemical, behavioral and histological consequences of ischemia. Brain Res. 1994; 641(2): 217-24., Goldberg MP, Monyer H, Weiss JH, Choi DW., Adenosine reduces cortical neuronal injury induced by oxygen or glucose deprivation in vitro. Neurosci Lett. 1988; 9(3): 323-7., Mori M, Nishizaki T, Okada Y" Protective effect of adenosine on the anoxic damage of hippocampal slice. Neuroscience. 1992; 46(2): 301-7., Von Lubitz DK, Beenhald er M, Lin RC, Carter MF, Paul IA, Bischofberger N, Jacobson KA., Reduction of postischemic brain damage and memory deficits following treatment with the "selective adenosine A 1 receptor agonist. Eur J Pharmacol. 1996; 302(1-3): 43-8. ), 但 A,R受体激 动剂严重的心血管副作用， 很大程度上限制了其临床应用 (Collis MG, Hourani SM.， Adenosine receptor subtypes. Trends Pharmacol Sci. 1993; 14(10): 360-6. Review., Daval JL, Nehlig A, Nicolas R, Physiological and pharmacological properties of adenosine: therapeutic implications. Life Sci. 1991; 49(20): 1435- 53. Review.)。 近年来， 国际医药界投入大量的人力、 物力， 试图 寻找神经保护作用确切， 心血管副作用小的选择性 受体激动剂。 本研究基于配体一受体竞争性结合试验的高通量筛选平台，首次发现芍药甙选择性竞争 结合 受体， 其化学结构区别于传统的 受体激动剂， 为一种新型的非嘌呤类 受 体选择性激动剂； 经典的试验动物模型证实， 芍药甙具有治疗和预防中风及帕金森病等神经 系统疾病的作用； 进一步的研究证明， 其心血管副作用小， 具有临床应用价值。
中风和帕金森病等神经系统疾病是对人类健康威胁最大的杀手之一，在老年人中患病率 和致残率居高不下。 仅以中风为例， 45— 89岁的群体中， 其发生率约为 0.35 %。 其中约 20 %中风患者的存活期低于 1个月； 存活期超过 6个月的患者中， 超过 30 %的生活不能自理。 个人、 家庭、 及社会承担着极大的心理上和经济上的双层负担。
本发明提供了一种治疗和预防中风及帕金森病等神经系统疾病的新型药物，具有巨大的 社会效益和经济效益。 发明内容
1. 本发明的目的在于公开芍药甙是一种非嘌呤类 A1受体选择性激动剂。
2. 本发明的又一目的在于提供芍药甙在预防和治疗中风和帕金森症等神经系统疾病中的 应用。 本发明采用化学方法从毛茛科植物白芍、 赤芍、 及牡丹的根中提取分离出一种天然活 性化合物芍药甙，经过药理试验发现芍药甙作为一种非嘌呤类腺苷受体一 1的选择性激动剂， 可以在制备预防和治疗中风和帕金森症等神经系统疾病药物中得到应用。
本发明采用白芍、 赤芍、 及牡丹的根为原料， 制备过程如下- 白芍、 赤芍、 或牡丹的根， 经乙醇回流提取， 得干浸膏， 浸膏加水充分溶解， 通过大孔树脂， 水洗脱至糖反应阴性， 加 2倍柱体积乙醇洗脱， 得浸膏， 经硅胶柱层析分离 得纯化合物芍药甙。
芍药甙化学结构如下：
Figure imgf000005_0001
它 的 分 子 式 为 ： C K Ou, 分 子 量 为 ' 480.48 化 学 名 为 β -D-Glucopyranoside,56-[(benzoyloxy)methyl]tdtrahydro-5-hydroxy-2-methyl-2,5-methano-lH-3,4
-dioxacyclobuta[cd]Pentalen-la(2H)-yl,[lar-cla α ,2 β ,3a α ,5 α ,5a α ,5b α ]]。
芍药甙的提取纯化方法参考文献（金继曙 都述虎 种明才 用大孔吸附树脂分离白芍总 甙， 中国中药杂志 1994年 01期 1994. VOL.19 NO. 1 ： 31 )。
本发明釆用上述方法提取获得的芍药甙进行了相关的药理药效试验。 一. 芍药甙的配体一受体竞争性结合试验：
釆用大鼠大脑皮层突触膜， 将大鼠断头后， 分离出大脑皮层， 随后在 1 : 15 ( W/V ) 的 蔗糖溶液中充分匀浆，离心，取上清离心。弃上清后，用 1 : 30 ( W/V)的 Tris-Hcl缓冲液 （50 mM， PH 7.4)重悬沉淀，匀浆洗涤后再离心， 洗涤过程重复 3次后，用 1 : 5 ( W/V )的 Tris-Hcl 缓冲液 (50 mM， PH 7.4)再次重悬沉淀， 分装保存于一 80°C， 并采用 Bradford法测定蛋白浓 度( (Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. ) , 上述操 作均在 4 °C进行。
用上述模型分别验证芍药甙与 和 A2aR 受体混合型激动剂 [3H]-乙基酰胺腺苷 ([3HJ-NECA) 及 受体选择性激动剂 [3H]-CCPA的竞争性结合。
实验证明芍药甙可竞争结合 [3H]-CCPA ， 同时对 [3H]-NECA 的竞争结合具有典型的双 位点竞争特性（ECSO!- ^nM, Ε。502=5.00μΜ)。 结果提示芍药甙同时结合 A!R和 A2AR受 体；并具有较高的选择性（EC502/ EC501=360倍），低浓度选择性结合 A,R，高浓度结合 A2AR。 二. 芍药甙预防和治疗中风的药效试验：
釆用可逆性大鼠中动脉栓塞法分别制成暂时性和永久性两种局灶脑缺血模型（Belayev L, Alonso OF, Busto R, Zhao W, Ginsberg MD. Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. Stroke, 1996; 27: 1616-22., Takano K, Tatlisumak T, Bergmann AG, Gibson DG 3rd, Fisher M. Reproducibility and reliability of middle cerebral artery occlusion using a silicone-coated suture (Koizumi) in rats. J Neurol Sci. 1997; 153: 8-11. ) ， 分别检验芍药甙对脑缺血大鼠神经缺损体征及对大脑损伤 体积的影响。 腹腔注射氯醛糖麻醉， 右股动脉插管监测血压， 保持 36.5〜37.5°C肛温， 颈部 切口， 游离颈外动脉， 电凝切断其分支动脉， 并在远端结扎， 取颈头部呈鼓锤状 4〜0号的单 丝尼龙线， 由颈外动脉经颈内动脉插入大脑前动脉以阻断大脑中动脉起始处， 同时也阻断了 大脑前动脉以阻断大脑中动脉起始处，同时也阻断了大脑前、后动脉及颈内动脉的侧支血流。 暂时性 MCAO模型组， 缺血 1.5hr后拔出栓线， 再灌注 22.5hr， 于缺血后 15分钟和 6小时， 分别 皮下注射生理盐水、不同剂量的芍药甙； 永久性 MCAO模型组， 不拔出栓线， 连续缺血 24hr， 于缺血后 15分钟和 6小时，分别皮下注射生理盐水、芍药甙、和 DPCPX +芍药甙，其中 DPCPX 在缺血前 15min预先给予。 .
根据 SyderffN3经缺损体征判别法进行综合等级评分 (Sydserff SG,Borelli AR,Green AR, Cross AJ.， Effect of NXY-059 on infarct volume after transient or permanent middle cerebral artery occlusion in the rat; studies on dose, plasma concentration and therapeutic time window. Br J Pharmacol. 2002;135(1):103-12.)。 根据前肢弯曲、 自发旋转、 及异侧搔挠刺激反应缺失的 程度， 分别评以 0— 2分 （0=正常， 2=严重） 。 此外， 肢体异侧旋转的扭矩也被作为一个单 独的指标， 评以 0— 2分 （0 =大扭矩， 2=小扭矩） 。 因此损伤评分值在 0— 8之间。
试验结果表明芍药甙在中剂量浓度（Smg.kg s.c. )下明显改善暂时性中动脉栓塞大鼠的 神经症状， 同时显著降低缺血引起的大脑皮层下核团及大脑皮层的损伤； 芍药甙剂量依赖性 勺减轻缺血引起的大脑皮层下核团及大脑皮层的损伤， 且此作用可被选择性 受体拮抗 剂 DPCPX所拮抗。 提示芍药甙可能通过激活 A,R受体， 发挥神经保护作用， 可用于中风的 临床治疗和预防。 三. 芍药甙在预防和治疗帕金森病 （PD) 的应用：
采用 N-甲基苯四氢吡啶（l-metliyl-4-phenyl-I,2,3,6-tetrahydro-pyridine, MPTP) 诱导的小 鼠 PD 模型，分别检测芍药甙对 PD模型小鼠爬杆能力及中脑黑质多巴胺神经元缺失的影响。 用 20-25g雄性 C57B1/6J小鼠，实验开始前测定按小鼠爬杆时间，以按爬杆时间随机分 5组， 每组 8只。 用低剂量和中剂量芍药甙分别连续 11天皮下注射给药， 于实验第 8天以 2小时 为间隔腹腔注射 （ip ) 4 次 N-甲基苯四氢吡啶 (l-methyl-4-phenyl-l,2,3,6-tetmhydro-pyridine, MPTP) 诱导小鼠 PD 模型。
通过爬杆法测定小鼠运动能力， 将小鼠头向下， 置于长 55cm， 直径为 8mm的垂直粗糙 的杆上， 记录小鼠从杆向下爬至四肢全部接触地面的时间 (Hamaue N, Minami M, Terado M, Hirafuji M, Endo T, Machida M, Hiroshige T, Ogata A, Tashiro K, Saito H, Parvez SH. Comparative study of the effects of isatin, an endogenous MAO- inhibitor, and selegiline on bradykinesia and dopamine levels in a rat model of Parkinson's disease induced by the Japanese encephalitis virus. Neurotoxicology. 2004 Jan;25(l-2):205-13.)。
试验结果表明芍药甙中剂量组 （5mg.kg— ^.c. ) 显著改善 MPTP 弓 1起的运动功能障碍， 对抗 MPTP诱发的多巴胺能神经元的缺失， 提示芍药甙可用于帕金森病的临床治疗和预防。 四 . 药甙对清醒大鼠血压的影响
以清醒大鼠为研究对象， 釆用尾套传感器， 将压力脉冲传至光电三极管， 转换为相应的 血压和心率值。 待大鼠恢复平静后， 记录其血压和心率， 作为基础值。 随后， 经尾静脉分别 "注射生理盐水、 不同剂量芍药甙和利血平 （reserpine)。
记录给药前 （O min)及给药后 （5min至 2hr)的血压和心率的变化。
试验结果显示不同剂量的芍药甙 （10, 40, 160mg kg— ', ί.ν. ) 均不影响大鼠血压和心率， 显然在其发挥治疗和预防中风及帕金森病等神经系统疾病的治疗剂量范围内，无显著的心血 管副作用。 附图说明- 图 1.1是分别用不同浓度的芍药甙、 CPA (选择性 A!R受体激动剂） 和 CV— 1808 (选 择性 A2AR受体激动剂） 竞争 [3H]-NECA (A^和 A2AR混合型激动剂） 在大鼠大脑皮层突 虫膜上的结合位点。 结果显示芍药甙剂量依赖性地竞争 [3H]-NECA 于大鼠大脑皮层突触膜 上的结合位点， 并具有双位点竞争特性 （EC50尸 13.9ηΜ， Ε。502=5.00μΜ)。
图 1.2是分别用不同浓度的芍药甙竞争 [3Η]- CCPA (选择性 受体激动剂） 在大鼠大 脑皮层突触膜上的结合位点。结果显示芍药甙剂量依赖性地竞争 [3H]-CCPA于大鼠大脑皮层 突触膜上的结合位点 （EC50=19.8nM)。
图 2.1 (a) 芍药甙 CS mgAg—1 ， S mg-kg "1 ) 对暂时性 MCACO大鼠神经症状的改善 (n=6, *p<0.05, **p<0.01 vs Sal) ; (b) 芍药甙 ( 2.5 mg-kg Λ ， 5 mg-kg ) 对暂时性 MCACO 大鼠皮层下核团及皮层的保护 （n=6, *p<0.05, **p<0.01 w Sal)。
图 2.2 芍药甙 G.Smg.kg-1 ， Smg-kg-1, 10mg-kg s.c.) 对暂时性 MCACO大鼠皮 层下核团及皮层的保护， DPCPX (0.25 mg-kg— 1 s.c.)阻断芍药甙的保护作用 （n=6, * p<0.05, ** p<0.01 vs Sal; # p<0.05 w芍药甙 10 mg-kg s.c.)。
图 3.1 芍药甙（PF) (2.5 mg-kg 1， 5 mg-kg s.c.)对 MPTP引起小鼠运动徐缓的影响。 图 3.2 芍药甙 （PF) (2.5 mg-kg"1, 5 mg-kg s.c.) 对 MPTP引起的多巴胺能神经元的 缺失及纹状体多巴胺神经纤维的减少的影响。
图 4.1 芍药甙 （10,40, 160mg kg—1, i.v.) 不影响清醒大鼠血压的影响 (n=6; *p<0.05,
**p<0.01)。
图 4.2· 芍药甙 （10,40, 160mg kg—1, i.v.) 不影响清醒大鼠心率的影响 (n=6; *p<0.05， **ρ<0·01)。
从上述实验可以看出， 本发明所提供的化合物芍药甙竞争结合 A!R受体， 并具有高选 择性。 就其化学结构而言， 区别于传统的 A!R受体激动剂， 为一种新型的非嘌吟类 A!R受 体激动剂。通过相关动物实验确证， 芍药甙具有治疗和预防中风及帕金森病等神经系统疾病 的作用； 进一步的研究证明， 其心血管副作用小， 具有临床应用价值， 可以在中风及帕金森 病等神经系统疾病的治疗和预防中得以应用。 具体实施方式
实施例 1： 芍药甙是一种非嘌呤类 受体的选择性激动剂
1.1. 大鼠大脑皮层突触膜制备
将大鼠断头后， 立即分离出大脑皮层， 置于冰冷的生理盐水。 随后， 在 1: 15 (W/V) 的蔗糖溶液 (0.32mol/L) 中充分匀浆， 离心 (l,000xg, 10分钟）， 取上清离心 (30,000xg, 30分钟）。 弃上清后， 用 1: 30 (W/V) 的 Tris-Hcl缓冲液 （50mM， PH 7.4)重悬沉淀， 匀浆 洗涤后再离心 （48,000xg， 10分钟）。 此洗涤过程重复 3次后， 用 1: 5 (W/V) 的 Tris-Hcl 缓冲液 (50 mM， PH 7.4)再次重悬沉淀， 分装保存于一 80°C， 并采用 Bradford法测定蛋白浓 度上述操作均在 4°C进行。
1.2. 配体一受体竞争结合 '
选用 和 A2aR受体混合型激动剂 [3H]- NECA (NEN; 15.3 Ci/mmol=0.57 TBq/mmol)以 及 AiR受体选择性激动剂 [3H]-CCPA (NEN; 30 Ci/mmol=2.9 PBq/ mol), 检验芍药甙与 A!R和 A2aR受体的结合。 反应体系为 200μΙ^， 其中预试药物 5μΙ^， 分别为芍药甙、 选择性激动剂 (CPA)和 A2aR受体选择性激动剂 (CV-1808) ， 终浓度均从 0, 1(Γ1ΰ到 10_4 moI /L; 大脑皮 层突触膜悬液 20μΙ^ 25nmol/L的 [3H]-NECA 或 0.2nmol/L的 [3H]-CCPA 20μ ^,分析缓冲液（50 mmol/L Tris-HCl, 1 mmol EDTA, 0.5% BSA, pH 7.4) 155μί。 样品于 25°C孵育 3小时后， 釆用 细胞收集器抽吸反应液， 使之透过 GF/C滤膜， 以中止反应， 再用过滤缓冲液 (50 mmol/ L Tiis-HCl, 0.1% BSA, pH 7.4)冲洗滤膜 3次， 随后于 40°C干燥 1小时。 再将滤膜装入密封袋， 加 入 5mL MicroScint 20， 压勾后加以封条， 进行放射活性计数 （Micro β, PerkinElmer)。 统计结 果按双位点竞争模型进行拟合。
1.3. 试验结果
芍药甙可竞争结合 [3H]-CCPA ， 其 EC50=19.8nM; 同时对 [3H]-NECA的竞争结合具有 典型的双位点竞争特性 （ECSO^ ^nM,
Figure imgf000009_0001
结果提示芍药甙同时结合 和 A2AR受体； 并具有较高的选择性 （EC502/ ECSO^SSO倍）， 低浓度选择性结合 A,R， 高 浓度结合 A2AR。 实施例 2: 芍药甙在预防和治疗中风中的应用
2.1 . 动物模型及给药方法
釆用可逆性大鼠中动脉栓塞法分别制成暂时性和永久性两种局灶脑缺血模型， 腹腔注 射氯醛糖 350mg · leg 麻醉， 右股动脉插管监测血压， 保持 36.5〜37.5°C肛温， 颈部切口， 游 离颈外动脉， 电凝切断其分支动脉，并在远端结扎，取颈头部呈鼓锤状 4〜0号的单丝尼龙线， 由颈外动脉经颈内动脉插入大脑前动脉以阻断大脑中动脉起始处，同时也阻断了大脑前动脉 以阻断大脑中动脉起始处， 同时也阻断了大脑前、 后动脉及颈内动脉的侧支血流。 暂时性 MCAO模型组， 缺血 1.5hr后拔出栓线， 再灌注 22.5hr, 于缺血后 15分钟和 6小时， 分别皮下注 射生理盐水 （ 2 mL' kg— 、 芍药甙 （2.5 mg'kg— 1 ， S mg-kg "1 ); 永久性 MCAO模型组， 不 拔出栓线， 连续缺血 24hr， 于缺血后 15分钟和 6小时， 分别皮下注射生理盐水（ 2 fnL. kg— ^、 芍药甙 (2.5 mg-kg ， 5 mg-kg ， 10 mg-kg )、和 DPCPX (2.5 mg-kg 1 ) +芍药甙 ( 10 mg-kg "' ), 其中 DPCPX在缺血前 15min预先给予。
2.2. 指标测定
2.2.1. 神经缺损评价标准
' 根据 Syderff神经缺损体征判别法进行综合等级评分 根据前肢弯曲、 自发旋转、及异侧 搔挠刺激反应缺失的程度， 分别评以 0— 2分 （0=正常， 2=严重） 。 此外， 肢体异侧旋转 的扭矩也被作为一个单独的指标， 评以 0— 2分 （0=大扭矩， 2=小扭矩） 。 因此损伤评分 值在 0— 8之间。
2.2.2. 脑缺血的功能性酶组织化学染色—— TTC染色
将大鼠断头后， 立即取出大脑， 一 20°C储存 15min， 连续冠状切片 （片厚 2mm)。 脑片 置于 1 %TTC (PH7.2) 染色， 37°C孵育 lOmin, 倾去染色液， 固定于 4%甲醛溶液。 数码相机 拍照， 对脑切片的皮层及皮层下损伤区进行象素点扫描（Adobe ImageReady 7.0)， 计算出皮 层损伤区， 皮层下损伤区及总损伤区所占的面积百分比， 进一步参照全脑体积， 推算皮层损 伤区， 皮层下损伤区及总损伤区的体积。
2.3. 试验结果
实验结果显示芍药甙（S mg g c.) 明显改善暂时性 MCAO大鼠的神经症状， 同时显 著降低缺血引起的大脑皮层下核团及大脑皮层的损伤； 芍药甙 （2.5, 5, 10 m kg S.C. ) 剂量 衣赖性的减轻缺血引起的大脑皮层下核团及大脑皮层的损伤， 且此作用可被选择性 A!R受 体拮抗剂 DPCPX所拮抗。 提示芍药甙可能通过激活 受体， 发挥神经保护作用， 可用于 中风及其后遗症中的临床治疗和预防。 实施例 3 : 芍药甙在预防和治疗帕金森病的应用
3.1 . 动物模型及给药方法
20-25g雄性 C57B1/6J小鼠， 实验开始前测定按小鼠爬杆时间， 以按爬杆时间随机分 5组， 每组 8只。 芍药甙 （2.5mg kg—1 , 5mg kg 1 ) 连续 11天皮下注射给药， 于实验第 8天以 小 时为间隔腹腔注射（ip) 4次 N-甲基苯四氢吡啶 (l-methyl-4-phenyl-l,2,3,6-tetrahydro-pyridine, MPTP) 诱导小鼠 PD 模型。 给药方案如下：
Figure imgf000010_0001
3.2. 指标测定
3.2.1 爬杆法测定小鼠运动能力
将小鼠头向下， 置于长 55cm， 直径为 8mm的垂直粗糙的杆上， 记录小鼠从杆向下爬至 四肢全部接触地面的时间
3.2.2中脑黑质多巴胺神经元免疫荧光染色——酪氨酸羟化酶 （Tyrosine hydroxylase, !!!) 染 色
心脏灌注 4%多聚甲醛进行前固定， 随后取出大脑， 4%甲醛溶液后固定一周。于一 20'C 连续冠状切片 （片厚 30 m)， 以酪氨酸羟化酶（Tyrosine hydroxylase,™)作为多巴胺神经 元的特异性标记。 一抗为单克隆小鼠 TH抗体 ( 1 :1000, Sigma), 二抗为 Alexa fluor 488 荧 光标记山羊抗小鼠（ 1 :1000， Molecular Probes)。 荧光显微镜（ Olympus DP70 )拍照， 并对黑 质 TH阳性细胞计数。
3.3. 试验结果
芍药甙 （S mgJcg -i s.c. ) 明对抗 MPTP引起的多巴胺能神经元的缺失及运动功能障碍， 提示芍药甙可用于帕金森病的临床治疗和预防。
,,实施例 4: 芍药甙不影响清醒大鼠血压
4.1 . 动物模型及给药方法
以清醒大鼠为研究对象， 釆用尾套传感器， 将压力脉冲传至光电三极管， 转换为相应的 血压和心率值。 待大鼠恢复平静后， 记录其血压和心率， 作为基础值。 随后， 经尾静脉分别 注射生理盐水、 芍药甙 （10， 40， 160mg kg"' ) 和利血平 （reserpine)。
4.2. 指标测定
记录给药前 （0 min)及给药后 （5min至 2hr)的血压和心率的变化。
4.3. 试验结果
芍药甙 （10, 40, 160mg kg—1, i.v. ) 不影响大鼠血压和心率， 显然在其发挥治疗和预防中 .风及帕金森病等神经系统疾病的治疗剂量范围内， 无显著的心血管副作用。

Claims

权 利 要 求
1、 一种从毛莨科植物白芍、 赤芍、 牡丹根中提取分离结构如下的芍药甙的医药用途
Figure imgf000012_0001
其特征在于芍药甙作为一种非嘌呤类 受体选择性激动剂在药物中的用途。
2. 根据权利要求 1所述的芍药甙的医药用途， 其特征在于在制备治疗和预防神经系统 疾病的药物中的应用。
3. 根据权利要求 2所述的芍药甙的医药用途， 其特征在于在制备治疗和预防中风和中 风后遗症的药物中的应用。
4. 根据权利要求 2所述的芍药甙的医药用途， 其特征在于在制备治疗和预防帕金森症 及其他相关的神经退行性病变的药物中的应用。
PCT/CN2005/000929 2004-07-02 2005-06-27 Utilisation medicale de paeoniflorine Ceased WO2006002589A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US11/631,123 US20080275226A1 (en) 2004-07-02 2005-06-27 Medical Use of Paeoniflorin
EP05757963A EP1767210A4 (en) 2004-07-02 2005-06-27 MEDICAL USE OF PAEONIFLORIN
JP2007519597A JP2008505128A (ja) 2004-07-02 2005-06-27 ペオニフロリンの医薬用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200410025717.8 2004-07-02
CNB2004100257178A CN100348200C (zh) 2004-07-02 2004-07-02 芍药甙的医药用途

Publications (1)

Publication Number Publication Date
WO2006002589A1 true WO2006002589A1 (fr) 2006-01-12

Family

ID=35782466

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/000929 Ceased WO2006002589A1 (fr) 2004-07-02 2005-06-27 Utilisation medicale de paeoniflorine

Country Status (5)

Country Link
US (1) US20080275226A1 (zh)
EP (1) EP1767210A4 (zh)
JP (1) JP2008505128A (zh)
CN (1) CN100348200C (zh)
WO (1) WO2006002589A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101338172B1 (ko) 2010-08-17 2013-12-09 한국화학연구원 작약 종자 추출물, 이의 분획물 또는 이로부터 분리한 화합물을 유효성분으로 함유하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물
CN102731522B (zh) * 2011-04-01 2014-10-22 中国科学院上海药物研究所 一种具有环氧化酶-2异常表达抑制活性的芍药苷类化合物，其制备方法及其用途
CN106565802B (zh) * 2016-09-26 2020-05-15 中国人民解放军北部战区总医院 白芍醇提物、4-o-没食子酰基白芍苷及其制备和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0772135B2 (ja) * 1986-07-23 1995-08-02 株式会社ツムラ 抗酸化剤
JP2729340B2 (ja) * 1992-03-02 1998-03-18 キッセイ薬品工業株式会社 痴呆症治療剤
CN1491661A (zh) * 2003-08-15 2004-04-28 凯 曹 一种芍药总甙制剂及其用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CLIN-EXP-PHARMACOL-PHYSIOL., vol. 26, no. 10, 1999, pages 815 - 816 *
DATABASE MEDLINE [online] CHENG J.-T. ET AL: "Paeoniflorin reverses guanethidine-Induced hypotension via activation of central adenosine A1 receptors In Wistar rats", XP008116244, Database accession no. (NLM10549407) *
JUN Y. ET AL: "Protective Effect of Total Paeony Glycoside Against Cerebral Ischemia-Reperfusion Injury In Mice", JOURNAL OF CHINESE MEDICINAL MATERIALS, vol. 23, no. 2, February 2002 (2002-02-01), pages 95 - 97 *
See also references of EP1767210A4 *
SIPING C. ET AL: "Determination of paeoniflorin which Is In Jianxing granule by HOLC", CHENISE TRADITIONAL PATENT MEDICINE, vol. 25, no. 7, July 2003 (2003-07-01), pages 596 *

Also Published As

Publication number Publication date
EP1767210A1 (en) 2007-03-28
CN1714797A (zh) 2006-01-04
JP2008505128A (ja) 2008-02-21
US20080275226A1 (en) 2008-11-06
CN100348200C (zh) 2007-11-14
EP1767210A4 (en) 2010-09-08

Similar Documents

Publication Publication Date Title
JP7706025B2 (ja) 多嚢胞性卵巣症候群の治療及び／又は予防するための製剤およびその製造方法
CN108175770A (zh) 一种通过作用腺苷受体来治疗肾衰竭的试剂
AU2009258384B2 (en) Extract of Coptidis rhizoma and use thereof in treating respiratory disease
CN1899367B (zh) 筋骨草提取物、其制备方法、用途和复方制剂
WO2006002589A1 (fr) Utilisation medicale de paeoniflorine
CN1692914B (zh) 知母皂苷bⅱ在制备用于防治脑卒中药物或产品中的用途
WO2006026926A1 (en) A medicinal composition for treating hepatic fibrosis and its preparation
CN103655544B (zh) 棕矢车菊素在制备预防或治疗慢性肝损伤和肝纤维化的药物中的应用
CN101843670B (zh) 一种清热解毒,利尿通淋的中药组合物及制备方法
CN101879151A (zh) 大黄素在制备p2x3介导神经病理痛/神经系统疾病药物中的应用
CN100584358C (zh) 一种治疗心脑血管病的中药组合物及其制剂和制备方法
CN117085095B (zh) 一种用于治疗失眠症的中药组合物及其应用
CN116098967B (zh) 一种平潜补利汤及其有效成分和应用
CN119112870B (zh) 治疗SARS-CoV-2感染致血糖升高的组合物及制备方法、应用和药物
CN100512827C (zh) 一种白花前胡总香豆素提取物中药片及其制备方法及应用
CN119097633A (zh) 一种中药组合物及其制备方法和应用
CN119499258A (zh) 人参二醇在制备预防和/或治疗帕金森病药物中的应用
CN119656254A (zh) 扁担藤水提取物在制备预防或治疗高尿酸血症及其相关的代谢性病症的药物中的应用
CN121401338A (zh) 一种痛泻要方调控blp+巨噬细胞亚群治疗ibs-d的应用
CN117771342A (zh) 一种治疗溃疡性结肠炎的中药组合物、制备方法和应用
CN105232729A (zh) 一种桑当总黄酮的制备方法及其应用
CN1330333C (zh) 一种中药组合物及其相关制剂
CN117398433A (zh) 一种丹参保心茶在制备防治认知功能障碍药物中的新应用
CN111821347A (zh) 一种延丹胶囊在制备治疗心肌缺血药物中的应用
CN115192645A (zh) 一种中药组合物及其制备方法和应用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

WWE Wipo information: entry into national phase

Ref document number: 2007519597

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2005757963

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2005757963

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 11631123

Country of ref document: US