Parathyroid hormone receptor 1 (PTHR1)[1]

Abaloparatide TFA（0-100 nM；40 分钟）可改善 MC3T3-E1 细胞中的 β-arrestin 募集和 Gs/cAMP 信号传导 [1]。 β-寄生虫 TFA (0-100 nM) 的 EC50 值为 0.8 nM，以剂量依赖性方式有效导致 U2OS 细胞中 PTHR1 内化 [1]。

特利帕肽和Abaloparatide/阿巴帕肽是甲状旁腺激素受体1 (PTHR1)类似物，治疗骨质疏松症的疗效差异不明。因此，我们比较了Abaloparatide/阿巴帕肽和特立帕肽对骨结构、代谢、核因子- κ B配体受体激活剂(RANKL)和骨保护素(OPG)水平的影响。野生型(WT)雌性小鼠每天注射载药或20-80µg/kg/天的特立帕肽或阿巴帕肽，持续30天。采用微计算机断层扫描检测股骨和脊柱，ELISA检测血清骨转换标志物RANKL和OPG水平。在20-80µg/kg/天的剂量下，这两种类似物同样增加了股骨远端分数小梁骨的体积、连通性和数量，并降低了结构模型指数(SMI)。然而，当剂量为20µg/kg/天时，只有Abaloparatide/阿巴帕肽的小梁厚度显著增加(13%)。股骨皮质评估显示Abaloparatide/阿巴帕肽比特立帕肽引起更大的剂量依赖性皮质厚度增加。特立帕肽和Abaloparatide/阿巴帕肽都增加了腰椎小梁连通性，但对其他指标没有影响或影响不大。生化分析表明，Abaloparatide/阿巴帕肽可促进前胶原1型完整n端前肽(骨形成标志物)和抗酒石酸酸性磷酸酶5b(骨吸收标志物)水平的升高，并降低RANKL/OPG比值。此外，PTHR1信号在0-100 nmol/L类似物处理的细胞中比较。有趣的是，Abaloparatide/阿巴帕肽的cAMP形成(2.3倍)和β-阻滞蛋白募集(1.6倍)的EC50明显低于特立帕肽。因此，Abaloparatide/阿巴帕肽肽改善的疗效可归因于增强骨形成和皮质结构，降低RANKL/OPG比率，增强Gs-cAMP和β-阻滞素信号[1]。

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Abaloparatide是一种新型的34个氨基酸的肽，被认为是甲状旁腺激素受体(PTH1R)信号通路的有效和选择性激活剂，与PTH(1-34)同源性为41%，与PTHrP(1-34)同源性为76%。对去卵巢成骨减少(OVX)大鼠进行了一项为期12个月的治疗研究，以表征Abaloparatide/阿巴帕肽增加骨量的机制。SD (Sprague-Dawley)大鼠在6月龄时接受OVX或假手术治疗，并不治疗3个月，以允许OVX引起的骨质流失。10只OVX大鼠在骨衰竭期后被安乐死，其余OVX大鼠每天皮下注射1、5或25 μg/kg/d (n = 18/剂量水平)的运载体(n = 18)或阿巴巴拉肽，持续12个月。假对照组(n = 18)每天接受车辆。纵向评估骨密度测定和骨形成和骨吸收生化指标，尸检时采集L3椎和胫骨进行组织形态学测定。鲍巴总苷增加生化骨形成标志物，但不增加骨吸收标志物或引起高钙血症。鲍巴肽增加骨小梁、皮质内和骨膜表面骨形成的组织形态学指标，而不增加破骨细胞或侵蚀表面。Abaloparatide/阿巴帕肽诱导骨小梁体积和密度的显著增加以及骨小梁微结构的改善。鲍巴肽刺激骨膜扩张和胫骨骨干皮质内骨移位，导致皮质骨体积和密度显著增加。服用Abaloparatide/阿巴帕肽(25 μg/kg) 12个月后，OVX-Vehicle对照组的全身骨密度(BMD)保持稳定，增加25%。组织形态学和生物标志物数据表明，皮质和小梁骨量的增加可归因于阿巴巴拉肽的选择性合成代谢作用，而没有证据表明刺激骨吸收。©2016美国骨与矿物研究协会。[2]

PathHunter®eXpress PTHR1 CHO‐K1 β‐抑制素GPCR检测[1]
为了评估Abaloparatide和teriparatide刺激PTHR1对β -抑制素向细胞膜募集的影响，采用PathHunter eXpress PTHR1中国仓鼠卵巢- K1 (CHO - K1) β -抑制素GPCR检测。该分析利用了酶片段互补技术。PTHR1与一个小的酶供体片段ProLink™(PK)融合在框架中，并在CHO - K1细胞中共表达，稳定地表达β -阻滞蛋白和较大的N端缺失β -半乳糖苷酶突变体(称为酶受体或EA)的融合蛋白。PTHR1的激活刺激β -阻滞蛋白与PK -标记的GPCR结合，并迫使两个酶片段互补，从而形成活性β -半乳糖苷酶。然后使用化学发光PathHunter检测试剂测量酶活性的增加。细胞播种、孵育和检测按照制造商的指示进行。简单地说，将细胞接种于透明底白色96孔板中，在37°C CO2培养箱中培养48小时。在37°C的CO2培养箱中，用对照物、特立帕肽或阿巴帕肽处理细胞60分钟。在孵育结束时，在室温下黑暗中添加β - gal酶底物60分钟。利用BMG Labtech PHERAstar FS发光平板阅读器测量β -半胱氨酸酶片段互补和β -抑制素/ PTHR1相互作用的光产生(相对光单位，RLU) [1]。

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PathHunter®eXpress PTHR1激活GPCR内化试验[1]
为了确定PTHR1的内化，我们使用了PathHunter eXpress PTHR1 U2OS激活GPCR内化实验。PTHR1激活的GPCR内化U2OS细胞系被设计为共表达一个未标记的PTHR1，一个EA标记的β -阻滞蛋白和一个定位于核内体的PK标记。激活未标记的PTHR1诱导β -抑制素募集，随后在PK -标记的内体中内化GPCR - β -抑制素- EA复合物。类似于β -抑制素分析格式，这种内化迫使两个β -半乳糖酶片段互补，形成水解底物以产生化学发光信号的功能酶。U2OS成骨细胞系播种、孵育和检测按照制造商的指示进行。在37°C的CO2培养箱中，用对照物、特立帕肽或阿巴帕肽处理细胞60分钟。在孵育结束时，在室温下黑暗中添加β - gal酶底物60分钟。利用BMG Labtech PHERAstar FS发光平板阅读器测量β - gal酶片段互补和β -抑制素/内体/PTHR1形成的光产生(RLU)。

细胞内cAMP生成的测定[1]
将MC3T3‐E1细胞以40000个细胞/孔的速度接种于含有500 μ L α‐MEM的24孔板上，其中α‐MEM中添加10%胎牛血清和1% PS。培养1周后，将培养基取出，并用250 μ L刺激培养基(α‐MEM中含有0.05%胎牛血清、0.1%牛血清sa、5 mmol/L hepes缓冲液和0.5 mmol/L IBMX)替换15分钟。IBMX是一种磷酸二酯酶抑制剂，可防止生成的cAMP降解。然后在250µL刺激培养基中加入载体、Abaloparatide和特立帕肽，最终浓度分别为0、0.01、0.1、1、10和100 nmol/L/孔。37°C孵育40分钟，然后取出培养基，在N3液体中快速冷冻，保存在- 80°C。细胞内cAMP的提取，加入100 mmol/L的Hcl，细胞在室温下孵育1小时。使用cAMP竞争性ELISA试剂盒，按照制造商的方案和说明检测细胞内cAMP。

Animal/Disease Models: Female SD (Sprague-Dawley) rats (age 22 weeks)[2]
Doses: 1 µg/kg, 5 µg/kg, 25 µg/kg
Route of Administration: subcutaneous (sc) injection; daily; for 12 months
Experimental Results: Increased biochemical bone formation markers, histomorphometric indices of bone formation on trabecular, endocortical, and periosteal surfaces. Induced substantial increases in trabecular bone volume and density and improvements in trabecular microarchitecture. Stimulated periosteal expansion and endocortical bone apposition at the tibial diaphysis, leading to marked increases in cortical bone volume and density. Whole-body bone mineral density (BMD) was increasing 25%.

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Animal/Disease Models: 16weeks old wild-type (WT) female C57BL/6J mice[1]
Doses: 20-80 µg/kg
Route of Administration: Sc; daily for 30 days
Experimental Results: Efficiently expanded cortical thickness (Ct. Th) at both doses of 20 and 80 µg/kg/day by 17% and 18%, respectively, increased P1NP levels to 227% and 407% at 20 and 80 µg /kg/day, respectively.

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16-week-old wild-type (WT) female C57BL/6J mice[1]
20-80 µg/kg
S.c.; daily for 30 days

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All experiments were conducted on 16‐week‐old wild‐type (WT) female C57BL/6J mice (Stock number 664). Vehicle (0.9% NaCl/10 mmol/L acetic acid) or 20–80 µg/kg/day teriparatide or abaloparatide was injected subcutaneously (SC) daily (except Sunday) and continued for 30 days. No peptide injection was performed on the day of animal sacrifice.[1]

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A total of 13 rats were euthanized or found dead between study days 117 to 358 before study termination: 6 in the sham control group, 2 in the OVX-Veh group, 3 in the OVX + abaloparatide 1 μg/kg/d group, 1 in the OVX + abaloparatide 5 μg/kg/d group, and 1 in the OVX + abaloparatide 25 μg/kg/d group. For these animals’ data, absolute values were reported if collected, and data based on % change from baseline were censored as required. Five deaths were likely secondary to complications from blood collection, whereas the remaining deaths were attributed to incidental age-related pathologies.[1]
Study design and dose selection[1]
After a 13-week postsurgical bone depletion period, one group of untreated OVX rats was euthanized as a pretreatment baseline group for histomorphometry data. The remaining groups were given daily s.c. injections of vehicle (Vehicle; 0.9% sodium chloride) or one of three dose levels of abaloparatide in a 0.1 mL/kg volume. Abaloparatide dose levels were 1 μg/kg/d (OVX-ABL1), 5 μg/kg/d (OVX-ABL5), and 25 μg/kg/d (OVX-ABL25), with dosing guided by weekly body weight measurements. Preliminary results from another rat study indicated that 6 weeks of abaloparatide at 1.25 μg/kg/d completely reversed OVX-induced bone loss (Radius Health, Inc., Waltham, MA, USA). This led to selection of 1 μg/kg as the low dose, and also 5- and 25-fold multiples of this dose to provide safety margins.[1]

Absorption
The absolute bioavailability of abaloparatide in healthy women after subcutaneous administration of an 80 mcg dose was 36%. Following subcutaneous administration of 80 mcg abaloparatide in postmenopausal women with osteoporosis for seven days, the mean (SD) Cmax was 812 (118) pg/mL and the AUC0-24 was 1622 (641) pgxhr/mL. The median Tmax was 0.51 hours, with a range from 0.25 to 0.52 hours.

Route of Elimination
The peptide fragments of abaloparatide are primarily eliminated through renal excretion.

Volume of Distribution
The volume of distribution was approximately 50 L.

Clearance
The mean apparent total plasma clearance for subcutaneous administration is 168 L/h in healthy subjects.

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Metabolism / Metabolites
Abaloparatide is metabolized into smaller peptide fragments via non-specific proteolytic degradation.

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Biological Half-Life
The mean half-life of abaloparatide is approximately one hour.

Protein Binding
In vitro, abaloparatide was approximately 70% bound to plasma proteins.

[1]. Sahbani K, et al. Abaloparatide exhibits greater osteoanabolic response and higher cAMP stimulation and β-arrestin recruitment than teriparatide. Physiol Rep. 2019 Oct;7(19):e14225.
[2]. Varela A, et al. One Year of Abaloparatide, a Selective Activator of the PTH1 Receptor, Increased Bone Formation and Bone Mass in Osteopenic Ovariectomized Rats Without Increasing Bone Resorption. J Bone Miner Res. 2017 Jan;32(1):24-33.

Abaloparatide is an N-terminal analog of parathyroid hormone-related protein (PTHrP) and an agonist at the parathyroid hormone type 1 (PTH1) receptor. It is a synthetic 34 amino acid peptide with 41% homology to human parathyroid hormone 1-34 and human PTHrP 1-34. Abaloparatide and PTHrP share the first 21 amino acids and the receptor-activating domain. Abaloparatide is an osteoanabolic agent that stimulates bone formation. It was first approved by the FDA on April 28, 2017, for the treatment of osteoporosis in postmenopausal women and is also used to increase bone density in men with osteoporosis. In October 2022, the EMA's Committee for Medicinal Products for Human Use (CHMP) recommended abaloparatide be granted marketing authorization in Europe and the drug was fully authorized by the European Commission on December 19, 2022.

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Abaloparatide is a 34 amino acid synthetic analog of human parathyroid hormone-related protein (PTHrP) (PTHrP(1-34) analog), with bone-growing and bone density conserving activities. Upon subcutaneous administration, abaloparatide acts similar to PTHrP and targets, binds to and activates parathyroid hormone 1 (PTH1) receptor (PTH1R), a G protein-coupled receptor (GPCR) expressed in osteoblasts and bone stromal cells. PTH1R activates the cyclic AMP (cAMP) signaling pathway and the bone anabolic signaling pathway, leading to bone growth, increased bone mineral density (BMD) and volume. This correlates with increased bone mass and strength and prevents or treats osteoporosis and decreases fractures.

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Drug Indication
Abaloparatide is indicated for the treatment of postmenopausal women with osteoporosis at high risk for fracture (defined as a history of osteoporotic fracture or multiple risk factors for fracture) or patients who have failed or are intolerant to other available osteoporosis therapy. In postmenopausal women with osteoporosis, abaloparatide reduces the risk of vertebral and nonvertebral fractures. Abaloparatide is also indicated to increase bone density in men with osteoporosis at high risk for fracture (defined as a history of osteoporotic fracture or multiple risk factors for fracture) or patients who have failed or are intolerant to other available osteoporosis therapy.

Treatment of osteoporosis in postmenopausal women at increased risk of fracture.
Treatment of osteoporosis.

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Pharmacodynamics
Abaloparatide stimulates bone formation on periosteal, trabecular, and cortical bone surfaces. It increases bone mineral density and bone formation markers in a dose-dependent manner. Abaloparatide causes transient and limited increases in osteoclast bone resorption and increases bone density. In rats and monkeys, abaloparatide exerted anabolic effects, increasing bone mineral density and mineral content correlating with increases in bone strength at vertebral and nonvertebral sites.

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Mechanism of Action
Abaloparatide is an agonist at the PTH1 receptor (PTH1R), a G-protein-coupled receptor (GPCR) that regulates bone formation and bone turnover, as well as mineral ion homeostasis. The PTH1R couples to Gs and Gq, which stimulates adenylyl cyclase (AC), which activates the cAMP/PKA signalling cascade, and phospholipase C (PLC), which activates the IP/PKC signalling cascade. Abaloparatide binds to the PTH1R in target cells to activate the Gs-protein-mediated cAMP signalling pathway, thereby stimulating osteoblastic activity. Abaloparatide also activates Gq and β-arrestin-1 pathway downstream of PTH1R as off-targets in target cells such as the testis and epididymis, which have been associated with anti-inflammatory effects and alleviation of epididymitis and orchitis symptoms. The PTH1R has two conformations with distinct ligand binding profiles. The R0 conformation is a G protein–independent high-affinity conformation, and upon binding, the ligand induces a longer-lasting signalling response that gradually increases cAMP. Due to the prolonged signalling response, ligands selectively binding to the R0 conformation are associated with a risk for increased calcium mobilization and hypercalcemia. Conversely, the RG conformation is G-protein–dependent (GTPγS-sensitive) with a shorter signalling response. Abaloparatide binds to the RG conformation with greater selectivity: it induces more transient signalling responses and favours net bone formation over bone resorption. The drug's relatively low risk for hypercalcemia and osteoclast resorption compared to [teriparatide] is attributed to the preferential binding of abaloparatide to the RG conformation.

C176H301N56F3O51

4074.61

Abaloparatide;247062-33-5

Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-{Aib}-Lys-Leu-His-Thr-Ala-NH2

AVSEHQLLHDKGKSIQDLRRRELLEKLL-{Aib}-KLHTA-NH2

Typically exists as White to off-white solid at room temperature

BIM-44058; Abaloparatide TFA; BA-058; ITM-058; BIM 44058; BA 058;ITM 058; BIM44058; BA058;ITM058; trade name: Tymlos

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~24.54 mM)

如何溶解多肽，详情请参考右上角《产品说明书》第3页：“多肽溶解指南”。

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注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。