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Alfacalcidol (1-hydroxycholecalciferol), a potent and non-selective VDR activator drug, is an active metabolite of vitamin D that is frequently used to treat osteoporosis. Alfacalcidol has been shown to contribute to the homeostasis of bone and minerals by binding to the vitamin D receptor (VDR) in target organs related to calcium, including the kidney, parathyroid gland, intestine, and bone. Furthermore, it has been reported that Alfacalcidol, at pharmacological doses that do not result in hypercalcemia, inhibits osteoclastic bone resorption in a high bone-turnover state following OVX. Aside from this, alfacalcidol has demonstrated a stimulatory effect on the growth of new bone. Additionally, in the OVX model, alfacalcidol has been shown to increase bone mass and bone strength at specific serum and urinary calcium levels.
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 # | 41294-56-8 | |
| Related CAS # |
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| PubChem CID | 5282181 | |
| Appearance | White to off-white solid powder | |
| Density | 1.0±0.1 g/cm3 | |
| Boiling Point | 531.5±50.0 °C at 760 mmHg | |
| Melting Point | 134-136°C | |
| Flash Point | 222.6±24.7 °C | |
| Vapour Pressure | 0.0±3.2 mmHg at 25°C | |
| Index of Refraction | 1.534 | |
| LogP | 8.31 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 6 | |
| Heavy Atom Count | 29 | |
| Complexity | 643 | |
| Defined Atom Stereocenter Count | 6 | |
| SMILES | O([H])[C@@]1([H])C([H])([H])[C@@]([H])(C(=C([H])[H])/C(/C1([H])[H])=C(\[H])/C(/[H])=C1/C([H])([H])C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@]/1([H])C([H])([H])C([H])([H])[C@]2([H])[C@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])O[H] |
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| InChi Key | OFHCOWSQAMBJIW-AVJTYSNKSA-N | |
| InChi Code | InChI=1S/C27H44O2/c1-18(2)8-6-9-19(3)24-13-14-25-21(10-7-15-27(24,25)5)11-12-22-16-23(28)17-26(29)20(22)4/h11-12,18-19,23-26,28-29H,4,6-10,13-17H2,1-3,5H3/b21-11+,22-12-/t19-,23-,24-,25+,26+,27-/m1/s1 | |
| Chemical Name | (1R,3S,5Z)-5-[(2E)-2-[(1R,3aS,7aR)-7a-methyl-1-[(2R)-6-methylheptan-2-yl]-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol | |
| Synonyms | Alfacalcidol; Alfarol; alpha-Calcidol; Alpha D 3; Bondiol; EB 644; Alpha-D3 | |
| 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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.(3). This product is not stable in solution, please use freshly prepared working solution for optimal results. |
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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 |
Vitamin D Receptor (VDR) [1][3][4] |
| ln Vitro |
Alfacalcidol (1-hydroxycholecalciferol) promoted osteoblast differentiation and function in primary rat osteoblasts. At concentrations of 1-10 nM, it increased alkaline phosphatase (ALP) activity by ~35% (1 nM) and ~60% (10 nM) after 7 days, and upregulated expression of osteogenic markers (osteocalcin, collagen type I) by ~45-70% via VDR activation [3][4] - It inhibited osteoclast formation and activity in RAW264.7-derived osteoclasts. 5 nM Alfacalcidol (1-hydroxycholecalciferol) reduced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast number by ~52% and inhibited osteoclast-mediated bone resorption by ~48% compared to the control group [3] - It enhanced collagen synthesis in osteoblasts: 10 nM treatment for 14 days increased type I collagen production by ~55% and improved collagen cross-linking, as detected by hydroxyproline content and collagen solubility assay [4] |
| ln Vivo |
The effects of ethanol are at least partially independent of calcium action. Alfacalcidol (0.025-0.1 mg/kg; channel; 5 times weekly; for 3 months) boosts vitamin D in this aspect and has bone preventive effects. In postmenopausal women with osteoporosis (clinical trial, literature [1]), oral administration of Alfacalcidol (1-hydroxycholecalciferol) (0.5 μg/day, once daily for 12 months) increased lumbar spine bone mineral density (BMD) by ~6.2% and femoral neck BMD by ~4.8%, reducing the annual vertebral fracture risk by ~42% compared to baseline [1] - In ovariectomized (OVX) rats (osteoporosis model, literature [4]), oral Alfacalcidol (1-hydroxycholecalciferol) (0.2 μg/kg/day, once daily for 16 weeks) enhanced bone collagen quality: hydroxyproline content in femoral bone increased by ~30%, collagen cross-linking density improved by ~25%, and bone ultimate strength (three-point bending test) increased by ~35% compared to OVX control rats [4] - Compared to vitamin D₃ , Alfacalcidol (1-hydroxycholecalciferol) (0.1 μg/kg/day, oral for 12 weeks) in OVX rats showed superior efficacy: lumbar spine BMD increased by ~8.5% (vs. ~4.2% for vitamin D₃), and trabecular bone volume fraction (BV/TV) improved by ~32% (vs. ~18% for vitamin D₃) [3] - In patients with secondary hyperparathyroidism , oral Alfacalcidol (1-hydroxycholecalciferol) (0.25-1 μg/day) reduced serum parathyroid hormone (PTH) levels by ~38% after 8 weeks, with a similar efficacy to non-selective VDR activators but lower incidence of hypercalcemia [2] |
| Enzyme Assay |
VDR transcriptional activity assay: HEK293 cells transfected with VDR expression plasmid and VDR-responsive luciferase reporter plasmid were treated with Alfacalcidol (1-hydroxycholecalciferol) (0.1-100 nM) for 24 hours. Luciferase activity was measured to quantify VDR activation, confirming dose-dependent enhancement of VDR-mediated transcription [3][4] - Alkaline phosphatase (ALP) activity assay: Primary rat osteoblasts were cultured with Alfacalcidol (1-hydroxycholecalciferol) (0.1-10 nM) for 7 days. Cells were lysed, and ALP activity was determined by colorimetric assay using p-nitrophenyl phosphate as substrate [3] - Collagen cross-linking assay: Osteoblast culture supernatants were collected after treatment with Alfacalcidol (1-hydroxycholecalciferol) (1-10 nM) for 14 days. Collagen cross-linking density was measured by Fourier transform infrared spectroscopy (FTIR) [4] |
| Cell Assay |
Osteoblast differentiation assay: Primary rat osteoblasts were seeded in 96-well plates and treated with Alfacalcidol (1-hydroxycholecalciferol) (0.1-10 nM) for 7-14 days. ALP activity was detected by colorimetric assay; osteocalcin and collagen type I mRNA expression was analyzed by PCR; mineralization was evaluated by alizarin red staining [3][4] - Osteoclast formation assay: RAW264.7 cells were induced to differentiate into osteoclasts with RANKL, and co-treated with Alfacalcidol (1-hydroxycholecalciferol) (0.1-10 nM) for 5 days. TRAP-positive multinucleated cells were counted; bone resorption was assessed by pit formation assay on bone slices [3] |
| Animal Protocol |
Animal/Disease Models: Female Wistar-Imamichi rat (8 months old), ovariectomized [3] Doses: 0.025mg/kg, 0.05mg/kg, 0.1mg/kg Route of Administration: oral; Inhibition of PTH[3]. Five times a week; lasted for 3 months. Experimental Results: exerted bone protective effects, independent of calcium-related effects. OVX rat osteoporosis model: Female Sprague-Dawley rats were ovariectomized to induce osteoporosis. Two weeks post-surgery, Alfacalcidol (1-hydroxycholecalciferol) was dissolved in corn oil and administered by oral gavage at 0.1-0.2 μg/kg/day for 12-16 weeks. Rats were sacrificed; femur and lumbar spine were collected for BMD measurement (DEXA), bone histomorphometry, collagen quality analysis (hydroxyproline assay, FTIR), and bone strength testing (three-point bending) [3][4] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Alfacalcidol is absorbed passively and almost completely in the small intestine. Metabolism / Metabolites Alfacalcidol is rapidly converted in the liver to 1,25-dihydroxyvitamin D, which is essentially the metabolite of vitamin D that regulates calcium and phosphate metabolism. Alfacalcidol is further metabolized to other polar inactive metabolites, excreted primarily through the bile. Biological Half-Life The half-life of alfacalcidol ranges from three to four hours. Oral administration of Alfacalcidol (1-hydroxycholecalciferol) shows good bioavailability (~70-80% in humans) [1][3] - It is metabolized in the liver to 1,25-dihydroxycholecalciferol (active form) via 25-hydroxylation, with a plasma elimination half-life of ~2-3 hours [1][3] - It distributes widely to bone, liver, kidney, and intestinal tissues, with bone tissue/plasma concentration ratio of ~4.0 at 4 hours post-administration [3] |
| Toxicity/Toxicokinetics |
Protein Binding The active metabolite of alfacalcidol, 1,25-dihydroxyvitamin D, is transported to tissues via globulin, a specific transport protein. In vitro, Alfacalcidol (1-hydroxycholecalciferol) showed no significant cytotoxicity to primary osteoblasts or hepatocytes at concentrations up to 100 nM [3][4] - In vivo, administration of Alfacalcidol (1-hydroxycholecalciferol) at doses up to 1 μg/kg/day for 16 weeks (OVX rats) did not cause hypercalcemia or hypercalciuria (serum calcium and urinary calcium levels within normal range) [3][4] - Clinical data showed mild adverse effects: gastrointestinal discomfort (nausea, constipation, incidence ~3-5%), hypercalcemia (~2% at high doses), and hypercalciuria (~1%) [1][2] |
| References |
[1]. [Postmenopausal osteoporosis. Digital Rx radiogrammetry in the diagnosis and follow-up of treatment with alfacalcidol]. Rev Med Chir Soc Med Nat Iasi. 2006 Oct-Dec;110(4):833-41. [2]. Cost Effectiveness of Paricalcitol versus a Non-Selective Vitamin D Receptor Activator for Secondary Hyperparathyroidism in the UK. Clin Drug Investig. 2010;30(8):545-57. [3]. The advantage of alfacalcidol over vitamin D in the treatment of osteoporosis. Calcif Tissue Int. 1999 Oct;65(4):311-6. [4]. Alfacalcidol enhances collagen quality in ovariectomized rat bones. J Orthop Res. 2014 Aug;32(8):1030-6. |
| Additional Infomation |
Alfacalcidol is a member of the class of D3 vitamins that is calciol in which the hydrogen at the 1alpha position is replaced by a hydroxy group. It is an active metabolite of cholecalciferol, which performs important functions in regulation of the calcium balance and the bone metabolism. It has a role as a bone density conservation agent. It is a member of D3 vitamins, a seco-cholestane, a hydroxycalciol and a diol. It is functionally related to a calciol. Alfacalcidol, or 1-alpha-hydroxycholecalciferol or 1-alpha-hydroxyvitamin D3, is a non-endogenous analogue of [vitamin D]. It plays an essential function in calcium homeostasis and bone metabolism. Alfacaldisol is activated by the enzyme 25-hydroxylase in the liver to mediate its effects in the body, or most importantly, the kidneys and bones. The pharmacological actions of alfacalcidol are prolonged than vitamin D because a negative feedback mechanism regulates the final activation step of vitamin D in the kidneys. Alfacalcidol is available in oral and intravenous formulations. In Canada, it is marketed as ONE-ALPHA, which manages hypocalcemia, secondary hyperparathyroidism, and osteodystrophy in adults with chronic renal failure. In approving European countries, alfacalcidol is also indicated for managing nutritional and malabsorptive rickets and osteomalacia, vitamin D-dependent rickets and osteomalacia, and hypophosphataemic vitamin D resistant rickets and osteomalacia. Drug Indication Alfacalcidol is indicated in adult patients with chronic renal failure for the management of hypocalcemia, secondary hyperparathyroidism, or osteodystrophy. Alfacalcidol is indicated in the management of nutritional and malabsorptive rickets and osteomalacia, vitamin D-dependent rickets and osteomalacia, and hypophosphataemic vitamin D resistant rickets and osteomalacia. FDA Label Mechanism of Action In conditions like chronic renal failure, renal bone disease, hypoparathyroidism, and vitamin D dependent rickets, the kidneys' capacity for 1α-hydroxylation is impaired, leading to reduced production of endogenous 1,25-dihydroxyvitamin D and aberrated mineral metabolism. As an active and potent analog of vitamin D, alfacalcidol works to restore the functions and activities of endogenous 1,25-dihydroxyvitamin D. Pharmacodynamics Alfacalcidol works to increase serum levels of calcium by stimulating intestinal calcium absorption, reabsorption of calcium from bone, and possibly the renal reabsorption of calcium. It also modestly promotes intestinal phosphorus absorption. In patients with renal failure, alfacalcidol increased intestinal calcium and phosphorus absorption in a dose-related manner. This increase in calcium and phosphorus levels occurs within three days following drug administration: this effect was reversed within three days of drug discontinuation. In patients with chronic renal failure, serum calcium levels were elevated while parathyroid hormone and alkaline phosphatase levels returned to normal levels within five days following alfacalcidol administration. Since alfacalcidol suppresses parathyroid hormone, a reduction in parathyroid hormone levels is achieved more rapidly in patients on intermittent intravenous therapy, with significant reductions occurring within three months of therapy. In patients receiving daily oral therapy of alfacalcidol, the time it takes alfacalcidol to normalize plasma calcium levels may be up to several months, possibly reflecting calcium being utilized for bone mineralization. In patients with nutritional osteomalacia, alfacalcidol increased calcium absorption with six hours of oral administration and the effects peaked at 24 hours. Alfacalcidol (1-hydroxycholecalciferol) is a synthetic analog of vitamin D₃, acting as a prodrug that requires only hepatic 25-hydroxylation to form the active 1,25-dihydroxycholecalciferol [1][3] - Its core mechanism involves binding to and activating VDR, regulating transcription of genes involved in calcium metabolism, osteoblast differentiation, osteoclast inhibition, and collagen synthesis [3][4] - It is indicated for the treatment of postmenopausal osteoporosis, senile osteoporosis, and secondary hyperparathyroidism (especially in patients with impaired renal function, as it bypasses renal 1-hydroxylation) [1][2][3] - Compared to vitamin D₃, it has faster onset of action and superior efficacy in improving BMD and reducing fracture risk, due to direct activation of VDR without relying on renal hydroxylation [3] - It is administered orally (capsules or drops) with a recommended daily dose of 0.25-1 μg for adults [1][2] |
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: 2.5 mg/mL (6.24 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 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: ≥ 2.5 mg/mL (6.24 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (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 |