CN117500820A - 钙敏感受体激动剂化合物的盐酸盐及药物组合物和其应用 - Google Patents
钙敏感受体激动剂化合物的盐酸盐及药物组合物和其应用 Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- OENLEHTYJXMVBG-UHFFFAOYSA-N pyridine;hydrate Chemical compound [OH-].C1=CC=[NH+]C=C1 OENLEHTYJXMVBG-UHFFFAOYSA-N 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
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- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
- A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
钙敏感受体激动剂化合物的盐酸盐及药物组合物和其应用。具体而言,提供了一种多肽钙敏感受体激动剂化合物的盐酸盐及药物组合物和其应用,其对人类钙敏感受体(Calcium‑sensing Receptor,CaSR)具有激动剂作用从而降低血浆甲状旁腺激素和血清钙离子水平,并可以用于继发性甲状旁腺功能亢进,肿瘤引发的高钙血症等代谢类疾病的治疗。
Description
本公开属于医药技术领域,具体涉及一种在人类钙敏感受体(CaSR)具有激动剂作用的化合物的盐酸盐及其组合物和其应用,并可用于代谢性疾病例如原发性甲状旁腺功能亢进,继发性甲状旁腺功能亢进和高钙血症等相关代谢类疾病的治疗。
钙敏感受体(Calsium-sensing Receptor,CaSR)是指分布在人体甲状旁腺器官细胞表面的一种A家族G-蛋白偶联受体(G-Protein Coupled Receptor,GPCR)。甲状旁腺激素的分泌受到甲状旁腺细胞表面钙敏感受体的高度调节从而维持人体矿物质的稳态水平,钙敏感受体通过持续监测人体内钙离子浓度的细微变化继而通过改变甲状旁腺激素的分泌水平进行相应的响应。
在慢性肾病患者中,体内对于实现钙离子和磷离子的稳态水平的需求导致了甲状旁腺激素持续地从甲状旁腺的分泌。这种持续的甲状旁腺激素的分泌一开始是适应性的,但随着慢性肾病的进展最终导致了甲状旁腺的增生以及体内过高的甲状旁腺激素水平并诱发了继发性甲状旁腺功能亢进的形成。有研究表明,持续的继发性甲状旁腺功能亢进会导致甲状旁腺细胞表面钙敏感受体和维生素D受体的缺失。这些由疾病引发的下游病理效应进一步导致了甲状旁腺对于体内矿物质稳态调节的失调。
拟钙剂泛指生理功能和作用机制类似于钙离子并可以直接激活甲状旁腺细胞表面钙敏感受体的化合物。盐酸西那卡塞是由安进公司开发的有机小分子拟钙剂,其可激活甲状旁腺器官表面的钙敏感受体,抑制甲状旁腺激素的分泌水平进而达到治疗继发性甲状旁腺功能亢进等相关代谢类疾病的目的。盐酸西那卡塞在临床上获批用于治疗慢性肾病透析患者中的继发性甲状旁腺功能亢进,患者采用口服给药的方式,使用频率为每天口服一到二次,最高剂量可为每次90毫克。盐酸西那卡塞在临床上展现出优异的降低继发性甲状旁腺功能亢进患者血浆甲状旁腺激素水平的疗效。然而,在患者使用过程中观测到明显的药物引发的副作用,例如与胃肠道副作用相关的恶心,呕吐以及腹泻。此外,盐酸西那卡塞口服给药的用药方式对于慢性肾病透析患者而言是一个较大的负担,并且盐酸西那卡塞已被证明可以抑制细胞色素450并诱发与此有关的药物与药物之间的相互作用。这些与盐酸西那卡塞使用相关的副作用在一定程度上降低了患者的依从性和顺应性。
因此,临床上急需具有可以通过静脉注射给药的钙敏感受体激动剂化合物, 其可以通过激活甲状旁腺细胞表面的钙敏感受体从而降低甲状旁腺激素的分泌进而达到治疗继发性甲状旁腺功能亢进等相关代谢类疾病的疗效。这类钙敏感受体激动剂化合物可以显著提升慢性肾病患者治疗的依从性和顺应性。
WO2021115272描述了一系列多肽钙敏感受体激动剂化合物,其中式(I)化合物对人类钙敏感受体具有激动剂作用从而降低血浆甲状旁腺激素和血清钙离子水平,并可以用于继发性甲状旁腺功能亢进,肿瘤引发的高钙血症等代谢类疾病的治疗。
发明内容
本公开提供一种钙敏感受体激动剂的盐酸盐及其药物组合物和应用。本公开提供的化合物的盐酸盐,溶解度高,稳定性高,不仅可有效降低血浆甲状旁腺激素,而且组胺释放水平低,副作用小。
本公开提供的化合物的盐酸盐,所述化合物的结构如式(I)所示:
本公开中的氨基酸序列采用氨基酸的标准单字母或三字母代码表示,即丙氨酸(Ala,A),半胱氨酸(Cys,C),精氨酸(Arg,R),D-2-氨基丁酸(D-Abu)。本公开式(I)化合物还可用以下序列表示:Ac-c(C)-r-r-(D-Abu)-r-a-r-NH
2。
在某些实施方式中,所述式(I)化合物盐酸盐中式(I)化合物结合盐酸的 个数可以为1-10(可以是1-10之间的任意数值,即平均值),优选盐酸个数为4-8,更优选4-5,可选盐酸个数包括但不限于1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10。
本公开提供一种上述的化合物的盐酸盐在制备用于降低受试者甲状旁腺激素水平,治疗继发性甲状旁腺功能亢进或肿瘤引发的高钙血症的药物中的用途。
本公开还提供一种上述的化合物的盐酸盐,所述盐酸盐用于降低受试者甲状旁腺激素水平,治疗继发性甲状旁腺功能亢进或肿瘤引发的高钙血症。
在一些实施方案中,所述甲状旁腺功能亢进为罹患慢性肾病的受试者的继发性甲状旁腺功能亢进。
本公开另一方面提供一种制备上述化合物盐酸盐的方法,包括将式(I)所示化合物与盐酸混合的步骤。
本公开提供一种制备上述化合物盐酸盐的方法,具体包括:(1)使用树脂通过固相合成依次偶联主链7个氨基酸和乙酸酐得到主链树脂肽;(2)与含有TFA/H
2O/TIS/DPDS的裂解液室温搅拌反应得到主链粗肽;(3)与H-L-Cys-OH在水溶液中反应得到粗肽;(4)经过高压制备纯化和纳滤,浓缩得到成品。
WO2021115272公开的内容全文引用到本公开中。
图1为式(I)化合物在体外针对人类红血细胞的溶血效应,其中*:阳性对照(聚乙二醇辛基苯基醚),#:PBS缓冲液;
图2为式(I)化合物在正常大鼠体内降低甲状旁腺激素水平的功效;
图3为式(I)化合物在正常大鼠体内降低血清钙离子水平的功效。
以下将结合实施例更详细地解释本公开,本公开的实施例仅用于说明本公开的技术方案,并非限定本公开的实质和范围,本公开所用药用辅料均可通过商业途径购得。
1、实验试剂
序号 | 试剂 | 来源 |
1 | Rink-amide MBHA树脂 | 西安蓝晓科技 |
2 | HCTU | 苏州昊帆科技 |
3 | 4-甲基吗啉 | TCI Chemicals |
4 | 乙腈(色谱级) | Sigma-Aldrich |
5 | 氮,氮-二甲基甲酰胺 | 国药试剂 |
6 | 二氯甲烷 | 国药试剂 |
7 | 三氟乙酸 | TCI Chemicals |
8 | 三异丙基硅烷 | TCI Chemicals |
9 | 甲基叔丁基醚 | TCI Chemicals |
10 | 4-甲基哌啶 | TCI Chemicals |
11 | L-半胱氨酸 | Sigma-Aldrich |
12 | Fmoc-D-Cys(Trt)-OH | 吉尔生化 |
13 | Fmoc-D-Arg(Pbf)-OH | 吉尔生化 |
14 | Fmoc-D-Ala-OH | 吉尔生化 |
15 | Fmoc-D-Abu-OH | 吉尔生化 |
16 | 2,2-二吡啶二硫醚 | 吉尔生化 |
2、实验仪器
序号 | 仪器 | 来源 |
1 | Prelude-X多通道多肽合成仪 | Protein Technology |
2 | H-CLASS分析型超高效液相色谱 | Waters |
3 | Xevo液相色谱/质谱联用 | Waters |
4 | Labconco多功能冻干机 | Thermo-Fisher Scientific |
5 | Prep150制备型高效液相色谱 | Waters |
6 | 多通道高速离心机 | 希格玛 |
实施例1
在Prelude-X全自动多肽合成仪上使用Fmoc/tBu合成策略进行固相肽合成,从Rink-amide MBHA树脂(0.1mmole)开始,其中使用10当量的用HCTU和4-甲基吗啉活化的氨基酸残基(HCTU、4-甲基吗啉和氨基酸残基三者摩尔比为1:2:1)在氮,氮-二甲基甲酰胺中在室温下进行25分钟偶联。
在完成上述肽-树脂的合成后,在含有90:5:5(v/v/v)的三氟乙酸:三异丙基硅烷:水和2,2-二吡啶二硫醚(1mmole)的溶液中,在室温,2小时同时完成多肽从固相树脂的切割,侧链保护基的去除以及D-Cys侧链巯基的活化。反应结束后过滤并用三氟乙酸洗涤树脂2次,合并滤液后加入大量冷冻甲基叔丁基醚析出固体,离心后除去上清液得到多肽的粗品并进行干燥称重。
将上述得到的多肽粗品和L-Cys(0.1mmole)溶解于PBS缓冲液(pH=7.4)中,在室温下振荡反应并用超高效液相色谱监测实施例编号1的产生。反应完成后往混合液加入三氟乙酸(300ul)猝灭反应并用于后续纯化。
将上述得到的混合液经过0.22um膜过滤后用WATERS Prep150制备型反相高效液相色谱系统进行分离,缓冲液为A(0.1%三氟乙酸,水溶液)和B(0.1%三氟乙酸,90%乙腈,水溶液)。其中,制备色谱柱为X-SELECT OBD C-18(WATERS)反相色谱柱,纯化过程中色谱仪检测波长设定为220nm,流速为15mL/min。收集产物相关馏分冻干后得到实施例编号1的多肽纯品,收率45%。多肽纯品通过分析型超高效液相色谱和超高效液相色谱/质谱联用确定纯度及化合物身份,其中化合物纯度为96.78%,化合物分子量为:1109.60Da。
化合物编号 | 序列 |
1 | Ac-c(C)-(D-Phg)-r-r-r-a-r-NH 2 |
“Ac-c(C)”表示氨基末端的D构型半胱氨酸(c)被乙酰化,并通过二硫键与L构型(C)的另一半胱氨酸连接;“r-NH
2”表示羧基末端的D构型精氨酸(r)被酰胺化。
实施例2
采用与实施例1类似的合成方案合成式(I)化合物,并用分析型超高效液相色谱和超高效液相色谱/质谱联用确定合成多肽的纯度和分子量,其中式(I)化合物纯度为95.79%,化合物分子量为:1062.29Da。
实施例3
式(I)化合物盐酸盐的制备
1、称取Rink Amide-AM树脂(1034.4g)加入到玻璃反应釜中,再加入DMF溶胀树脂。加入20%PIP/DMF(V/V)溶液,分别反应脱除Fmoc,然后用DMF洗涤,茚三酮检测呈蓝色。称取Fmoc-D-Arg(Pbf)-OH(1167.6g)、Oxyma(383.6g),使用DMF(5.0L)和DCM(5.0L)溶解,然后加入DIC(340.5g)。搅拌混匀加入到玻璃反应釜中反应,以茚三酮检测反应终点(如果树脂无色透明就终止反应,如果树脂有颜色则延长反应时间,下同),反应结束后,依次使用DMF、IPA、DMF、IPA,DMF洗涤树脂。称取Ac
2O(368.1g)、DIEA(467.4g)加入DMF(5.0L)和DCM(5.0L)。搅拌混匀加入到玻璃反应釜中反应,取少量树脂茚三酮检测至反应终点,反应结束后,依次使用DMF、IPA、DMF、IPA,DMF洗涤树脂。
2、按照相同的偶联方法,依次偶联后续的氨基酸,得 N-Ac-D-Cys(Trt)-D-Arg(Pbf)-D-Arg(Pbf)-D-Abu-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Rink Amide Resin 2.5Kg。
3、将TFA/TIS/H2O(V:V:V=97:2.5:0.5)裂解液加入到50L反应器中,再加入DPDS(793.2g),搅拌溶解后。将2中的产物(2.5Kg)加入到反应器中,室温搅拌反应,过滤。滤液加入到IPE/ACN(V:V=7:1)中,收集前体多肽850g,收率75.4%。
4、将6.73L水加入到20L反应器中,加入H-L-Cys-OH.HCl.H
2O(118.4g),搅拌溶解后,加入3的前体多肽(850g)反应。结束后缓慢冲析入0.01moL/L的HCl/IPA溶液中,搅拌后离心,收集滤饼。干燥后得到目标多肽粗品700g,收率79%。
5、取4中的粗肽700g,使用汉邦纯化系统,波长254nm,填料C18,流动相0.1%TFA/H
2O和0.1%TFA/乙腈,进行纯化,收集目标峰馏分,得到纯化液90L,工序收率64.1%。
6、使用纳滤系统,流加盐酸溶液,同时除去样品溶液中的三氟乙酸根,得到式(I)化合物盐酸盐溶液,浓缩和冻干后,得到式(I)化合物盐酸盐240g,收率90%,质谱信号为1061.5367Da(理论值1061.5447Da),HPLC纯度98.33%,总收率34.4%。
根据中国药典2020年版四部通则0701电位滴定法,测得式(I)化合物结合盐酸的个数为4.4。
实施例4
式(I)化合物盐酸盐的理化性质考察
考察实施例1制得的式(I)化合物盐酸盐的理化性质和溶解度,结果见下表。
实施例5
式(I)化合物盐酸盐的稳定性考察
将实施例1制得的式(I)化合物盐酸盐分别在40℃±2℃和RH75%±5%、25℃和总照度大于1.2×106Lux·hr、近紫外能量大于200w·hr/m
2、25℃、RH60%条件下分别放置一段时间,考察其稳定性,结果见下表。
结论:在高温条件下,放置30天,水分、盐酸含量指标均基本不变,杂质总量增加。在高湿条件下,放置10天,水分增加明显,杂质总量也有增加。在光照条件下放置30天,水分、盐酸含量均基本不变,杂质总量增加明显。
将实施例1制得的式(I)化合物盐酸盐在5℃±3℃条件下进行加速试验,结果见下表。
结论:在加速和长期条件下考察3个月,三批样品的各项指标均基本不变,可见在5℃±3℃条件下,式I化合物的稳定性良好。
实施例6
以下结合本公开中的具体实施例进一步描述解释本公开,但这些实施例并非意味着限制本公开的范围。
1、体外,体内生物学测试评价所需实验试剂
序号 | 试剂 | 来源 |
1 | FBS,500ml | ThermoFisher Scientific |
2 | DMEM,High Glucose,GlutaMAX,500ml | ThermoFisher Scientific |
3 | Penicillin-Streptomycin,Liquid,100ml(100X) | ThermoFisher Scientific |
4 | 1X PBS pH 7.2-7.4(500ml) | Solarbio |
5 | 1X TrypLE Express Enzyme,no phenol red(500ml) | ThermoFisher Scientific |
6 | Hygromycin B Gold solution(5g,1x 50ml,100mg/ml) | Invivogen |
7 | HEPES,1M | Gibco |
8 | MgCl 2,1M | Sigma-Aldrich |
9 | KCl,1M | Sigma-Aldrich |
10 | NaCl,5M | Sigma-Aldrich |
11 | Glucose | Sigma-Aldrich |
12 | LiCl,8M | Sigma-Aldrich |
13 | CaCl 2,1M | Sigma-Aldrich |
14 | IP-One-Gq Kit(1,000tests) | Cisbio |
2、实验仪器
序号 | 仪器 | 来源 |
1 | EnVision检测器 | Perkin Elmer |
评估式(I)化合物对人类钙敏感受体(CaSR)的激动剂活性
实验方法:
将HEK293/CaSR稳转细胞株(来源:康龙化成)培养在完全培养基(成分:DMEM,high glucose+10%FBS+2mM GlutaMAX+1X Penicillin-Streptomycin+200μg/ml Hygromycin B),于37℃,5%CO2环境中孵育至70%-90%融合度。细胞株经TrypLE消化处理后接种于384孔细胞培养板,在37℃,5%CO2中培养过夜。细胞换液后,加入刺激缓冲液(HEPES 10mM,MgCl2 0.5mM,KCl 4.2mM,NaCl 146mM,葡萄糖5.5mM,LiCl 50mM,CaCl2 1.2mM)和不同浓度的待测实施例化合物,并于37℃下孵育60分钟,按照Cisbio IP-One Tb试剂盒说明书中的步骤检测细胞中IP-One的产生。收集各实施例的原始数据后利用软件计算各待测实施例在人类钙敏感受体的EC50值,并以此评价实施例针对人类钙敏感受体的激动剂活性。
实验数据处理方法:
使用EnVision检测器进行HTRF的信号读取,激发波长为320nm,发射波长为620nm和665nm。计算信号比值(665nm/620nm*10,000),并在GraphPad Prism 6中将信号比值与样品浓度使用四参数方程进行非线性拟合,得出待测实施例1的EC50值,具体数值见下表1。
表1体外钙敏感受体激动剂活性
实施例编号 | 序列 | 钙敏感受体EC 50(uM) |
式(I)化合物 | Ac-c(C)-r-r-(D-Abu)-r-a-r-NH 2 | 6.28 |
依特卡肽 | Ac-c(C)-a-r-r-r-a-r-NH 2 | 6.78 |
依特卡肽类似物 | Ac-c(C)-r-r-a-r-a-r-NH 2 | 6.74 |
本公开的式(I)化合物具有优良的体外功效,对应于在体外人类钙敏感受体激动剂活性评估中低于10uM的EC50值,与阳性药依特卡肽的活性相当。
评估式(I)化合物在大鼠腹膜肥大细胞上的体外组胺释放活性
实验方法和数据处理:
为评估部分待测实施例的体外组胺释放水平,通过灌洗缓冲液(含肝素5U/mL的冷HBSS+25mM HEPES,pH 7.4)灌洗大鼠腹膜分离大鼠腹膜肥大细胞。分离后,离心细胞,去除灌洗缓冲液,加入刺激缓冲液(HBSS+25mM HEPES+1mM CaCl2,pH 7.4)重悬细胞并洗涤两次。按105细胞/孔密度铺板(200μl/孔),分别加入阳性对照Compound 48/80(终浓度4μg/mL)、待测实施例化合物(最终浓度10μM)或溶媒对照,37℃孵育15min。离心,取细胞上清液,按照LDN Histamine ELISA试剂盒(BAE-1000)说明书检测上清液的组胺浓度。具体数值见下表2。
表2体外组胺释放副反应水平
实施例编号 | 体外相对组胺释放倍数 |
PBS缓冲液 | 1.00 |
式(I)化合物 | 0.97 |
依特卡肽 | 1.70 |
依特卡肽会较明显地引起体外大鼠腹膜肥大细胞的组胺释放,具体体现在相对于PBS缓冲液时相对组胺释放倍数高于1.50。令人意想不到的是,式(I)化合物相对依特卡肽体外大鼠腹膜肥大细胞组胺释放水平大大降低。
评估式(I)化合物在体外对人红血细胞的溶血效应
实验方法和数据处理:
为评估实施例化合物在体外对红血细胞的溶血效应,取人全血(100ul)并与磷酸盐缓冲液混匀,在4℃条件下离心10分钟后丢弃上清液。用PBS缓冲液(900ul)重悬红细胞后在4℃条件下离心10分钟后丢弃上清液并重复上述步骤一次。将式(I)化合物溶解在1×PBS缓冲液中,终浓度为100ug/ml。分别利用待测实施例化合物溶液,聚乙二醇辛基苯基醚-100溶液以及PBS缓冲液重悬红细胞并在37℃条件下孵育1小时。孵育后在4℃条件下离心10分钟并抽取上清液(100ul),转移至96孔板后在540nm处测定吸光度并以此评估式(I)化合物在体外对红血细胞的溶血效应。
3.3.3实验结果
式(I)化合物在100ug/ml的浓度下未观测到明显的红细胞溶血效应,与之相对应聚乙二醇辛基苯基醚-100溶液在实验条件下观测到明显的红细胞溶血效 应,见附图1。
评估式(I)化合物在正常大鼠模型上单次给药后的体内药效
实验方法和数据处理:
试验选用SPF级正常成年大鼠(Sprague Dawley,SD),体重为250~350克,在动物房中正常饮食恢复7天。大鼠随机分组,每组6只,雌雄各半,分别编号。实验开始前一天,每只大鼠采血540μL,检测血浆甲状旁腺素水平和血清钙离子浓度作为给药前的对照值。血浆分离方法为采用K2-EDTA抗凝、经颈静脉采血,采集后置于冰上,随后将全血在2~8℃条件下以6,800rpm离心6分钟,轻轻取出上层即为血浆,2~8℃保存。血清分离方法为经颈静脉采血,将全血室温静置1小时,然后在室温,3,500rpm转速下离心10分钟,轻轻取出上层,即为血清,室温保存。实验前一天开始,动物禁食过夜,自由饮水。采血后次日,式(I)化合物和依特卡肽溶于磷酸盐缓冲液(Phosphate buffered saline,PBS,Gibco),每只大鼠静脉注射给予式(I)化合物及依特卡肽3mg/kg或等体积的PBS缓冲溶液,随后按照如下方法采血测定相应指标。给药后1小时、2小时、4小时分别采血100μL,按照上述方法分离血浆,采用Rat Intact PTH ELISA Kit(Quidel-Immunotopics,Cat.#:60-2500),按照试剂盒说明书进行血浆甲状旁腺激素水平的测定(ELISA:Enzyme-linked immunosorbent assay,酶联免疫吸附测定)。详细步骤为:采用试剂盒提供的抗生蛋白链菌素预铺的反应条,每孔中加入25μL的标准品、对照品或血浆样品。按照1:1混合生物素化的大鼠甲状旁腺激素抗体和大鼠甲状旁腺激素/HRP结合抗体,并在每孔中加入100μL的此混合溶液。使用密封薄膜封闭反应条,并使用铝箔包裹反应条以避光保存,室温条件下在水平振荡器上振荡以220rpm的转速振荡3h。移去孔中溶液,以350μL的清洗工作液洗涤每孔,再移去孔中溶液;以同样方法洗涤总共5次,最后吸净每孔中的溶液。在每个孔中加入150μL的辣根过氧化物酶ELISA底物。使用密封薄膜封闭反应条,并使用铝箔包裹反应条以避光保存,室温条件下在水平振荡器上以180~220rpm的转速振荡30min。在每个孔中加入100μL的ELISA终止液,室温条件下在水平振荡器上以180~220rpm的转速振荡1分钟。在加入ELISA终止液后的10min内,在450纳米处读取每个孔的吸光度,同时以620nm处的吸光度作为背景扣除。以150μL的辣根过氧化酶ELISA底物加上100μL的ELISA终止液 作为吸光度测量的空白对照。根据标准品的吸光度绘制标准曲线,再将其它样品的吸光度结合标准曲线计算出血浆甲状旁腺激素的实际浓度,血清钙离子浓度的测定按相关试剂盒的步骤进行。
实验结果
式(I)化合物在3mg/kg的剂量下4小时内完全降低正常大鼠血浆甲状旁腺激素水平,血清钙离子水平也相应的得到降低,见附图2和附图3。
Claims (6)
- 一种化合物的盐酸盐,所述化合物的结构如式(I)所示:
- 一种药物组合物,其包含如权利要求1中所述化合物的盐酸盐。
- 如权利要求1所述的化合物的盐酸盐或如权利要求2所述的药物组合物在制备用于治疗甲状旁腺激素水平异常相关疾病的药物中的用途。
- 如权利要求3所述用途,其特征在于,所述的甲状旁腺激素水平异常相关疾病为甲状旁腺功能亢进。
- 如权利要求4所述用途,其特征在于,所述的甲状旁腺功能亢进为罹患慢性肾病的受试者的继发性甲状旁腺功能亢进。
- 一种制备如权利要求1所述化合物盐酸盐的方法,包括将式(I)所示化合物与盐酸混合的步骤。
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