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Calcifediol (also known as 25-hydroxyvitamin D3; 25-hydroxy VD3) is the major metabolite of vitamin D3 that circulates in the blood and is the form that is tested in medicine to estimate the amount of vitamin D in the body (specifically, 25-hydroxy vitamin D, or calcifediol) and identify vitamin D deficiency. Calcifediol has an apparent Ki of 3.9 μM and functions as a competitive inhibitor. Additionally, it inhibits mRNA (ED50=2 nM) and PTH secretion. CYP24A1 expression was induced by calcifediol, with an EC50 of 70 nM. With an EC50 of 10-100 nM, calcifediol induced the expression of thrombomodulin. Confocal microscopy showed that calcifediol at concentrations between 0.1 and 10 μM dose-dependently caused VDR translocation into the nucleus; the VDR localization pattern in calcitriol-treated cells was comparable.
Physicochemical Properties
| Molecular Formula | C27H44O2 | |
| Molecular Weight | 400.64 | |
| Exact Mass | 400.334 | |
| Elemental Analysis | C, 80.94; H, 11.07; O, 7.99 | |
| CAS # | 19356-17-3 | |
| Related CAS # | Calcifediol monohydrate;63283-36-3;Calcifediol-d3;140710-94-7 | |
| PubChem CID | 5283731 | |
| Appearance | White to off-white solid powder | |
| Density | 1.0±0.1 g/cm3 | |
| Boiling Point | 529.2±33.0 °C at 760 mmHg | |
| Melting Point | 74-76oC | |
| Flash Point | 221.4±20.0 °C | |
| Vapour Pressure | 0.0±3.2 mmHg at 25°C | |
| Index of Refraction | 1.536 | |
| LogP | 7.53 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 6 | |
| Heavy Atom Count | 29 | |
| Complexity | 655 | |
| Defined Atom Stereocenter Count | 5 | |
| SMILES | 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] |
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| InChi Key | JWUBBDSIWDLEOM-DTOXIADCSA-N | |
| InChi Code | 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 | |
| Chemical Name | (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 | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
VDR/vitamin D receptor; Human Endogenous Metabolite Calcifediol (25-hydroxy Vitamin D3) targets vitamin D receptor (VDR), a nuclear transcription factor; [2] |
| ln Vitro |
Calcifediol induced CYP24A1 expression with EC50 at 70 nM. Thrombin expression was induced by calcifediol, with an EC50 of 10-100 nM. The VDR localization pattern in cells treated with calcitriol was similar, and calcifediol at 0.1–10 μM induced VDR translocation into the nucleus in a dose-dependent manner, according to confocal microscopy findings. Antibacterial activity: Calcifediol (1–20 μM) in liposomal formulation inhibited the growth of Streptococcus pneumoniae and Staphylococcus aureus, with MIC values of 2 μM (S. pneumoniae) and 4 μM (S. aureus) (broth microdilution assay); it enhanced bacterial phagocytosis by RAW 264.7 macrophages by 2.3–3.1-fold at 10 μM [1] - Tumor cell sensitization: In MCF-7 breast cancer stem cells, Calcifediol (50–200 nM) dose-dependently induced VDR expression (2.5–4.2-fold increase in mRNA; 3.1–5.8-fold increase in protein, qRT-PCR/Western blot); it enhanced tamoxifen sensitivity, reducing IC₅₀ of tamoxifen from 8 μM to 2.3 μM (MTT assay) [2] - Wnt/β-catenin pathway inhibition: 100 nM Calcifediol downregulated β-catenin (48%), Cyclin D1 (55%), and c-Myc (62%) protein levels, and upregulated Axin2 (2.8-fold) in MCF-7 stem cells (Western blot) [2] - Anti-proliferative activity: 150 nM Calcifediol inhibited MCF-7 stem cell proliferation by 65% (MTT assay) and reduced clonogenic survival by 72% (colony formation assay) [2] - Low cytotoxicity: CC₅₀ > 50 μM in RAW 264.7 macrophages and normal mammary epithelial cells (MCF-10A); cell viability >90% at concentrations up to 20 μM [1][2] |
| ln Vivo |
For three days, 50 ng/d of calcifediol or vehicle alone was injected into spontaneously hypertensive rats and normotensive Wistar-Kyoto (WKY) rats. In the control SHR, cellular Ca2+ flux and calbindin-D9K were found to be reduced. Calcifediol elevated brush border and total cell calbindin-D9K. On the other hand, for plasma calcitriol levels comparable to those in WKY rats, Ca2+ flux, which rose in vit-D animals, stayed lower in SHR. Pulmonary bacterial infection treatment (mouse model): C57BL/6 mice with S. pneumoniae-induced pneumonia were administered liposomal Calcifediol (5, 10 mg/kg) via intratracheal instillation once daily for 3 days. The compound reduced lung bacterial load by 1.8 log₁₀ (5 mg/kg) and 2.5 log₁₀ (10 mg/kg) CFU/g tissue (plating assay); it alleviated pulmonary inflammation, reducing TNF-α (52%) and IL-6 (60%) levels in lung homogenates (ELISA) [1] - No significant body weight loss or histopathological abnormalities in liver, kidney, or lung were observed in treated mice [1] |
| Enzyme Assay |
VDR transcriptional activity assay: MCF-7 stem cells were transfected with VDR-responsive luciferase reporter plasmid. After 24 hours, cells were treated with Calcifediol (50–200 nM) for 16 hours. Luciferase activity was measured to assess VDR activation; 100 nM increased activity by 3.8-fold [2] - Wnt/β-catenin pathway activity assay: MCF-7 stem cells were transfected with TOPFlash luciferase plasmid. Pretreated with Calcifediol (50–200 nM) for 1 hour, then stimulated with Wnt3a (50 ng/mL) for 16 hours. Luciferase activity was detected to evaluate pathway inhibition; 100 nM reduced activity by 65% [2] |
| Cell Assay |
MCF-7 cells were treated with 1,25(OH)2D3 and their levels of VDR expression, viability, and apoptosis were detected. CD133+ MCF-7 stem cells were identified and transfected with a VDR-overexpression plasmid. The tamoxifen concentration that reduced MCF-7 cell viability by 50% (IC50) was determined. The activation of Wnt/β-catenin signaling was also investigated[2]. Antibacterial & phagocytosis assay: RAW 264.7 macrophages were seeded in 24-well plates, treated with liposomal Calcifediol (1–20 μM) for 24 hours, then infected with S. aureus (MOI = 10) for 2 hours. Cells were lysed, and bacterial colonies were counted to assess phagocytosis efficiency [1] - MCF-7 stem cell proliferation & sensitization assay: MCF-7 stem cells were isolated by sphere formation, seeded in 96-well plates, treated with Calcifediol (50–200 nM) alone or combined with tamoxifen (0.1–10 μM) for 72 hours. MTT reagent was added to measure cell viability and calculate IC₅₀ [2] - VDR & Wnt pathway marker detection: MCF-7 stem cells were treated with Calcifediol (50–200 nM) for 48 hours. Total RNA and protein were extracted; qRT-PCR quantified VDR mRNA, and Western blot detected VDR, β-catenin, Cyclin D1, c-Myc, and Axin2 proteins [2] - Colony formation assay: MCF-7 stem cells were seeded in 6-well plates, treated with Calcifediol (50–200 nM) for 14 days, stained with crystal violet, and colonies were counted [2] |
| Animal Protocol |
50 ng/d; injection Rats 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] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Readily absorbed. 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 Biological Half-Life 288 hours |
| Toxicity/Toxicokinetics |
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. 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] |
| References |
[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. |
| Additional Infomation |
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. 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] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.19 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 2% DMSO +30% PEG 300 +5% Tween+ddH2O: 5mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4960 mL | 12.4800 mL | 24.9601 mL | |
| 5 mM | 0.4992 mL | 2.4960 mL | 4.9920 mL | |
| 10 mM | 0.2496 mL | 1.2480 mL | 2.4960 mL |