WO2018072348A1 - 骨形成蛋白-9在制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物中的用途 - Google Patents

骨形成蛋白-9在制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物中的用途 Download PDF

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WO2018072348A1
WO2018072348A1 PCT/CN2017/070610 CN2017070610W WO2018072348A1 WO 2018072348 A1 WO2018072348 A1 WO 2018072348A1 CN 2017070610 W CN2017070610 W CN 2017070610W WO 2018072348 A1 WO2018072348 A1 WO 2018072348A1
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bone morphogenetic
morphogenetic protein
bmp
mice
thiamine
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PCT/CN2017/070610
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French (fr)
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钟春玖
王子高
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复旦大学附属中山医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose

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  • the present invention relates to bone morphogenetic protein-9, and in particular to the use of bone morphogenetic protein-9 as a pharmaceutical composition for the treatment and/or prevention of diseases associated with degeneration of the cholinergic system.
  • AD Alzheimer's disease
  • the main clinical manifestation is progressive cognitive decline, which causes serious harm to individuals, families and society. So far, the pathogenesis of AD is still not fully understood, and various hypotheses have been formed, such as the A ⁇ hypothesis, the tau protein hypothesis, and the cholinergic hypothesis.
  • a ⁇ hypothesis the tau protein hypothesis
  • tau protein hypothesis the tau protein hypothesis
  • Clinically widely used cholinesterase inhibitors do not prevent the progression of AD, so it is necessary to rethink the pathogenesis of AD.
  • thiamine deficiency can cause brain glucose metabolism disorder in the brain by inducing a decrease in enzyme activity involved in glucose metabolism in the brain, and ultimately promote the development of AD.
  • the thiamine-deficient mouse model can better mimic the pathophysiological characteristics of human AD brain glucose metabolism disorder, cholinergic neurodegeneration and cognitive impairment. Therefore, the present invention conducts related research by simulating human AD with a thiamine deficiency model.
  • BFCS basal forebrain Cholinergic system
  • cholinergic drugs have been shown to improve cognitive impairment in patients with mild-to-moderate AD, but it is only a symptomatic treatment, and can not reverse the degeneration process of BFCS, so find BFCS degeneration in AD patients. The reasons for contributing to the development of more effective drugs.
  • ceramide deficiency-induced brain glucose metabolism disorder may be an important cause of AD
  • thiamine deficiency-induced brain glucose metabolism disorder may also be the root cause of BFCS degeneration in AD patients.
  • it is unclear how thiamine deficiency causes the above-mentioned degeneration of BFCS.
  • Bone morphogenetic protein-9 also known as growth differentiation factor 2 (GDF2), is one of the members of the transforming growth factor- ⁇ superfamily. BMP-9 mainly binds to type I and type II receptors on the cell membrane, which in turn activates smad1/5/8 in the cytoplasm, and phosphorylated smad1/5/8 forms a complex with smad4 and translocates into the nucleus. Regulate the transcription of target genes and exert corresponding biological effects.
  • bone morphogenetic protein-9 as a treatment for diseases associated with degeneration of the cholinergic system has not yet been reported.
  • bone morphogenetic protein-9 which can be used as a pharmaceutical composition for the treatment and/or prevention of diseases associated with degeneration of the cholinergic system
  • bone morphogenetic protein- 9 can significantly increase the number of cholinergic neurons and increase the content and activity of acetylcholine transferase.
  • the present invention provides the use of bone morphogenetic protein-9 as a pharmaceutical composition for the treatment and/or prevention of a disease associated with degeneration of the cholinergic system.
  • the cholinergic system is the central cholinergic nervous system.
  • the diseases associated with degeneration of the cholinergic system include Alzheimer's disease.
  • the bone morphogenetic protein-9 is any one or two of human bone morphogenetic protein-9, animal bone morphogenetic protein-9 or genetically engineered bone morphogenetic protein-9.
  • the bone morphogenetic protein-9 has an effect of increasing the number of cholinergic neurons.
  • the bone morphogenetic protein-9 has an effect of increasing acetylcholine transferase and increasing the activity of acetylcholine transferase.
  • the bone morphogenetic protein-9 has an effect of increasing the release of acetylcholine.
  • the pharmaceutical composition consists of the bone morphogenetic protein-9 and a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutical composition is any one of a tablet, a capsule, a pill, a suppository, an oral solution, a suspension or an injection.
  • the bone morphogenetic protein-9 is administered nasally.
  • the use of the bone morphogenetic protein-9 provided by the present invention as a pharmaceutical composition for treating and/or preventing a disease associated with degeneration of the cholinergic system has the following advantages:
  • BMP-9 can promote the differentiation of medial septal nucleus neurons into cholinergic neurons, and promote the synthesis and secretion of acetylcholine in a dose- and time-dependent manner; BMP-9 can make the inner septum nucleus ChAT (acetylcholine transferase)-positive bile Alkali-energy neuron counts increased; BMP-9 can significantly improve the memory and cognitive ability of dementia mice; BMP-9 can bypass the blood-brain barrier by nasal administration, has brain-targeting, and avoids the liver's first Over-effect, and the nasal administration is convenient, non-invasive, and easy to promote.
  • ChAT acetylcholine transferase
  • Figure 1 is a road map of a mouse searching platform for the Morris water maze test of Experimental Example 2 of the present invention.
  • FIG. 2 is a line drawing of a route length, a swimming speed, and an escape latency and time of a mouse detected by the Morris water maze of Experimental Example 2 of the present invention (a is a route length map, b is a swimming speed map, and c is an escape latency map).
  • Figure 3 is a bar graph of the number of times the mouse passes through the platform position, the time from the incubation period to the platform, and the time of occurrence in the target quadrant detected by the Morris water maze of Experimental Example 2 of the present invention (a is the number of times of crossing the platform position, b is the The time chart of the incubation period to the platform, c is the time chart appearing in the target quadrant)
  • Figure 4 is a graph showing the fluorescence of immunofluorescence staining and the number of cholinergic neurons of medial septal nucleus in Experimental Example 3 of the present invention (a is a fluorescence map, b is a graph of the number of cholinergic neurons in the medial septal nucleus, n is the number of mice) .
  • Fig. 5 is a gel strip diagram and a ChAT content map of the experimental Bundle (Western Blot) assay of Experimental Example 4 of the present invention (a is a gel strip diagram, b is a ChAT content map, and n is the number of mice).
  • Fig. 6 is a bar graph of ChAT activity (n is the number of mice) measured in Experimental Example 5 of the present invention.
  • Figure 7 is a bar graph of the content of Ach (acetylcholine) measured in Experimental Example 6 of the present invention (n is a mouse) number).
  • the present invention is useful for providing the use of bone morphogenetic protein-9 as a pharmaceutical composition for the treatment and/or prevention of diseases associated with degeneration of the cholinergic system, mainly in the central cholinergic nervous system, especially for the treatment and prevention
  • the bone morphogenetic protein-9 is either or both of human bone morphogenetic protein-9, animal bone morphogenetic protein-9 or genetically engineered bone morphogenetic protein-9. Nasal route of administration.
  • the pharmaceutical composition is composed of the bone morphogenetic protein-9 and a pharmaceutically acceptable carrier or adjuvant, and may be any of a tablet, a capsule, a pill, a suppository, a soft capsule, an oral liquid, a suspension or an injection.
  • a pharmaceutically acceptable carrier or adjuvant may be any of a tablet, a capsule, a pill, a suppository, a soft capsule, an oral liquid, a suspension or an injection.
  • bone morphogenetic protein-9 has the function of increasing the number of cholinergic neurons, can increase acetylcholine transferase, and increase the activity of acetylcholine transferase, and can also increase the release of acetylcholine.
  • mice Forty male C57 mice (inbred mice) of 12 weeks old were randomly divided into control group (Con), thiamine deficiency group (PTD), BMP-9 group, thiamine deficiency + BMP-9 group. (PTD+BMP-9), 10 mice per group, all animals were housed in SPF (no pathogen-specific) room at room temperature and 25 °C, free to ingest and drink.
  • Con control group
  • PTD thiamine deficiency group
  • BMP-9 thiamine deficiency + BMP-9
  • SPF no pathogen-specific
  • the control group was given normal diet + intraperitoneal injection of normal saline 5 ⁇ L / (gd); thiamine deficiency group was given thiamine deficiency + intraperitoneal injection of pyridine thiamine 0.5 ⁇ g / (gd); BMP group was given normal diet + intraperitoneal injection of normal saline 5 ⁇ L/(gd)+ nasal drops 1ng/(gd) BMP-9; thiamine deficiency+BMP-9 group given thiamine deficiency + intraperitoneal injection of pyrithione 0.5 ⁇ g/(gd)+ nasal drops 1 ng/(gd) of recombinant human BMP-9, wherein recombinant human BMP-9 was composed of BMP-9 powder (3209-BP-010, It was formulated with 4 mM HCl containing 0.1% BSA (bovine serum albumin) to prepare a recombinant human BMP-9 at a concentration of 20 ⁇ g/mL.
  • mice were returned to the cage, they were administered nasally once a day, and the other side of the nasal cavity was replaced every other day.
  • the administration method was the same as before, for a total of 10 days.
  • Training stage put the mouse head into the pool wall, record the time when the mouse reaches the underwater platform, the distance to reach the platform and the average swimming speed. If the mouse still does not find the platform within 60s, then The mice were guided to the platform, and the mice were removed after staying on the platform for 20 s. If the mice reached the platform within 60 s, they were allowed to stay on the platform for 15 s, and then they were taken out, and each mouse was trained 8 times a day. The training interval is 30 minutes and the training is 5 days.
  • mice look for platforms can be roughly divided into four types: edge, random, trend, and linear.
  • Different search methods represent mice searching for platforms with different strategies.
  • the search strategy of normal animals shows the change pattern of “edge ⁇ random ⁇ trend ⁇ linear” with the increase of training times, and the search strategy of dementia animals Abnormal changes will occur, as shown in Figure 1, the search route of the control mice is random; the search route of the mice in the thiamine-deficient group is marginal, and the search route is disordered; the search route of the BMP-9 mice It is simpler and presents a trend; the search route of the thiamine deficiency + BMP-9 group presents a random-to-trend change, indicating that the mice in the thiamine-deficient group have poor learning ability.
  • the distance from the mouse to the destination platform can be seen as the shortest route distance between the BMP-9 mice, followed by the control group and the thiamine deficiency + BMP-9 group, thiamine
  • the route of the lack of mice was the longest and the platform position was not found, indicating that the mice in the thiamine-deficient group had the worst cognitive function, while the thiamine deficiency + BMP-9 group was comparable to the control group, indicating that BMP-9 was prevented.
  • the lack of thiamine affects cognitive function.
  • the swimming speeds of the control group, the thiamine deficiency group, the BMP-9 group, and the thiamine deficiency + BMP-9 group were basically the same, and the mice were excluded.
  • mice in the thiamine-deficient group had the longest escape latency after training for 5 days, and the time to escape latency was significantly shortened in the thiamine-deficient + BMP-9 group.
  • the control group and BMP- The shortest time to escape latency was observed in the 9 groups of mice, indicating that the mice with thiamine deficiency had the worst learning and memory ability, further indicating that BMP-9 can prevent the damage of cognitive function caused by thiamine deficiency.
  • the data of the control group, the thiamine deficiency group, the BMP-9 group, and the thiamine deficiency + BMP-9 group in a, b, and c of Fig. 3 are the average values of each group of mice.
  • the control mice passed the platform twice, the thiamine-deficient mice never passed through the platform, and the BMP-9 and thiamine-deficient + BMP-9 mice passed through the platform. The number of times was the same, both passed through the platform, but the swimming route was close to the platform, indicating that the mice in the thiamine-deficient group had poor memory.
  • the control, BMP-9, and thiamine-deficient + BMP-9 mice ranged from latency to platform for 20 s, while thiamine-deficient mice from latency to platform The time was 50-60 s, and it can be seen that the mice in the thiamine-deficient group took longer to find the platform.
  • the probability of the control group and the BMP-9 group appearing in the target quadrant is close to 50%, and the probability of the thiamine-deficient group appearing in the target quadrant is about 20%, thiamine deficiency +
  • the probability of BMP-9 mice appearing in the target quadrant was 40%. It can be seen that BMP-9 can significantly improve the memory of thiamine-deficient mice.
  • the thoracic was intubated to the ascending aorta, first perfused with 60 mL of normal saline, and then perfused with 4% paraformaldehyde 100 mL.
  • the brain was decapitated and placed in 4% paraformaldehyde and fixed overnight. Placed in 20% and 30% sucrose solution until the bottom of the tissue, OCT (Optimal Cutting Temperature Compound, a water-soluble mixture of polyethylene glycol and polyvinyl alcohol) was embedded and crowned on a cryostat.
  • OCT Optimal Cutting Temperature Compound, a water-soluble mixture of polyethylene glycol and polyvinyl alcohol
  • the brain slices were rinsed with 0.01 M PBS (phosphate buffered saline solution) and then diafiltered with 0.3% Triton-X100 (polyethylene glycol octylphenyl ether) for 30 min, and 5% normal sputum serum was blocked for 1 h.
  • PBS phosphate buffered saline solution
  • Triton-X100 polyethylene glycol octylphenyl ether
  • the amount of fluorescence in the BMP-9 group was slightly higher than that in the control group, and the amount of fluorescence in the thiamine-deficient + BMP-9 group was significantly smaller than that in the control group and the BMP-9 group.
  • the thiamine-deficient group had the least amount of fluorescence.
  • the order of the number of medial septal nucleus cholinergic neurons in mouse brain tissue is BMP-9 group > control Group > thiamine deficiency + BMP-9 group > thiamine deficiency group, indicating that BMP-9 can increase the number of medial septal nucleus cholinergic neurons and improve the number of cholinergic neurons caused by thiamine deficiency .
  • mice After the mice were anesthetized, the brain was quickly decapitated, 10 mg of basal forebrain tissue was taken, total protein was extracted, total protein content was determined, and 20 ⁇ g of protein was taken, and 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) was used. Glue electrophoresis) Separation gel and 5% concentrated gel 100V electrophoresis for 90min, 300mA transfer film for 60min.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel
  • the amount of ChAT in the control and BMP-9 groups is basically the same, thiamine deficiency + The BMP-9 group was slightly lower, while the amount of ChAT in the thiamine-deficient group was significantly lower, further confirming that the expression level of ChAT in the brain tissue of the thiamine-deficient group was significantly decreased.
  • mice were anesthetized, the brain was quickly decapitated, 10 mg of hippocampus tissue was taken, rinsed with pre-cooled PBS, and 0.5% Triton X-100 and 0.1 mg/mL PMSF (phenylmethylsulfonyl fluoride) were added.
  • PMSF phenylmethylsulfonyl fluoride
  • the standard curve was prepared using the standard in the ChAT Active Kit.
  • the initial concentration of the standard was 100 U, and the dilution ratios were 1, 2, 4, 8, 16, and 32 times, respectively.
  • the concentrations were 100 U, 50 U, and 25 U.
  • the OD value of the sample calculates the ChAT activity of the sample.
  • the activity of ChAT in the brain tissue of BMP-9 mice is 15-20 U/mg protein, control group and thiamine.
  • the activity of ChAT in brain tissue of mice lacking +BMP-9 group was 10U/mg protein, and the activity of ChAT in brain tissue of mice exposed to thiamine was 5U/mg protein, indicating that BMP-9 can significantly increase the activity of ChAT. And can significantly increase the activity of ChAT in brain tissue of thiamine-deficient mice.
  • the standard curve was prepared using the standard in the Ach kit.
  • the initial concentration of the standard was 500 pmol, and the dilution ratio was 1,1.25, 1.33, 2.5, and 5 times, respectively.
  • the concentrations were 500 pmol, 400 pmol, 300 pmol, and 200 pmol.
  • the present invention is useful for providing bone morphogenetic protein-9 as a pharmaceutical composition for treating and/or preventing diseases associated with degeneration of the cholinergic system, and BMP-9 can be significantly increased by nasal administration.
  • the number of cholinergic neurons in the medial septal nucleus, the expression of acetylcholine transferase in the brain, the activity of acetylcholine transferase, and the release of acetylcholine and can significantly improve the cognitive and memory ability of cholinergic neurons in degenerative mice, fully It was confirmed that BMP-9 administration can significantly reverse the BFCS degeneration induced by thiamine deficiency, and can be used as a drug for treating Alzheimer's disease.

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Abstract

本发明公开了骨形成蛋白-9在制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物中的用途。该胆碱能系统为中枢胆碱能神经系统，上述的与胆碱能系统退变相关疾病包括阿尔茨海默病。该骨形成蛋白-9经鼻给药。

Description

[根据细则37.2由ISA制定的发明名称]　骨形成蛋白-9在制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物中的用途 技术领域
本发明涉及骨形成蛋白-9，具体涉及骨形成蛋白-9作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是当今世界最常见的神经系统退行性疾病，主要临床表现为进行性认知功能减退，给个人、家庭和社会产生严重的危害。迄今为止，AD的发病机理仍然不完全清楚，目前已形成了多种假说，如Aβ假说、tau蛋白假说、胆碱能假说等。然而，目前基于Aβ假说及tau蛋白假说的所有临床试验均以失败而告终，临床广泛使用的胆碱酯酶抑制剂并不能阻止AD病情的进展，所以必须对AD的发病机制进行重新思考。
越来越多的研究证实AD患者脑内葡萄糖与能量代谢显著降低，且明显早于临床症候的出现及特征性病理损害的形成。进一步研究发现，AD患者脑内参与脑细胞糖和能量代谢的α-酮戊二酸脱氢酶、丙酮酸脱氢酶和转酮醇酶活性显著下降，而这三种酶均以二磷酸硫胺素作为辅酶。研究表明，AD患者血浆中硫胺素活性成分二磷酸硫胺素含量显著低于认知功能正常的人群。动物实验研究发现，硫胺素缺乏导致的病理学损害特征与AD惊人地相似：如脑区选择性神经元丢失、海马及其邻近脑区tau蛋白异常磷酸化、Aβ分泌增多和异常沉积等。因此，硫胺素缺乏能够通过诱导参与脑内糖代谢的酶活性下降而导致脑内脑糖代谢障碍，最终促进AD的发生发展。硫胺素缺乏小鼠模型能较好地模拟人类AD脑葡萄糖代谢障碍、胆碱能神经退变和认知功能损害的病理生理特点。因此，本发明以硫胺素缺乏模型模拟人类AD开展有关研究。
大量研究表明AD患者存在显著的基底前脑胆碱能系统(basal forebrain  cholinergic system，BFCS)退变，主要表现为胆碱能神经元的丢失、乙酰胆碱转移酶及乙酰胆碱酯酶活性下降、乙酰胆碱受体活性降低及含量减少、乙酰胆碱释放减少等。目前，拟胆碱能药物虽已被证实可改善轻-中度AD患者的认知功能损害，但它仅为一种对症治疗，并不能逆转BFCS的退变过程，因此寻找AD患者BFCS退变的原因有助于更有效的药物的开发。考虑到硫胺素缺乏诱导的脑内糖代谢障碍可能是导致AD发病的重要原因，硫胺素缺乏诱导的脑内糖代谢障碍也可能是AD患者BFCS发生退变的根本原因。研究工作发现硫胺素缺乏可显著减少BFCS中内侧隔核区域中胆碱能神经元的数目，抑制乙酰胆碱转移酶的活性，减少乙酰胆碱的释放，最终诱导认知功能损害。然而，硫胺素缺乏是如何导致BFCS发生上述退变的原因目前尚不明了。
骨形成蛋白-9(bone morphogenetic protein-9,BMP-9)又名生长分化因子2(growth differentiation factor 2,GDF2)，是转化生长因子-β超家族成员之一。BMP-9主要与细胞膜上的I型及II型受体相结合，继而激活胞浆中的smad1/5/8，磷酸化的smad1/5/8与smad4形成复合体转位至细胞核内，进而调控靶基因的转录，发挥相应的生物学效应。
但目前尚未报道骨形成蛋白-9作为治疗与胆碱能系统退变相关疾病的应用。
发明的公开
本发明的目的是提供一种骨形成蛋白-9的新用途，骨形成蛋白-9能够作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途，骨形成蛋白-9能够显著提高胆碱能神经元的数目，增加乙酰胆碱转移酶的含量和活性。
为了达到上述目的，本发明提供了骨形成蛋白-9作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途。
所述的胆碱能系统为中枢胆碱能神经系统。
所述的与胆碱能系统退变相关疾病包括阿尔茨海默病。
所述的骨形成蛋白-9为人源骨形成蛋白-9、动物骨形成蛋白-9或基因工程表达的骨形成蛋白-9中的任意一种或两种。
所述的骨形成蛋白-9具有增加胆碱能神经元数目的作用。
所述的骨形成蛋白-9具有增加乙酰胆碱转移酶，并提高乙酰胆碱转移酶的活性的作用。
所述的骨形成蛋白-9具有增加乙酰胆碱释放的作用。
所述的药物组合物由所述的骨形成蛋白-9和药学上可接受的载体或辅料组成。
所述的药物组合物为片剂、胶囊、丸剂、栓剂、口服液、混悬剂或注射液中的任意一种。
所述的骨形成蛋白-9通过经鼻给药。
本发明提供的骨形成蛋白-9作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途，具有以下优点：
BMP-9可促进内侧隔核神经元向胆碱能神经元分化，并可剂量和时间依赖性地促进其合成及分泌乙酰胆碱；BMP-9可使内侧隔核ChAT(乙酰胆碱转移酶)阳性的胆碱能神经元计数增加；BMP-9能够明显提高痴呆小鼠的记忆和认知能力；BMP-9经鼻给药，可以绕过血脑屏障，具有脑靶向性，而且避免了肝脏的首过效应，而且该经鼻给药方式便捷，无创伤，易于推广。
附图的简要说明
图1为本发明实验例2Morris水迷宫检测的小鼠寻找平台的路线图。
图2为本发明实验例2Morris水迷宫检测的小鼠的路线长度、游泳速度和逃避潜伏期与时间的折线图(a为路线长度图，b为游泳速度图，c为逃避潜伏期图)。
图3为本发明实验例2Morris水迷宫检测的小鼠穿越平台位置的次数、从潜伏期到平台的时间和出现在目标象限的时间的条形图(a为穿越平台位置的次数图，b为从潜伏期到平台的时间图，c为出现在目标象限的时间图)
图4为本发明实验例3免疫荧光染色的荧光图和内侧隔核胆碱能神经元数目图(a为荧光图，b为内侧隔核胆碱能神经元数目图，n为小鼠数目)。
图5为本发明实验例4Western Blot(蛋白印迹)检测的凝胶条带图和ChAT含量图(a为凝胶条带图，b为ChAT含量图，n为小鼠数目)。
图6为本发明实验例5测定的ChAT活性条形图(n为小鼠数目)。
图7为本发明实验例6测定的Ach(乙酰胆碱)含量条形图(n为小鼠 数目)。
实现本发明的最佳方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。
本发明用于提供骨形成蛋白-9作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途，主要在中枢胆碱能神经系统中的用途，尤其是治疗和预防阿尔茨海默病的用途，该骨形成蛋白-9为人源骨形成蛋白-9、动物骨形成蛋白-9或基因工程表达的骨形成蛋白-9中的任意一种或两种，可通过经鼻给药途径使用。
该药物组合物由所述的骨形成蛋白-9和药学上可接受的载体或辅料组成，可以为片剂、胶囊、丸剂、栓剂、软胶囊、口服液、混悬剂或注射液中的任意一种。
其中，骨形成蛋白-9具有增加胆碱能神经元数目的作用，能够增加乙酰胆碱转移酶，并提高乙酰胆碱转移酶的活性的作用，还能增加乙酰胆碱的释放。
实验例1建立胆碱能神经元(BFCS)退变的小鼠模型
选取12周龄雄性C57小鼠(近交系小鼠)40只，随机分为对照组(Con)、硫胺素缺乏组(PTD)、BMP-9组、硫胺素缺乏+BMP-9组(PTD+BMP-9)，每组10只小鼠，所有动物饲养于明、暗12h相间，室温25℃的SPF(无特定病原体)级室舍中，自由摄食和饮水。
对照组给予普通饮食+腹腔注射生理盐水5μL/(g.d)；硫胺素缺乏组给予缺乏硫胺素饲料+腹腔注射吡啶硫胺0.5μg/(g.d)；BMP组给予普通饮食+腹腔注射生理盐水5μL/(g.d)+经鼻滴入1ng/(g.d)BMP-9；硫胺素缺乏+BMP-9组给予缺乏硫胺素饲料+腹腔注射吡啶硫胺0.5μg/(g.d)+经鼻滴入1ng/(g.d)的重组人BMP-9，其中重组人BMP-9由BMP-9粉剂(3209-BP-010，
Figure PCTCN2017070610-appb-000001
)和含0.1％BSA(牛血清白蛋白)的4mM HCl配制，配制得到的重组人BMP-9的浓度为20μg/mL。
经鼻滴入BMP-9的实验操作：
左手抓取并固定小鼠，使其头部竖立朝上，充分暴露鼻孔，用微量移液器取1ng/(g.d)的重组人BMP-9，小心滴至一侧鼻孔，维持小鼠体位直立5min 后将小鼠放回笼中，每日经鼻给药一次，隔日更换另一侧鼻腔给药，给药方法同前，共计10天。
实验例2Morris水迷宫检测
经鼻给药第5天，开始进行Morris水迷宫检测，具体检查方法如下：
(1)训练阶段：将小鼠头朝池壁放入水中，记录小鼠到达水下平台的时间、到达平台游经的路程及平均游泳速度，如小鼠在60s内仍未找到平台，则将小鼠引导至平台，待其在平台停留20s后取走小鼠，如小鼠在60s内达到平台，则让其在平台停留15s，而后将其取出，每只小鼠每天训练8次，每次训练时间间隔30min，训练共计5天。
(2)撤台实验：将平台撤离，开始60s的空间探索训练，记录小鼠寻找到隐藏在水面下平台的时间(逃避潜伏期)、到达目的象限(原先平台所在象限)的时间、在该象限停留的时间、穿越原先平台位置的次数以及游泳速度。
Morris水迷宫检测结果：
小鼠寻找平台的方式大致可分为四种：边缘式、随机式、趋向式及直线式。不同的搜索方式代表小鼠以不同的策略寻找平台，正常动物的搜索策略随着训练次数的增加而呈现“边缘式→随机式→趋向式→直线式”的变化规律，而痴呆动物的搜索策略会发生异常变化，如图1所示，对照组小鼠的搜索路线呈现随机式；硫胺素缺乏组小鼠的搜索路线呈现边缘式，并且搜索路线混乱；BMP-9组小鼠的搜索路线较简单，呈现趋向式；硫胺素缺乏+BMP-9组的搜索路线呈现由随机式到趋向式的变化，表明硫胺素缺乏组小鼠的学习能力差。
如图2的a所示，从小鼠到达目的平台位置的路线距离可以看出，BMP-9组小鼠的路线距离最短，其次为对照组和硫胺素缺乏+BMP-9组，硫胺素缺乏组小鼠的路线距离最长且未找到平台位置，说明硫胺素缺乏组小鼠的认知功能最差，而硫胺素缺乏+BMP-9组和对照组相当，表明BMP-9防止了硫胺素缺乏对认知功能的损害。
如图2的b所示，在5天训练过程中，对照组、硫胺素缺乏组、BMP-9组和硫胺素缺乏+BMP-9组小鼠的游泳速度基本相同，排除了小鼠的游泳速度对逃避潜伏期时间长短的影响。
如图2的c所示，在训练5天后，硫胺素缺乏组小鼠逃避潜伏期的时间最长，硫胺素缺乏+BMP-9组小鼠逃避潜伏期的时间明显缩短，对照组和BMP-9组小鼠逃避潜伏期的时间最短，说明硫胺素缺乏组小鼠的学习记忆能力最差，进一步表明BMP-9能够防止了硫胺素缺乏对认知功能的损害。
图3的a、b和c中对照组、硫胺素缺乏组、BMP-9组和硫胺素缺乏+BMP-9组的数据均为每组小鼠各自的平均值。
如图3的a所示，对照组小鼠穿过平台2次，硫胺素缺乏组小鼠始终未穿过平台，BMP-9组和硫胺素缺乏+BMP-9组小鼠穿过平台的次数相同，均穿过1次平台，但游泳路线均接近平台，表明硫胺素缺乏组小鼠的记忆力差。
如图3的b所示，对照组、BMP-9组和硫胺素缺乏+BMP-9组小鼠从潜伏期到平台的时间均为20s，而硫胺素缺乏组小鼠从潜伏期到平台的时间为50～60s，可以看出硫胺素缺乏组小鼠寻找平台的时间较长。
如图3的c所示，对照组和BMP-9组小鼠出现在目标象限的概率接近50％，硫胺素缺乏组小鼠出现在目标象限的概率为20％左右，硫胺素缺乏+BMP-9组小鼠出现在目标象限的概率为40％，可以看出BMP-9能够明显改善硫胺素缺乏小鼠的记忆力。
综合上述Morris水迷宫实验的结果，表明BMP-9能够逆转硫胺素缺乏所引起的记忆力和学习能力下降的情况。
实验例3免疫荧光染色
在小鼠麻醉后，开胸经插管至升主动脉，先予60mL生理盐水灌流，再予4％多聚甲醛100mL灌注固定，断头取脑后置于4％多聚甲醛后固定过夜，依次置于20％和30％蔗糖溶液直至组织沉底，OCT(Optimal Cutting Temperature Compound，是一种聚乙二醇和聚乙烯醇的水溶性混合物)包埋后在冰冻切片机上进行冠状切片，采取漂片法进行免疫荧光染色，脑片用0.01M PBS(磷酸缓冲盐溶液)漂洗后用0.3％Triton-X100(聚乙二醇辛基苯基醚)透膜30min，5％正常驴血清封闭1h，加山羊抗ChAT(乙酰胆碱转移酶)(1:200，
Figure PCTCN2017070610-appb-000002
)，4℃孵育过夜，经0.01M PBS漂洗后加TRITC(四甲基异硫氰酸罗丹明)标记的猴抗山羊IgG(1:200，
Figure PCTCN2017070610-appb-000003
)，室温避光孵育1h，经0.01M PBS漂洗后贴片，甘油封片(甘油：PBS＝1:1)，荧光显微镜下观察拍照。
免疫荧光染色结果：
如图4的a所示，BMP-9组小鼠的荧光数量略高于对照组，硫胺素缺乏+BMP-9组小鼠的荧光数量明显小于对照组和BMP-9组小鼠，而硫胺素缺乏组小鼠的荧光数量最少。如图4的b(n为小鼠数目，统计的数据为3只小鼠的平均值)所示，小鼠脑组织的内侧隔核胆碱能神经元数目的顺序为BMP-9组>对照组>硫胺素缺乏+BMP-9组>硫胺素缺乏组，表明BMP-9可以增加内侧隔核胆碱能神经元数目，改善硫胺素缺乏引起的胆碱能神经元数目减少的情况。
实验例4Western Blot(蛋白印迹)检测
在小鼠麻醉后迅速断头取脑，取10mg基底前脑组织，提取总蛋白，测定总蛋白含量，各取20μg蛋白，经10％SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)分离胶和5％浓缩胶100V电泳90min，300mA转膜60min。5％脱脂牛奶封闭1h，分别加山羊抗ChAT(乙酰胆碱转移酶)(1:500)；小鼠抗β-Actin(β-肌动蛋白)(1:1000)，4℃孵育过夜，TBST(三羟甲基氨基甲烷缓冲液+吐温20)洗膜10min×3次后，加入对应的荧光二抗：
Figure PCTCN2017070610-appb-000004
800CW(绿色荧光蛋白)猴抗山羊IgG(免疫球蛋白G)(1:1000)、
Figure PCTCN2017070610-appb-000005
680CW山羊抗小鼠IgG(1:1000)，室温孵育1h，用TBST洗膜10min×3次，最后利用Odyssey近红外荧光成像仪进行扫膜，结果用凝胶图像分析仪进行灰度值分析。
Western Blot检测结果：
如图5的a和图5的b(n为小鼠数目，统计的数据为3只小鼠的平均值)所示，对照组、BMP-9组ChAT的量基本相当，硫胺素缺乏+BMP-9组略低，而硫胺素缺乏组ChAT的量明显很低，进一步证实硫胺素缺乏组小鼠脑组织中ChAT的表达量显著降低。
实验例5ChAT(乙酰胆碱转移酶)活性测定
参照ChAT活性试剂盒
Figure PCTCN2017070610-appb-000006
的步骤进行，在小鼠麻醉后迅速断头取脑，取10mg海马组织，用预冷的PBS冲洗，加入含0.5％Triton X-100和0.1mg/mL PMSF(苯甲基磺酰氟)的TBS(三羟甲基氨基甲烷缓冲液)1mL，充分匀浆，14000r/min离心5min，取20μL上清液，依次加入试剂盒中的100μL检测试剂A、100μL检测试剂B，而后加入100μL四甲基联苯胺，37℃避光孵育20min，用分光光度计在450nm处测得吸光度，根据标准曲线计 算出ChAT活性(由于脑组织内ChAT活性较低，本实验中样品未做稀释直接检测)。
标准曲线的制作：
标准曲线是用ChAT活性试剂盒中的标准品来制作的，标准品的初始浓度为100U,稀释倍数依次为1，2，4，8，16和32倍，得到浓度依次为100U，50U，25U，12.5U，6.25U，3.125U和1.563U的标准品，根据这些不同浓度标准品的OD(光密度)值制作标准曲线，以标准品的浓度为横坐标，OD值为纵坐标，根据样品的OD值计算出样品的ChAT活性。
ChAT活性测定结果：
如图6(n为小鼠数目，统计的数据为3只小鼠的平均值)所示，BMP-9组小鼠脑组织中ChAT的活性为15～20U/mg protein，对照组和硫胺素缺乏+BMP-9组小鼠脑组织中ChAT的活性为10U/mg protein，硫胺素缺乏组小鼠脑组织中ChAT的活性为5U/mg protein，表明BMP-9能够明显提高ChAT的活性，并且能够显著增加硫胺素缺乏小鼠脑组织中的ChAT的活性。
实验例6Ach(乙酰胆碱)含量测定
参照Ach含量试剂盒
Figure PCTCN2017070610-appb-000007
的步骤进行，在小鼠麻醉后迅速断头取脑，取10mg海马组织，用预冷的PBS冲洗，加入500μL样品裂解液，充分匀浆，10000r/min离心5min，取20μL上清液，用样品稀释液(乙酰胆碱检测缓冲液，Choline Assay Buffer)稀释至50μL，而后依次加入44μL样品稀释液，依此加入2μL胆碱探针、2μL乙酰胆碱酯酶及2μL胆碱酶复合物，室温避光孵育30min，用分光光度计在570nm处测得吸光度，根据标准曲线计算出Ach含量。
标准曲线的制作：
标准曲线是用Ach含量试剂盒中的标准品来制作的，标准品的初始浓度为500pmol，稀释倍数依次为1，1.25，1.33，2.5和5倍，得到浓度依次为500pmol，400pmol，300pmol，200pmol及100pmol的标准品，根据这些不同浓度的标准品的OD值，以标准品的浓度为横坐标，以OD为纵坐标，得到标准曲线，而后根据样品的OD值计算出样品的Ach含量。
Ach含量测定结果：
如图7(n为小鼠数目，统计的数据为3只小鼠的平均值)所示，对照组 (Con)、硫胺素缺乏组(PTD)、BMP-9组、硫胺素缺乏+BMP-9组(PTD+BMP-9)中Ach的含量CAch分别为10000pmol/mg，CAch<5000pmol/mg，CAch>10000pmol/mg，5000pmol/mg<CAch<10000pmol/mg，可以看出BMP-9组小鼠脑组织中释放的Ach含量略高于对照组，硫胺素缺乏组小鼠脑组织中释放的Ach含量明显低于对照组，但是硫胺素缺乏+BMP-9组脑组织中释放的Ach含量与硫胺素缺乏组相比显著增加，表明BMP-9可以促进脑组织中释放Ach，尤其能够改善由于硫胺素缺乏引起的Ach含量过低的情况。
综上所述，本发明用于提供骨形成蛋白-9作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途，BMP-9通过经鼻给药，能够显著增加内侧隔核胆碱能神经元的数目、脑内乙酰胆碱转移酶表达含量、乙酰胆碱转移酶活性以及乙酰胆碱释放量，并且能够明显提高胆碱能神经元退变小鼠的认知和记忆能力，充分地证实了BMP-9给药可显著逆转硫胺素缺乏所诱导的BFCS退变，能够作为治疗阿尔茨海默病的药物。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。

Claims (10)

  1. 骨形成蛋白-9作为制备治疗和/或预防与胆碱能系统退变相关疾病的药物组合物的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述的胆碱能系统为中枢胆碱能神经系统。
  3. 根据权利要求1或2所述的用途，其特征在于，所述的与胆碱能系统退变相关疾病包括阿尔茨海默病。
  4. 根据权利要求3所述的用途，其特征在于，所述的骨形成蛋白-9通过经鼻给药。
  5. 根据权利要求4所述的用途，其特征在于，所述的骨形成蛋白-9为人源骨形成蛋白-9、动物骨形成蛋白-9或基因工程表达的骨形成蛋白-9中的任意一种或两种。
  6. 根据权利要求5所述的用途，其特征在于，所述的骨形成蛋白-9具有增加胆碱能神经元数目的作用。
  7. 根据权利要求5所述的用途，其特征在于，所述的骨形成蛋白-9具有增加乙酰胆碱转移酶，并提高乙酰胆碱转移酶的活性的作用。
  8. 根据权利要求5所述的用途，其特征在于，所述的骨形成蛋白-9具有增加乙酰胆碱释放的作用。
  9. 根据权利要求1或2所述的用途，其特征在于，所述的药物组合物由所述的骨形成蛋白-9和药学上可接受的载体或辅料组成。
  10. 根据权利要求1或2所述的用途，其特征在于，所述的药物组合物为片剂、胶囊、丸剂、栓剂、口服液、混悬剂或注射液中的任意一种。
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CN115737787A (zh) * 2022-12-15 2023-03-07 四川大学 乳铁蛋白联合胆碱在制备预防和/或治疗阿尔兹海默症的药物中的用途

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CN103305458A (zh) * 2012-03-06 2013-09-18 中国科学院上海生命科学研究院 从多潜能干细胞诱导胆碱能神经元的方法

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CN115737787A (zh) * 2022-12-15 2023-03-07 四川大学 乳铁蛋白联合胆碱在制备预防和/或治疗阿尔兹海默症的药物中的用途
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