Vitamin D Receptor (VDR) [1][3][4]

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]

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.

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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]

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]

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/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.

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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]

Absorption, Distribution and Excretion
Alfacalcidol is passively absorbed in the small intestine and is almost completely absorbed. Metabolism/Metabolites Alfacalcidol is rapidly converted to 1,25-dihydroxyvitamin D in the liver, which is the main metabolite of vitamin D and regulates calcium and phosphorus metabolism. Alfacalcidol is further metabolized into other polar, inactive metabolites, which are mainly excreted via bile. Biological Half-Life The half-life of alfacalcidol is three to four hours.

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Oral administration of alfacalcidol (1-hydroxycholecalciferol) shows good bioavailability (approximately 70-80% in humans)[1][3]
-It is metabolized in the liver by 25-hydroxylation to 1,25-dihydroxycholecalciferol (the active form), with a plasma elimination half-life of approximately 2-3 hours[1][3]
-It is widely distributed in bone, liver, kidney, and intestinal tissues, with a bone/plasma concentration ratio of approximately 4.0 4 hours after administration[3]

Protein Binding
Alfacalcidol's active metabolite, 1,25-dihydroxyvitamin D, is transported to tissues via a specific transporter protein, globulin. In vitro studies have shown that concentrations up to 100 nM of alfacalcidol (1-hydroxycholecalciferol) have no significant cytotoxicity to primary osteoblasts or hepatocytes [3][4]. In vivo studies have shown that daily administration of alfacalcidol (1-hydroxycholecalciferol) at doses up to 1 μg/kg for 16 consecutive weeks (ovariectomized rats) did not induce hypercalcemia or hypercalciuria (serum and urinary calcium levels were within the normal range) [3][4]. Clinical data show that adverse reactions are mild: gastrointestinal discomfort (nausea, constipation, incidence approximately 3-5%), hypercalcemia (approximately 2% at high doses), and hypercalciuria (approximately 1%) [1][2].

[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.

Alfacalcidol is a member of the vitamin D3 family, belonging to the calcium alcohol class, with a hydroxyl group replacing the hydrogen at the 1α position. It is the active metabolite of cholecalciferol and plays an important role in regulating calcium homeostasis and bone metabolism. It helps maintain bone density. Alfacalcidol belongs to the vitamin D3 family and is an open-ring cholesterol alkyl group, hydroxycalciferol, and diol. Functionally, it is related to calcium alcohols. Alfacalcidol, also known as 1-α-hydroxycholecalciferol or 1-α-hydroxyvitamin D3, is a non-endogenous analog of vitamin D. It plays a crucial role in calcium homeostasis and bone metabolism. After activation by 25-hydroxylase in the liver, alfacalcidol exerts its effects in the body, particularly in the kidneys and bones. The pharmacological effects of alfacalcidol are more persistent than those of vitamin D because the final activation step of vitamin D in the kidneys is regulated by a negative feedback mechanism. Alfacalcidol is available in both oral and intravenous formulations. In Canada, it is marketed under the brand name ONE-ALPHA for the treatment of hypocalcemia, secondary hyperparathyroidism, and osteomalacia in adults with chronic renal failure. In European countries where it has been approved, alfacalcidol is also indicated for the treatment of malnutrition-related rickets and osteomalacia, vitamin D-dependent rickets and osteomalacia, and hypophosphatemic vitamin D-resistant rickets and osteomalacia.

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Drug Indications
Alfacalcidol is indicated for the treatment of hypocalcemia, secondary hyperparathyroidism, or osteomalacia in adults with chronic renal failure.Alfacalcidol is indicated for the treatment of malnutrition-related rickets and osteomalacia, vitamin D-dependent rickets and osteomalacia, and hypophosphatemic vitamin D-resistant rickets and osteomalacia.
FDA Label
Mechanism of Action
In diseases such as chronic renal failure, renal osteodystrophy, hypoparathyroidism, and vitamin D-dependent rickets, the kidneys' 1α-hydroxylation capacity is impaired, leading to reduced endogenous 1,25-dihydroxyvitamin D production and abnormal mineral metabolism. As a potent vitamin D analog, alfacalcidol restores the function and activity of endogenous 1,25-dihydroxyvitamin D.
Pharmacodynamics
Alfacalcidol increases serum calcium levels by stimulating intestinal calcium absorption, bone reabsorption of calcium, and possibly renal reabsorption of calcium. It also slightly promotes intestinal phosphorus absorption. In patients with renal failure, alfacalcidol increases intestinal calcium and phosphorus absorption in a dose-dependent manner. This increase in calcium and phosphorus levels occurs within three days of administration; this effect is reversed within three days of discontinuation.
In patients with chronic renal failure, serum calcium levels increased after taking alfacalcidol, while parathyroid hormone and alkaline phosphatase levels returned to normal within five days. Because alfacalcidol inhibits parathyroid hormone secretion, parathyroid hormone levels decreased more rapidly in patients receiving intermittent intravenous administration, usually within three months of treatment. In patients taking alfacalcidol orally daily, plasma calcium levels may take several months to return to normal, which may reflect that calcium is used for bone mineralization. In patients with nutritional osteomalacia, calcium absorption was increased 6 hours after oral administration of alfacalcidol and peaked at 24 hours. Alfacalcidol (1-hydroxycholecalciferol) is a synthetic analog of vitamin D₃ and, as a prodrug, requires only 25-hydroxylation in the liver to form active 1,25-dihydroxycholecalciferol [1][3].
- Its core mechanism involves binding to and activating the vitamin D receptor (VDR), thereby regulating the 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 for patients with impaired renal function, as it bypasses renal 1-hydroxylation)[1][2][3].
- It has a faster onset of action and is more effective in improving bone density than vitamin D₃. It reduces the risk of fractures by directly activating the vitamin D receptor (VDR) without relying on renal hydroxylation[3].
- The recommended daily dose for adults is 0.25–1 μg, orally (capsules or drops)[1][2].

C27H44O2

400.64

400.334

C, 80.94; H, 11.07; O, 7.99

41294-56-8

5282181

White to off-white solid powder

1.0±0.1 g/cm3

531.5±50.0 °C at 760 mmHg

134-136°C

222.6±24.7 °C

0.0±3.2 mmHg at 25°C

1.534

8.31

2

2

6

29

643

6

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]

OFHCOWSQAMBJIW-AVJTYSNKSA-N

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

(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

Alfacalcidol; Alfarol; alpha-Calcidol; Alpha D 3; Bondiol; EB 644; Alpha-D3

2934.99.9001

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.

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

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)