CN110179828B - 含有痘苗病毒接种炎症组织提取物的n-乙酰基半乳糖胺转移酶表达促进剂 - Google Patents
含有痘苗病毒接种炎症组织提取物的n-乙酰基半乳糖胺转移酶表达促进剂 Download PDFInfo
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Abstract
本发明的目的在于提供痘苗病毒接种炎症组织提取物在制造N‑乙酰基半乳糖胺转移酶表达促进剂中的应用等。在本发明中,显示痘苗病毒接种炎症组织提取物促进椎间盘细胞的N‑乙酰基半乳糖胺转移酶的表达。因此,痘苗病毒接种炎症组织提取物或含有该提取物的制剂作为椎间盘细胞的N‑乙酰基半乳糖胺转移酶表达促进剂等有用。
Description
技术领域
本发明涉及痘苗病毒接种炎症组织提取物(以下,有时记作“本发明提取物”。)的新的医药用途等。更具体而言,涉及含有本发明提取物的N-乙酰基半乳糖胺转移酶(GalNAcT)表达促进剂等。
背景技术
椎间盘由髓核、纤维环、软骨终板这样的3种具有不论是生物化学还是物理学上都不同的特性的组织构成,通过髓核内丰富的蛋白多糖保持大量的水,具有椎间盘的约80%为水分的特征。椎间盘变性是腰痛的主要原因之一,并且关系到椎间盘突出或椎管狭窄症等的发病。伴随年龄增长的椎间盘变性的个人差异大,但据认为与髓核部中构成细胞的变化和由其造成的基质变化、之后所发生的纤维环结构的破裂与椎间盘变性有密切关系。人在60岁以后,椎间盘中的蛋白多糖含量从十多岁的65%降低到一半以下的30%左右,其结果,椎间盘所含的水分减少。伴随于此,容易发生由外部原因造成的结构破裂。
蛋白多糖是在核心蛋白的丝氨酸残基结合有糖质的四糖结合区域结合有多条以硫酸软骨素等二糖单元连接而成的多糖体(糖胺聚糖、GAG)的化合物。作为生物体成分具有多种功能性的蛋白多糖除了在主要的各种脏器、脑、皮肤、身体全体的组织中的细胞外基质和细胞表面存在以外,还作为软骨的主要成分存在。
GAG根据二糖的基本骨架的不同,被分类为(1)肝素、硫酸乙酰肝素、(2)硫酸软骨素、硫酸皮肤素、(3)硫酸角质素、(4)透明质酸的四个种类。它们的基本骨架受到以硫酸化为主的各种修饰。在大多数情况下,GAG以结合于核心蛋白的形式、即作为蛋白多糖存在。已知各种各样的糖转移酶和硫酸基转移酶参与GAG的生物合成。
硫酸软骨素是GAG的一种,作为聚集蛋白聚糖的侧链丰富地存在,其中,聚集蛋白聚糖为软骨的细胞外基质的主要构成成分。已知在椎间盘变性疾病或变形性关节病(OA)患者的软骨中,硫酸软骨素减少。
硫酸软骨素是N-乙酰基半乳糖胺(GalNAc)和葡糖醛酸(GlcUA)的二糖交替重复聚合而成的直链状的硫酸化糖链。该硫酸软骨素的糖链骨架的生物合成中,有2种N-乙酰基半乳糖胺转移酶参与。1种是将GalNAc转移到上述结合区域的末端的GlcUA残基上的N-乙酰基半乳糖胺转移酶1(GalNAcT1),另一种是将GalNAc转移到二糖重复结构的GlcUA末端的N-乙酰基半乳糖胺转移酶2(GalNAcT2)。硫酸软骨素的生物合成从GalNAc由GalNAcT1转移到上述结合区域的末端的GlcUA残基开始。然后GlcUA由葡糖醛酸转移酶、接着GalNAc由GalNAcT2被交替转移,合成二糖的重复区域,糖链伸长。并且最后构成糖由硫酸基转移酶硫酸化而成为硫酸软骨素。这样,可知在硫酸软骨素的生物合成中,GalNAcT1参与糖链的合成开始,GalNAcT2参与糖链的伸长。即,GalNAcT1不参与硫酸软骨素的糖链的伸长,而与糖链数的增加相关。
关于本发明涉及的N-乙酰基半乳糖胺转移酶表达促进剂所含有的痘苗病毒接种炎症组织提取物(本发明提取物)或含有该提取物的制剂,已知有镇痛作用、镇静作用、抗应激作用、抗过敏作用、免疫促进作用、抗癌作用、肝硬化抑制作用、对特发性血小板减少性紫癜的治疗效果、对带状疱疹后神经痛、脑水肿、痴呆、脊髓小脑变性症等的治疗效果、对雷诺氏综合征、糖尿病性神经障碍、亚急性视神经脊髓病(Smon)后遗症等的治疗效果、激肽释放酶产生抑制作用、末梢循环障碍改善作用、骨萎缩改善作用、对败血症、内毒素休克的治疗有效的一氧化氮产生抑制作用、对骨质疏松症的治疗效果、基于Nef作用抑制作用、趋化因子产生抑制作用的艾滋病治疗效果、对脑梗塞等的缺血性疾病的治疗效果、对纤维肌痛症的治疗效果、对感染症的治疗效果、对由抗癌剂引起的末梢神经障碍的预防或减轻作用、慢性前列腺炎、间质性膀胱炎和/或排尿障碍的治疗效果、BDNF等的神经营养因子的产生促进作用、肝的保护作用、多能性干细胞(Muse细胞)迁移促进作用、肌肉损伤的预防或治疗效果等非常多样的作用、效果。除此以外,还已知本发明提取物或含有该提取物的制剂具有促进软骨细胞中的胶原蛋白和蛋白多糖的合成的作用(参照专利文献1)。然而,目前为止并不知道本发明提取物或含有该提取物的制剂促进GalNAcT的表达、相比于GalNAcT2更强地促进GalNAcT1的表达等。相比于GalNAcT2更强地促进GalNAcT1的表达,是指糖链数的增加作用比硫酸软骨素的糖链的伸长作用强。目前为止并不知道具有那样作用的药剂。
现有技术文献
专利文献
专利文献1:国际公开WO2012/051173号公报
发明内容
发明所要解决的课题
本发明提供含有本发明提取物的GalNAcT表达促进剂等。
用于解决课题的方法
本发明的发明人对本发明提取物的药理作用进行了深入研究,结果发现,本发明提取物具有优异的GalNAcT表达促进作用,从而完成了本发明。
发明的效果
本发明提取物具有促进GalNAcT的表达的作用,因此含有本发明提取物的制剂能够成为伴随椎间盘变性的疾病或OA的优异的治疗或预防剂。特别是含有本发明提取物的制剂作为副作用等问题少的安全性高的药剂被长年使用,因此本发明的有用性极高。
具体实施方式
本发明提取物是含有从接种痘苗病毒并发痘的动物的炎症组织提取分离的非蛋白性的活性物质的提取物。本发明提取物在提取的状态下为液体,但也能够通过干燥制成固体。本发明制剂作为医药品非常有用。作为本发明制剂,在申请人在日本制造并销售的具体的商品中,有“含有接种痘苗病毒的家兔的炎症皮肤的提取液的制剂”(商品名:神经妥乐平/NEUROTROPIN〔注册商标〕)(以下称为“神经妥乐平”)。神经妥乐平有注射剂和片剂,均为医疗用医药品(ethical drug)。
神经妥乐平的注射剂的适应症为“腰痛症、颈肩综合征、症状性神经痛、伴随皮肤疾病(湿疹、皮肤炎、荨麻疹)的瘙痒、过敏性鼻炎、亚急性脊髓视神经病(SMON)后遗症状的寒冷感、感觉异常、疼痛”。神经妥乐平的片剂的适应症为“带状疱疹后遗神经痛、腰痛症、颈肩综合征、肩周炎、变形性关节症”。本发明制剂是申请人创制并作为医药品开发出来的,其有效性和安全性上的优异特长受到赞赏,已经销售多年,并已经在日本医药品市场中确立了稳固的地位。
本发明中的痘苗病毒接种炎症组织提取物能够通过如下操作得到:接种痘苗病毒,将发痘的炎症组织破碎,加入提取溶剂,除去组织碎片后,进行除蛋白处理,使其吸附于吸附剂,然后将有效成分洗脱。即,例如为如下的工序。
(A)收集接种痘苗病毒并使其发痘的兔、小鼠等的皮肤组织等,将发痘组织破碎,加入水、苯酚水、生理盐水或苯酚加甘油水等提取溶剂后,通过过滤或离心分离,得到提取液(滤液或上清液)。
(B)将上述提取液调整为酸性的pH并加热,进行除蛋白处理。接着,将除蛋白后的溶液调整为碱性并加热后,进行过滤或离心分离。
(C)使所得到的滤液或上清液为酸性,使其吸附于活性炭、高岭土等吸附剂。
(D)在上述吸附剂中加入水等提取溶剂,调整为碱性的pH,将吸附成分洗脱,由此能够得到痘苗病毒接种炎症组织提取物。然后,根据希望,也能够通过适当将洗脱液在减压下蒸发干燥固化或冻结干燥,制成干燥固化物。
作为用于接种痘苗病毒得到炎症组织的动物,能够使用兔、牛、马、绵羊、山羊、猴、大鼠、小鼠等感染痘苗病毒的各种动物,作为炎症组织,优选兔的炎症皮肤组织。兔只要是属于兔目即可,可以使用任何兔子。作为例子,有:穴兔(Oryctolagus cuniculus)、家兔(驯养的穴兔)、野兔(日本野兔)、鼠兔和雪兔等。其中,适合使用家兔。在日本,有从过去被饲养的作为家畜或实验用动物而频繁使用的称为家养兔(イエウサギ)的兔,这也是家兔的别称。在家兔中存在多个品种(breed),能够优选使用称为日本白色种和新西兰白色种(新西兰白)的品种。
痘苗病毒(vaccinia virus)可以是任意株的痘苗病毒。作为例子,可以列举利斯特(Lister)株、大连(Dairen)株、池田(Ikeda)株、EM-63株、纽约市公众卫生局(New YorkCity Board of Health)株等。
关于上述本发明提取物的基本的提取工序(A)~(D),更详细而言,例如,能够作为如下的工序实施。
关于工序(A)
在兔的皮肤中将痘苗病毒进行皮内接种使其发痘,收集炎症皮肤组织。收集的皮肤组织以苯酚溶液等进行清洗、消毒。将该炎症皮肤组织破碎,加入其1至5倍量的提取溶剂。其中,所谓破碎是指使用绞碎机等细细地破碎为肉末状。另外,作为提取溶剂,能够使用蒸馏水、生理盐水、弱酸性至弱碱性的缓冲液等,也可以适当添加苯酚等的杀菌-防腐剂、甘油等的稳定剂、氯化钠、氯化钾、氯化镁等的盐类等。此时,也能够通过冷冻融化、超声波、细胞膜溶解酶或表面活性剂等的处理来破坏细胞组织,使提取变得容易。将得到的悬浮液放置5日至12日。在此期间,可以边适当搅拌或不搅拌边加温到30至45℃。通过对得到的液体进行固液分离(过滤或离心分离等)除去组织片,得到粗提取液(滤液或上清液)。
关于工序(B)
对在工序(A)中得到的粗提取液进行除蛋白处理。除蛋白能够由通常进行的公知方法实施,能够应用加热处理、利用蛋白质变性剂(例如酸、碱、脲、胍、丙酮等有机溶剂等)的处理、等电点沉淀、盐析等的方法。接着,利用除去不溶物的通常方法,例如使用滤纸(纤维素、硝酸纤维素等)、玻璃过滤器、硅藻土制品、塞茨过滤板(Seitz filter)等的过滤、超滤、离心分离等,得到除去了析出的不溶蛋白质的滤液或上清液。
关于工序(C)
将在工序(B)中得到的滤液或上清液调整为酸性、优选调整为pH3.5至5.5,进行向吸附剂的吸附操作。作为能够使用的吸附剂,能够列举活性碳、高岭土等,在提取液中添加吸附剂进行搅拌,或使提取液通过吸附剂填充柱,能够使有效成分吸附于该吸附剂。在提取液中添加吸附剂时,通过过滤或离心分离等除去溶液,能够得到吸附有活性成分的吸附剂。
关于工序(D)
为了使活性成分从工序(C)中得到的吸附剂中洗脱(脱离),在该吸附剂中加入洗脱溶剂,调整为碱性、优选调整为pH9至12,在室温或适当加热、或者搅拌进行洗脱,以过滤或离心分离等通常的方法除去吸附剂。作为所使用的洗脱溶剂,能够使用碱性溶剂,例如,能够使用调整为碱性pH的水、甲醇、乙醇、异丙醇等或它们的适当的混合溶液,能够优选使用调整为pH9至12的水。洗脱溶剂的量能够适当设定。为了将这样操作得到的洗脱液作为原药使用,能够将pH适当地调整到中性附近等,最终得到痘苗病毒接种兔炎症皮肤提取物(本发明提取物)。
本发明提取物在制成时为液体,因此也能够通过适当浓缩、稀释制成所希望浓度的提取物。从本发明提取物制造制剂时,优选实施加热灭菌处理。为了制成注射剂,例如,能够加入氯化钠等配制与生理盐水等渗的溶液。另外,也能够通过以液体或凝胶等的状态口服给药,也能够通过对本发明提取物进行适当的浓缩干燥固化等的操作,制造片剂等的口服用固体制剂。从本发明提取物制造这样的口服用固体制剂的具体的方法记载于日本专利第3818657号和日本专利第4883798号的说明书中。这样操作得到的注射剂和口服用制剂等是本发明制剂的例子。
以下,例示本发明提取物的制造方法的例子、以及关于本发明提取物的新的药理作用、N-乙酰基半乳糖胺转移酶的表达促进作用的药理试验结果,但本发明不受这些实施例的记载任何限制。
实施例
实施例1本发明提取物的制造
在健康成年家兔的皮肤中皮内接种痘苗病毒,切下并收集发痘的皮肤。收集的皮肤以苯酚溶液进行清洗、消毒后,除去多余的苯酚溶液,进行破碎,加入苯酚溶液混合,放置3~7日后,进而边搅拌3~4日边加热到35~40℃。然后,用盐酸将固液分离得到的提取液调整到pH4.5~5.2,以90~100℃加热处理30分钟后,过滤除去蛋白。再用氢氧化钠将滤液调整到pH9.0~9.5,以90~100℃加热处理15分钟后,进行固液分离。
用盐酸将得到的除蛋白液调整到pH4.0~4.3,加入除蛋白液质量的2%量的活性碳,搅拌2小时后,进行固液分离。在收集的活性碳中加水,用氢氧化钠调整到pH9.5~10,以60℃搅拌90~100分钟后,进行离心分离得到上清液。在以离心分离沉淀的活性碳中再添加水后,用氢氧化钠调整到pH10.5~11,以60℃搅拌90~100分钟后,进行离心分离得到上清液。合并两上清液,用盐酸中和,得到本发明提取物。
实施例2(试验方法和试验结果)
接着,表示显示上述实施例1中得到的本发明提取物的椎间盘细胞的GalNAcT表达促进作用的药理试验的试验方法和试验结果。
细胞和试剂
在试验例1至3中,受到了东海大学医学部实验伦理委员会的承认,使用以以下程序制备的人髓核细胞。从男性3人、女性2人合计5人的椎间盘突出患者(年龄29至38岁),在获得同意的前提下在术中收集髓核组织。将髓核组织切成小片,用TrypLE Express(Gibco)处理1小时,然后用0.25mg/ml的Collagense-P(Roche)处理2小时。在37℃将分离得到的细胞用α-MEM培养基(Wako Chemical)清洗2次,以约5×103个/cm2的密度接种。将细胞在添加了10%胎牛血清(FBS,Sigma-Aldrich)、100U/ml青霉素(Gibco)和100mg/ml链霉素(Gibco)的α-MEM培养基中,在2%O2的低氧条件下和5%CO2条件下在37℃培养。培养基每周更换2次,在细胞达到汇合之前用胰蛋白酶(Gibco)处理,传代培养。在各个实验中使用从第三次传代得到的细胞。
将髓核细胞以5000个/cm2的密度接种于25cm2的烧瓶,在添加本发明提取物之前在含有10%FBS的α-MEM培养基中培养过夜。然后,将细胞用添加了本发明提取物、10%FBS和170μM的抗坏血酸的α-MEM培养基进行了处理。一边维持2周培养,一边将培养基每隔一天进行更换。将达到了汇合的髓核细胞用于以下的试验。
统计解析
关于统计解析,进行重复测量方差分析。在得到低于0.05的p值时,进行Bonferroni事后检验。
试验例1对GAG表达的效果
GAG和DNA分析
将培养的细胞用Dulbecco′s磷酸缓冲生理盐水(DPBS,DS-Pharma)清洗,用含有25mg/ml的木瓜蛋白酶(Sigma-Aldrich)、8mg/ml的乙酸钠(Wako Chemical)、4mg/ml的乙二胺四乙酸(Sigma-Aldrich)和1.57mg/ml的L-半胱氨酸(Sigma-Aldrich)的缓冲液,在65℃处理过夜。关于硫酸化GAG含量,通过使用作为分光光度计的SPECTRA MAX i3(MolecularDevices),以6-硫酸软骨素(Biocolor)为标准,使用1,9-二甲基-亚甲基蓝(Biocolor),测定在656nm的吸光度来算出。关于DNA量,使用在480nm的激发和在520nm的发光与上述同样用分光光度计,通过PicoGreen检测(ThermoFisher Scientific Waltham,MA)来算出。算出GAG相对于DNA量的比率(平均值±标准误差)。将上述试验的结果的一例表示于表1。
【表1】
**:p<0.01vs对照
相对于对照,GAG/DNA值在0.1mNU/mL给药组中增加到1.7倍,在1.0mNU/mL添加组中增加到1.4倍,本发明提取物0.1mNU/mL给药组中确认到显著的GAG产生的增强(表1)。
试验例2对CSGALNACT1、ANG1和IGF基因的表达产生的效果(实时定量PCR法)
添加本发明提取物1.0mNU/mL的1周后,收集细胞,在溶解缓冲液中匀浆,使用SVTotal RNA分离系统(Promega),制备Total RNA(tRNA)。对于各样品,使用High CapacityRNA-to-cDNA试剂盒(Applied Biosystems),将2μg的tRNA逆转录成cDNA。关于GALNACT1的mRNA量,以甘油醛-3-磷酸脱氢酶[GAPDH:制品名,pre-developed TaqMan Assay Reagents(Applied Biosystems)]为内部标准,通过比较CT法算出。引物和探针(AppliedBiosystems)使用以下的物质。IGF1(TaqMan Assay ID:Hs03986524_m1)、ANGPT1(TaqManAssay ID:Hs00181613_m1)、CSGALNACT1(TaqMan Assay ID:Hs00218054_m1)。算出将对照中的CSGALNACT1、ANGPT1或IGF1的表达量设为1时各组的比率(平均值±标准误差)。将上述试验的结果的一例表示于表2至表4。
【表2】
*:p<0.05vs对照**:p<0.01vs对照
【表3】
**:p<0.01vs对照
【表4】
**:p<0.01vs对照
确认到CSGALNACT1的表达量通过添加0.1mNU/mL和1.0mNU/mL的本发明提取物被显著增强(表2)。另外,确认到ANGPT1和IGF1的mRNA表达量通过添加0.1mNU/mL和1.0mNU/mL的本发明提取物被显著增强(表3和表4)。
试验例3对CSGALNACT1和CSGALNACT2的mRNA表达产生的效果(微阵列法)
使用微阵列比较本发明提取物1.0mNU/mL处理和未处理的患者由来髓核细胞的基因表达。添加本发明提取物和170μM的抗坏血酸。与试验例2同样制备tRNA,使用Low InputQuick Amp Labeling Kit(Agilent Technology)制备Cy3标记cRNA。对所得到的Cy3标记cRNA,使用SurePrint G3Human GE 8x60K v2Microarray(Agilent Technology)和GeneExpression Hybridization Kit(Agilent Technology)进行杂交。然后使用Agilent DNA微阵列扫描仪(Agilent Technology,G2600D SG13164306),按照AgilentG3_HiSen_GX_1Color(Agilent Technology)操作规程进行分析。
各探针的荧光强度使用数值化变换软件Agilent Feature Extraction11.5.1.1(Agilent Technology)转变为表达值。使用基因表达解析软件Gene Spring ver.13(Agilent Technology),检测出表达量多的基因。参与GAG合成的基因使用Database forAnnotation,Visualization and Integrated Discovery(DAVID)2017Tool和KyotoEncyclopedia of Genes and Genomes(KEGG)PATHWAY Database选择。算出将不添加本发明提取物地进行相同时间培养的情况(对照)的表达量设为1时的、添加后的表达量的比率。对相同基因的来自阵列上的多个探针的信号进行平均,作为1个数据使用。将上述试验的结果的一例表示于表5。
【表5】
在大多数参与GAG合成的基因中,确认到通过添加1.0mNU/mL的本发明提取物而表达被增强(表5)。在CSGALNACT中,CSGALNACT1表达增强为1.54倍、CSGALNACT2表达增强为1.12倍(表5)。
由以上可知,作为本发明的优选的实施方式列举了以下的方式,但并不限于这些实施方式。
(1)一种含有痘苗病毒接种炎症组织提取物的N-乙酰基半乳糖胺转移酶表达促进剂。
(2)如(1)所述的表达促进剂,其中,N-乙酰基半乳糖胺转移酵为N-乙酰基半乳糖胺转移酶1。
(3)如(1)所述的表达促进剂,其中,N-乙酰基半乳糖胺转移酶为N-乙酰基半乳糖胺转移酶2。
(4)如(1)至(3)中任一项所述的表达促进剂,其中,N-乙酰基半乳糖胺转移酶1的表达促进作用比N-乙酰基半乳糖胺转移酶2的表达促进作用强。
(5)如(1)至(4)中任一项所述的表达促进剂,其中,炎症组织为兔的炎症皮肤组织。
(6)如(1)至(5)中任一项所述的表达促进剂,其为注射剂。
(7)如(1)至(5)中任一项所述的表达促进剂,其为口服剂。
(8)一种痘苗病毒接种炎症组织提取物或含有该提取物的制剂的判定或评价方法,其以椎间盘细胞中的N-乙酰基半乳糖胺转移酶的表达促进作用作为指标。
(9)如(8)所述的判定或评价方法,其中,N-乙酰基半乳糖胺转移酶为N-乙酰基半乳糖胺转移酶1。
(10)如(8)所述的判定或评价方法,其中,N-乙酰基半乳糖胺转移酶为N-乙酰基半乳糖胺转移酶2。
(11)如(8)至(10)中任一项所述的判定或评价方法,其中,对N-乙酰基半乳糖胺转移酶1和N-乙酰基半乳糖胺转移酶2的表达增加率进行比较,确认N-乙酰基半乳糖胺转移酶1的表达增加率大。
(12)如(8)至(10)中任一项所述的判定或评价方法,其中,炎症组织为兔的炎症皮肤组织。
(13)通过进行上述(8)至(12)中任一项所述的判定或评价来保证痘苗病毒接种炎症组织提取物或含有该提取物的制剂的品质规格的方法。
(14)如上述(13)所述的保证品质规格的方法,其中,制剂为注射剂或口服剂。
(15)痘苗病毒接种炎症组织提取物在制造N-乙酰基半乳糖胺转移酶表达促进剂中的应用。
(16)如(15)所述的应用,其中,N-乙酰基半乳糖胺转移酶为N-乙酰基半乳糖胺转移酶1。
(17)如(15)所述的应用,其中,N-乙酰基半乳糖胺转移酶为N-乙酰基半乳糖胺转移酶2。
(18)如(15)至(17)中任一项所述的应用,其特征在于,N-乙酰基半乳糖胺转移酶表达促进剂对N-乙酰基半乳糖胺转移酶1的表达促进作用比对N-乙酰基半乳糖胺转移酶2的表达促进作用强。
(19)如(15)至(18)中任一项所述的应用,其中,炎症组织为兔的炎症皮肤组织。
(20)如(15)至(19)中任一项所述的应用,其中,N-乙酰基半乳糖胺转移酶表达促进剂为注射剂。
(21)如(15)至(19)中任一项所述的应用,其中,N-乙酰基半乳糖胺转移酶表达促进剂为口服剂。
产业上的可利用性
如上所述,本发明提取物具有N-乙酰基半乳糖胺转移酶的表达促进作用,特别是相比于N-乙酰基半乳糖胺转移酶2,具有更强地促进N-乙酰基半乳糖胺转移酶1的表达的作用。由此可知,含有本发明提取物的N-乙酰基半乳糖胺转移酶表达促进剂作为伴随椎间盘变性的疾病或OA的治疗或预防剂有用。
Claims (3)
1.一种痘苗病毒接种兔炎症皮肤组织提取物或含有所述提取物的制剂的判定或评价方法,其特征在于:
对培养的椎间盘细胞用所述痘苗病毒接种兔炎症皮肤组织提取物或含有所述提取物的制剂进行处理,对处理后的椎间盘细胞中的N-乙酰基半乳糖胺转移酶1和N-乙酰基半乳糖胺转移酶2的表达增加率进行比较,确认N-乙酰基半乳糖胺转移酶1的表达增加率大。
2.通过进行权利要求1所述的判定或评价来保证痘苗病毒接种兔炎症皮肤组织提取物或含有所述提取物的制剂的品质规格的方法。
3.如权利要求2所述的保证品质规格的方法,其特征在于:
制剂为注射剂或口服剂。
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US20220079995A1 (en) | 2022-03-17 |
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