Denosumab
(Synonyms: 迪诺苏单抗,D03684; Denosumab; Denosumab (usan)) 目录号 : GC19535
地诺单抗是一种人单克隆抗体，可结合并抑制 RANKL 的受体激活剂 (TNFSF11)。地诺单抗可以降低椎体、非椎体和髋部骨折的风险，还具有抗癌活性。
Cas No.:615258-40-7
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
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Purity: ≥99.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
RAW 264.7, MCF-7 cells |
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Preparation Method |
RAW 264.7 and MCF-7 cells were all cultured in Dulbecco’s Modified Eagle Medium (DMEM) solutions, into which 10% fetal bovine serum was added, while RAW 264.7 cells were co-cultured with MCF-7 cells in a non-contact system. |
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Reaction Conditions |
1 mg/mL, 5 days |
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Applications |
Denosumab inhibits MCF-7 cell line-induced spontaneous osteoclastogenesis, and the inhibition of denosumab was found to be more pronounced after MALAT1 downregulation and miR-124 overexpression |
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References: [1]. Feng, Qi et al. Denosumab inhibits MCF-7 cell line-induced spontaneous osteoclastogenesis via the RANKL/MALAT1/miR-124 axis. Translational cancer research vol. 9,4 (2020): 2482-2491. |
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Denosumab is a fully human IgG2 monovlonal antibody that binds human RANKL with a high affinity, exhibiting a dissociation equilibrium binding constant (Kd) of 3 pM as determined by immunoaffinity exclusion chromatography. Denosumab binds both soluble and membrane-bound primate RANKL but fails to recognize mouse or rat RANKL- a finding that is supported by sequence analysis of RANKL from diverse mammalian species. [2]
Initial screening of RANKL antibodies for in vivo bioactivity leveraged the crossreactivity of Denosumab with cynomolgus RANKL. Single-dose testing in cynomolgus monkeys revealed that infrequent dosing regimens in humans may be possible. The primary in vivo testing in primates accelerated the developmental timeframe of this molecule, which normally would have been preceded by extensive testing in animal models of bone loss in which recombinant OPG was active. [2]
Denosumab does not bind to other TNF family members, such as TRAIL, CD40 ligand (CD40L), TNFα and THFβ. Denosumab binds to the DE loop region of human RANKL, which is one of the surface loop structures that forms contacts with RANK on responding cells. Owing to the different terminology used for the loop regions, the human DE loop region corresponds to the CD loop regions of the murine RANKL structure. Both Denosumab and OPG-Fc bind to soluble or membrane-bound human RANKL and block it from binding to and oligomerizing its receptor, RANK. Denosumab, however, is more specific than human OPG-Fc because Denosumab recognizes only human and non-human primate RANKL, in contrast to OPG, which also binds to mouse and rat RANKL as well as TRAIL. [1,2]
References:
[1]. Feng, Qi et al. Denosumab inhibits MCF-7 cell line-induced spontaneous osteoclastogenesis via the RANKL/MALAT1/miR-124 axis.” Translational cancer research vol. 9,4 (2020): 2482-2491.
[2]. Lacey, David L et al. Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of Denosumab. Nature reviews. Drug discovery vol. 11,5 (2012): 401-19.
Denosumab 是一种全人 IgG2 单克隆抗体，它以高亲和力结合人 RANKL，通过免疫亲和排阻色谱法测定其解离平衡结合常数 (Kd) 为 3 pM。 Denosumab 结合可溶性和膜结合的灵长类动物 RANKL，但无法识别小鼠或大鼠 RANKL - 这一发现得到了来自不同哺乳动物物种的 RANKL 序列分析的支持。 [2]
RANKL 抗体的体内生物活性初步筛选利用了地诺单抗与食蟹猴 RANKL 的交叉反应性。在食蟹猴中进行的单剂量试验表明，人类可能采用不频繁的给药方案。在灵长类动物中进行的主要体内测试加速了该分子的发育时间框架，通常在重组 OPG 活跃的骨质流失动物模型中进行广泛测试之前。 [2]
Denosumab 不与其他 TNF 家族成员结合，例如 TRAIL、CD40 配体 (CD40L)、TNFα 和 THFβ。 Denosumab 结合人 RANKL 的 DE 环区域，它是与反应细胞上的 RANK 形成接触的表面环结构之一。由于用于环区域的不同术语，人 DE 环区域对应于鼠 RANKL 结构的 CD 环区域。 Denosumab 和 OPG-Fc 均与可溶性或膜结合的人 RANKL 结合，并阻止其与其受体 RANK 结合并寡聚化。然而，Denosumab 比人 OPG-Fc 更具特异性，因为 Denosumab 仅识别人和非人灵长类动物 RANKL，与 OPG 相比，OPG 还结合小鼠和大鼠 RANKL 以及 TRAIL。 [1,2]
| Cas No. | 615258-40-7 | SDF | |
| 别名 | 迪诺苏单抗,D03684; Denosumab; Denosumab (usan) | ||
| 分子式 | C6404H9908N1724O2004S50 | 分子量 | 144716.86 |
| 溶解度 | 储存条件 | Store at 4°C, Do not freeze | |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.0069 mL | 0.0346 mL | 0.0691 mL |
| 5 mM | 0.0014 mL | 0.0069 mL | 0.0138 mL |
| 10 mM | 0.0007 mL | 0.0035 mL | 0.0069 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Stopping Denosumab
Curr Osteoporos Rep2019 Feb;17(1):8-15.PMID: 30659428DOI: 10.1007/s11914-019-00502-4
Purpose of review: Denosumab discontinuation is associated with a rebound effect manifesting by an increased risk of multiple spontaneous vertebral fractures. The purpose of this review is to (1) better characterize this risk and (2) find solutions to avoid it.
Recent findings: In the absence of a potent bisphosphonate prescription at denosumab discontinuation, the frequency of multiple vertebral fractures is common or frequent (≥ 1/100 and < 1/10). In five recent case series, the median number of vertebral fractures was 5 within 7 to 20 months (median 11) after the last denosumab injection. Prescribing bisphosphonate before starting denosumab and/or after stopping denosumab may reduce this risk. However, only small case series have evaluated these strategies. After the second denosumab dose, there is a rebound effect with an increased risk of multiple vertebral fractures. A potent bisphosphonate prescribed at denosumab discontinuation could reduce this risk. As denosumab discontinuation is characterized by many uncertainties, denosumab is a second-line treatment for osteoporosis. Studies are urgently needed to define the management of denosumab discontinuation.
Denosumab: A Review in Postmenopausal Osteoporosis
Drugs Aging2018 Feb;35(2):163-173.PMID: 29435849DOI: 10.1007/s40266-018-0525-7
Denosumab (Prolia®; Pralia®) is a human monoclonal antibody targeting the key bone resorption mediator RANKL. The drug is administered via subcutaneous injection once every 6 months and is approved for various indications, including the treatment of postmenopausal (PM) women with osteoporosis at increased/high risk of fracture or failure/intolerance of other osteoporosis therapies (indications featured in this review). Denosumab showed benefit in several phase 3 or 4 studies in PM women with osteoporosis or low bone mineral density (BMD), including the pivotal 3-year double-blind FREEDOM trial and its 7-year open-label extension. Denosumab reduced the risk of vertebral, nonvertebral and hip fractures and increased BMD across skeletal sites versus placebo in FREEDOM, with these benefits maintained over up to 10 years' therapy in the extension. The drug was also more effective in improving BMD than bisphosphonates, including in women switched from a bisphosphonate regimen, in 1-year trials; however, whether these differences translate into differences in anti-fracture efficacy is unclear. Denosumab was generally well tolerated over up to 10 years' treatment, although an increased risk of multiple vertebral fractures was observed after discontinuation of the drug. Thus, denosumab is a key treatment option for PM women with osteoporosis who have an increased/high risk of fracture or failure/intolerance of other osteoporosis therapies, although the potential for multiple vertebral fractures to occur after discontinuation of the drug requires consideration of subsequent management options.
Discontinuation of Denosumab therapy for osteoporosis: A systematic review and position statement by ECTS
Bone2017 Dec;105:11-17.PMID: 28789921DOI: 10.1016/j.bone.2017.08.003
Introduction: The optimal duration of osteoporosis treatment is controversial. As opposed to bisphosphonates, denosumab does not incorporate into bone matrix and bone turnover is not suppressed after its cessation. Recent reports imply that denosumab discontinuation may lead to an increased risk of multiple vertebral fractures.
Methods: The European Calcified Tissue Society (ECTS) formed a working group to perform a systematic review of existing literature on the effects of stopping denosumab and provide advice on management.
Results: Data from phase 2 and 3 clinical trials underscore a rapid decrease of bone mineral density (BMD) and a steep increase in bone turnover markers (BTMs) after discontinuation of denosumab. Clinical case series report multiple vertebral fractures after discontinuation of denosumab and a renewed analysis of FREEDOM and FREEDOM Extension Trial suggests, albeit does not prove, that the risk of multiple vertebral fractures may be increased when denosumab is stopped due to a rebound increase in bone resorption.
Conclusion: There appears to be an increased risk of multiple vertebral fractures after discontinuation of denosumab although strong evidence for such an effect and for measures to prevent the occurring bone loss is lacking. Clinicians and patients should be aware of this potential risk. Based on available data, a re-evaluation should be performed after 5years of denosumab treatment. Patients considered at high fracture risk should either continue denosumab therapy for up to 10years or be switched to an alternative treatment. For patients at low risk, a decision to discontinue denosumab could be made after 5years, but bisphosphonate therapy should be considered to reduce or prevent the rebound increase in bone turnover. However, since the optimal bisphosphonate regimen post-denosumab is currently unknown continuation of denosumab can also be considered until results from ongoing trials become available. Based on current data, denosumab should not be stopped without considering alternative treatment in order to prevent rapid BMD loss and a potential rebound in vertebral fracture risk.
A Review on the Role of Denosumab in Fracture Prevention
Drug Des Devel Ther2020 Oct 1;14:4029-4051.PMID: 33061307DOI: 10.2147/DDDT.S270829
Denosumab is a receptor activator of nuclear factor kappa-Β ligand inhibitor, which suppresses the bone resorption process to preserve bone mass. It is usually recommended to postmenopausal women and men with high fracture risk. With the recent publication of the results from FREEDOM study and its extension, the long-term effect of denosumab in preventing fragility fractures has been put forward. This review aims at summarising the evidence of denosumab in reducing fracture risk and its safety derived from clinical studies. Most of the evidence are derived from FREEDOM trials up to 10 years of exposure. Denosumab is reported to prevent vertebral and non-vertebral fractures. It is also proven effective in Japanese women, patients with chronic kidney diseases and breast cancer patients receiving antineoplastic therapy. Denosumab discontinuation leads to high remodeling, loss of bone mineral density and increased fracture risk. These negative effects might be preventable by bisphosphonate treatment. The safety profile of denosumab is consistent with increased years of exposure. In conclusion, denosumab is a safe and effective option for reducing fracture risk among patients with osteoporosis.
10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomised FREEDOM trial and open-label extension
Lancet Diabetes Endocrinol2017 Jul;5(7):513-523.PMID: 28546097DOI: 10.1016/S2213-8587(17)30138-9
Background: Long-term safety and efficacy of osteoporosis treatment are important because of the chronic nature of the disease. We aimed to assess the long-term safety and efficacy of denosumab, which is widely used for the treatment of postmenopausal women with osteoporosis.
Methods: In the multicentre, randomised, double-blind, placebo-controlled, phase 3 FREEDOM trial, postmenopausal women aged 60-90 years with osteoporosis were enrolled in 214 centres in North America, Europe, Latin America, and Australasia and were randomly assigned (1:1) to receive 60 mg subcutaneous denosumab or placebo every 6 months for 3 years. All participants who completed the FREEDOM trial without discontinuing treatment or missing more than one dose of investigational product were eligible to enrol in the open-label, 7-year extension, in which all participants received denosumab. The data represent up to 10 years of denosumab exposure for women who received 3 years of denosumab in FREEDOM and continued in the extension (long-term group), and up to 7 years for women who received 3 years of placebo and transitioned to denosumab in the extension (crossover group). The primary outcome was safety monitoring, comprising assessments of adverse event incidence and serious adverse event incidence, changes in safety laboratory analytes (ie, serum chemistry and haematology), and participant incidence of denosumab antibody formation. Secondary outcomes included new vertebral, hip, and non-vertebral fractures as well as bone mineral density (BMD) at the lumbar spine, total hip, femoral neck, and one-third radius. Analyses were done according to the randomised FREEDOM treatment assignments. All participants who received at least one dose of investigational product in FREEDOM or the extension were included in the combined safety analyses. All participants who enrolled in the extension with observed data were included in the efficacy analyses. The FREEDOM trial (NCT00089791) and its extension (NCT00523341) are both registered with ClinicalTrials.gov.
Findings: Between Aug 3, 2004, and June 1, 2005, 7808 women were enrolled in the FREEDOM study. 5928 (76%) women were eligible for enrolment in the extension, and of these, 4550 (77%) were enrolled (2343 long-term, 2207 crossover) between Aug 7, 2007, and June 20, 2008. 2626 women (1343 long-term; 1283 crossover) completed the extension. The yearly exposure-adjusted participant incidence of adverse events for all individuals receiving denosumab decreased from 165·3 to 95·9 per 100 participant-years over the course of 10 years. Serious adverse event rates were generally stable over time, varying between 11·5 and 14·4 per 100 participant-years. One atypical femoral fracture occurred in each group during the extension. Seven cases of osteonecrosis of the jaw were reported in the long-term group and six cases in the crossover group. The yearly incidence of new vertebral fractures (ranging from 0·90% to 1·86%) and non-vertebral fractures (ranging from 0·84% to 2·55%) remained low during the extension, similar to rates observed in the denosumab group during the first three years of the FREEDOM study, and lower than rates projected for a virtual long-term placebo cohort. In the long-term group, BMD increased from FREEDOM baseline by 21·7% at the lumbar spine, 9·2% at total hip, 9·0% at femoral neck, and 2·7% at the one-third radius. In the crossover group, BMD increased from extension baseline by 16·5% at the lumbar spine, 7·4% at total hip, 7·1% at femoral neck, and 2·3% at one-third radius.
Interpretation: Denosumab treatment for up to 10 years was associated with low rates of adverse events, low fracture incidence compared with that observed during the original trial, and continued increases in BMD without plateau.
Funding: Amgen.