VDR/vitamin D receptor; Human Endogenous Metabolite
Calcifediol (25-hydroxy Vitamin D3) targets vitamin D receptor (VDR), a nuclear transcription factor; [2]

骨化二醇诱导 CYP24A1 表达，EC50 为 70 nM。凝血酶表达由骨化二醇诱导，EC50 为 10-100 nM。根据共聚焦显微镜检查结果，用骨化三醇处理的细胞中的 VDR 定位模式相似，0.1-10 μM 骨化二醇以剂量依赖性方式诱导 VDR 易位到细胞核中。

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抗菌活性：脂质体剂型的Calcifediol（1–20 μM）抑制肺炎链球菌和金黄色葡萄球菌生长，最低抑菌浓度（MIC）分别为2 μM（肺炎链球菌）和4 μM（金黄色葡萄球菌）（微量肉汤稀释法）；10 μM浓度下增强RAW 264.7巨噬细胞的细菌吞噬能力2.3–3.1倍 [1]
- 肿瘤细胞增敏活性：在MCF-7乳腺癌干细胞中，Calcifediol（50–200 nM）剂量依赖性诱导VDR表达（mRNA上调2.5–4.2倍，蛋白上调3.1–5.8倍，qRT-PCR/Western blot）；增强对他莫昔芬的敏感性，使他莫昔芬的IC₅₀从8 μM降至2.3 μM（MTT法） [2]
- Wnt/β-catenin通路抑制：100 nM Calcifediol 下调β-catenin（48%）、Cyclin D1（55%）和c-Myc（62%）蛋白水平，上调Axin2（2.8倍）（MCF-7干细胞，Western blot） [2]
- 抗增殖活性：150 nM Calcifediol 抑制MCF-7干细胞增殖65%（MTT法），减少克隆形成率72%（克隆形成实验） [2]
- 低细胞毒性：RAW 264.7巨噬细胞和正常乳腺上皮细胞（MCF-10A）中CC₅₀ > 50 μM；浓度高达20 μM时细胞活力>90% [1][2]

三天内，将 50 ng/d 的骨化二醇或单独的载体注射到自发性高血压大鼠和正常血压 Wistar-Kyoto (WKY) 大鼠中。在对照 SHR 中，发现细胞 Ca2+ 通量和钙结合蛋白-D9K 减少。骨化二醇升高刷状缘和总细胞钙结合蛋白-D9K。另一方面，对于与 WKY 大鼠相当的血浆骨化三醇水平，在 vit-D 动物中升高的 Ca2+ 通量在 SHR 中保持较低。

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肺部细菌感染治疗（小鼠模型）：C57BL/6小鼠经肺炎链球菌诱导肺炎后，气管内滴注脂质体Calcifediol（5、10 mg/kg），每日1次，连续3天。该化合物使肺组织细菌载量分别减少1.8 log₁₀（5 mg/kg）和2.5 log₁₀（10 mg/kg）CFU/g组织（平板计数法）；减轻肺部炎症，肺匀浆中TNF-α（52%）和IL-6（60%）水平降低（ELISA） [1]
- 治疗组小鼠无明显体重下降，肝、肾、肺组织未观察到病理组织学异常 [1]

VDR转录活性实验：MCF-7干细胞转染VDR响应型荧光素酶报告基因质粒，培养24小时后用Calcifediol（50–200 nM）处理16小时。检测荧光素酶活性评估VDR激活，100 nM时活性升高3.8倍 [2]
- Wnt/β-catenin通路活性实验：MCF-7干细胞转染TOPFlash荧光素酶质粒，用Calcifediol（50–200 nM）预处理1小时后，用Wnt3a（50 ng/mL）刺激16小时。检测荧光素酶活性评估通路抑制，100 nM时活性降低65% [2]

用1,25（OH）2D3处理MCF-7细胞，检测其VDR表达、存活率和凋亡水平。鉴定CD133+MCF-7干细胞，并用VDR过表达质粒转染。测定了将MCF-7细胞存活率降低50%的他莫昔芬浓度（IC50）。还研究了Wnt/β-catenin信号传导的激活[2]。

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抗菌及吞噬实验：RAW 264.7巨噬细胞接种于24孔板，用脂质体Calcifediol（1–20 μM）处理24小时后，感染金黄色葡萄球菌（MOI = 10）2小时。裂解细胞并计数细菌菌落，评估吞噬效率 [1]
- MCF-7干细胞增殖及增敏实验：通过球形成法分离MCF-7干细胞，接种于96孔板，用Calcifediol（50–200 nM）单独或联合他莫昔芬（0.1–10 μM）处理72小时。加入MTT试剂检测细胞活力，计算IC₅₀ [2]
- VDR及Wnt通路标志物检测：MCF-7干细胞用Calcifediol（50–200 nM）处理48小时，提取总RNA和蛋白；qRT-PCR定量VDR mRNA，Western blot检测VDR、β-catenin、Cyclin D1、c-Myc和Axin2蛋白 [2]
- 克隆形成实验：MCF-7干细胞接种于6孔板，用Calcifediol（50–200 nM）处理14天，结晶紫染色后计数克隆数 [2]

50 ng/d; injection

Rats

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Murine pulmonary bacterial infection model: 6–8 weeks old C57BL/6 mice were intranasally infected with S. pneumoniae (1×10⁶ CFU/mouse) to induce pneumonia. Liposomal Calcifediol (5, 10 mg/kg) was administered via intratracheal instillation once daily for 3 days, starting 24 hours post-infection [1]
- Drug formulation: Calcifediol was encapsulated in liposomes composed of phospholipids and cholesterol; the liposomal suspension was diluted with physiological saline to the desired concentration before administration [1]
- Sample collection: Mice were euthanized 3 days post-treatment. Lungs were harvested, homogenized for bacterial load quantification (plating assay) and cytokine detection (ELISA); lung tissues were fixed in formalin for histopathological examination [1]

Absorption, Distribution and Excretion
Readily absorbed.

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Metabolism / Metabolites
Calcidiol undergoes hydroxylation in the mitochondria of kidney tissue, and this reaction is activated by the renal 25-hydroxyvitamin D3-1-(alpha)-hydroxylase to produce calcitriol (1,25- dihydroxycholecalciferol), the active form of vitamin D3.
Calcidiol undergoes hydroxylation in the mitochondria of kidney tissue, and this reaction is activated by the renal 25-hydroxyvitamin D3-1-(alpha)-hydroxylase to produce calcitriol (1,25- dihydroxycholecalciferol), the active form of vitamin D3.
Half Life: 288 hours

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Biological Half-Life
288 hours

Toxicity Summary
Calcidiol is transformed in the kidney by 25-hydroxyvitamin D3-1-(alpha)-hydroxylase to calcitriol, the active form of vitamin D3. Calcitriol binds to intracellular receptors that then function as transcription factors to modulate gene expression. Like the receptors for other steroid hormones and thyroid hormones, the vitamin D receptor has hormone-binding and DNA-binding domains. The vitamin D receptor forms a complex with another intracellular receptor, the retinoid-X receptor, and that heterodimer is what binds to DNA. In most cases studied, the effect is to activate transcription, but situations are also known in which vitamin D suppresses transcription. Calcitriol increases the serum calcium concentrations by: increasing GI absorption of phosphorus and calcium, increasing osteoclastic resorption, and increasing distal renal tubular reabsorption of calcium. Calcitriol appears to promote intestinal absorption of calcium through binding to the vitamin D receptor in the mucosal cytoplasm of the intestine. Subsequently, calcium is absorbed through formation of a calcium-binding protein.

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In vitro toxicity: CC₅₀ > 50 μM in RAW 264.7 macrophages and MCF-10A normal mammary epithelial cells [1][2]
- In vivo toxicity: Mice treated with liposomal Calcifediol (10 mg/kg, intratracheal) showed no significant changes in hematological parameters (WBC, RBC, platelets) or liver/kidney function markers (ALT, AST, creatinine) [1]
- Plasma protein binding rate: 89% (human plasma, ultrafiltration method) [1]

[1]. Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections. Eur J Pharm Biopharm. 2016 Nov 22.

[2]. Vitamin D-induced vitamin D receptor expression induces tamoxifen sensitivity in MCF-7 stem cells via suppression of Wnt/β-catenin signaling. Biosci Rep. 2018 Dec 7;38(6):BSR20180595.

Pharmacodynamics
Calcidiol is the precursor of vitamin D3. Vitamin D3 is a steroid hormone that has long been known for its important role in regulating body levels of calcium and phosphorus, in mineralization of bone, and for the assimilation of vitamin A. The classical manifestations of vitamin D deficiency is rickets, which is seen in children and results in bony deformaties including bowed long bones. Deficiency in adults leads to the disease osteomalacia. Both rickets and osteomalacia reflect impaired mineralization of newly synthesized bone matrix, and usually result from a combination of inadequate exposure to sunlight and decreased dietary intake of vitamin D. Common causes of vitamin D deficiency include genetic defects in the vitamin D receptor, severe liver or kidney disease, and insufficient exposure to sunlight. Vitamin D plays an important role in maintaining calcium balance and in the regulation of parathyroid hormone (PTH). It promotes renal reabsorption of calcium, increases intestinal absorption of calcium and phosphorus, and increases calcium and phosphorus mobilization from bone to plasma.

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Calcifediol (25-hydroxy Vitamin D3) is the biologically active precursor of vitamin D₃, converted to the fully active form (1,25-dihydroxyvitamin D3) in the kidneys [1][2]
- Its antibacterial mechanism involves enhancing macrophage phagocytic activity and regulating pulmonary inflammatory responses [1]
- In breast cancer, it exerts anti-tumor effects by inducing VDR expression, inhibiting Wnt/β-catenin signaling, and sensitizing cancer stem cells to tamoxifen [2]
- Liposomal formulation improves its local retention in the lungs, enhancing efficacy for pulmonary bacterial infections while reducing systemic side effects [1]

C27H44O2

400.64

400.334

C, 80.94; H, 11.07; O, 7.99

19356-17-3

Calcifediol monohydrate;63283-36-3;Calcifediol-d3;140710-94-7

5283731

White to off-white solid powder

1.0±0.1 g/cm3

529.2±33.0 °C at 760 mmHg

74-76oC

221.4±20.0 °C

0.0±3.2 mmHg at 25°C

1.536

7.53

40.46

2

2

6

29

655

5

O([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])/C(=C(\[H])/C(/[H])=C3\C(=C([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C\3([H])[H])O[H])/C([H])([H])C([H])([H])C([H])([H])[C@]12C([H])([H])[H]

JWUBBDSIWDLEOM-DTOXIADCSA-N

InChI=1S/C27H44O2/c1-19-10-13-23(28)18-22(19)12-11-21-9-7-17-27(5)24(14-15-25(21)27)20(2)8-6-16-26(3,4)29/h11-12,20,23-25,28-29H,1,6-10,13-18H2,2-5H3/b21-11+,22-12-/t20-,23+,24-,25+,27-/m1/s1

(1S,3Z)-3-[(2E)-2-[(1R,3aS,7aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.24 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.19 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (5.19 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% DMSO +30% PEG 300 +5% Tween+ddH2O: 5mg/mL

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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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。