VDR/vitamin D receptor

When NHEK cell cultures were stimulated with IL-17A or IL-22, capecitatriol either had no effect (2–20 nM) or modestly increased (0.2 nM) the expression of IL-8 mRNA. Our earlier research was validated by the addition of IL-17A and IL-22, which greatly raised the mRNA expression of IL-8. Doses of 2, 20, and 40 nM of calcipotriol monohydrate dye-wise blocked this increased expression of IL-8 mRNA [1]. Medication administration to natural killer (NK) cells can alter the NK cytotoxic uptake or KIR expression. In under four hours, human NK cells were treated with 100, 10, or 1 ng/mL of 1,25 (OH) 2D3, calcipotriol monohydrate, or FTY720. Four hours following amino acids, the expression of NKp30 on the surface of NK cells was considerably up-regulated by three doses of 1, 25 (OH) 2D3, calcipotriol monohydrate, and FTY720 [2].

All animals except for the diclofenac plus DFMO plus calcipotriol monohydrate group collapsed, and one of the thirty-two clofenac animals in each group perished. The groups were equally divided in terms of survival. When compared to gradient (linear regression model), body weight gain was significantly lower in the diclofenac plus calcipotriol monohydrate and diclofenac plus DFMO plus calcipotriol monohydrate treatment groups [3].

1. DNA-Dependent Limited Protease Digestion Assay
This assay is used to analyze the conformational stability of the VDR upon ligand binding, both in solution and when bound to DNA response elements.
Procedure:
1. Reagent Preparation: The VDR protein is produced via in vitro translation and labeled with [³⁵S]-methionine. The response element (VDRE, e.g., rat ANF DR3-type) is prepared as unlabeled double-stranded DNA.
2. Complex Formation: The labeled VDR is incubated with RXR protein and the VDRE DNA in the presence of varying concentrations of Calcipotriol (or control ligands) to form a stable complex.
3. Protease Digestion: Trypsin is added to the reaction mix. The binding of the ligand protects specific domains of the VDR from proteolytic cleavage.
4. Analysis: The digestion products are separated by SDS-PAGE. The stabilization of specific fragments (e.g., the 28 kDa c1LPD fragment representing the agonistic conformation and the 23 kDa c3LPD fragment) is quantified. The ratio of these fragments indicates whether the ligand acts as an agonist.
5. Interpretation: For MC903, this assay reveals an EC₅₀ of approximately 0.2–0.3 nM for stabilizing the VDR within the VDR-RXR-VDRE complex, comparable to the natural hormone.

2. Ligand-Dependent Gel Shift Assay (Electrophoretic Mobility Shift Assay - EMSA)
This method visually confirms the stabilization and binding of the VDR-RXR heterodimer to DNA in the presence of the ligand.
Procedure:
1. Protein-DNA Complex: In vitro translated VDR and RXR proteins are mixed with a radiolabeled VDRE DNA probe and increasing concentrations of Calcipotriol.
2. Electrophoresis: The reaction mixtures are loaded onto a native polyacrylamide gel.
3. Detection: The migration of the protein-DNA complex is slower than that of free DNA. The intensity of the shifted band (representing the VDR-RXR-VDRE complex) is measured via autoradiography.
4. Quantification: The EC₅₀ is calculated based on the ligand concentration required to achieve half-maximal complex formation.
5. Result: MC903 promotes the formation of the heterodimer complex at concentrations as low as 0.1 nM, demonstrating high binding affinity.

3. Dissociation Kinetics Assay (Off-rate Analysis)
This test measures the stability or duration of the ligand-receptor complex over time.

Procedure:
1. Saturation Binding: VDR-RXR heterodimers are bound to the VDRE and saturated with a high concentration of Calcipotriol.
2. Time-Course Digestion: The complexes are exposed to trypsin for extended periods (from 15 minutes up to 24 hours).
3. Half-life (t½) Calculation: The amount of protected VDR fragment remaining is quantified at each time point to determine the half-life of the ligand-receptor complex.
4. Stability Profile: MC903 shows a dissociation rate faster than the natural hormone (t½ for MC903 is approximately 260-438 minutes, compared to 660 minutes for 1α,25(OH)₂D₃).

In this study, researchers describe here the effects of three drugs that are either approved or have the potential for treating multiple sclerosis (MS) patients through the in vitro activities of human natural killer (NK) cells and dendritic cells (DCs). Our results indicate that 1,25(OH)2D3, the biologically active metabolite of vitamin D3, calcipotriol and FTY720 augment IL-2-activated NK cell lysis of K562 and RAJI tumor cell lines as well as immature (i) and mature (m) DCs, with variable efficacies. These results are corroborated with the ability of the drugs to up-regulate the expression of NK cytotoxicity receptors NKp30 and NKp44, as well as NKG2D on the surfaces of NK cells. Also, they down-regulate the expression of the killer inhibitory receptor CD158. The three drugs down-regulate the expression of CCR6 on the surface of iDCs, whereas vitamin D3 and calcipotriol tend to up-regulate the expression of CCR7 on mDCs, suggesting that they may influence the migration of DCs into the lymph nodes. Finally, vitamin D3, calcipotriol and FTY720 enhance NK17/NK1 cell lysis of K562 cells, suggesting that a possible mechanism of action for these drugs is via activating these newly described cells. In conclusion, our results show novel mechanisms of action for vitamin D3, calcipotriol and FTY720 on cells of the innate immune system.[1]

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Interleukins (IL)-17A and -22 are involved in the patho-genesis of psoriasis. Cathelicidin LL37 serves as not only antimicrobial peptide but also as autoinflammatory mediator. 1,25-Dihydroxyvitamin D3 analogues, such as calcipotriol, are used as topical treatment for psoriasis. However, the effect of calcipotriol on the mRNA expression/production of human cathelicidin antimicrobial protein (hCAP18) and LL37 peptide by IL-17A/IL-22-stimulated keratinocytes remains controversial. To evaluate the modulatory action of calcipotriol on the production of hCAP18 and LL37, we analysed hCAP18 mRNA expression and hCAP18/LL37 peptide production in IL-17A/IL-22-stimulated cultured human keratinocytes by real-time qPCR, ELISA, western blotting, and immunocytostaining. By western blotting, hCAP18 protein was detected in keratinocytes cultured for 72 h with IL-17/IL-22. Calcipotriol increased hCAP18 mRNA expression in IL-17/IL-22-stimulated keratinocytes. However, LL37 peptide in the culture supernatants was reduced by calcipotriol. Immunostaining revealed that the overproduced LL37 resides within the cells. LL37 promotes psoriasis via interaction with extracellular DNA, but may suppress psoriasis by interfering cytosolic DNA.[2]

A total of 160 SKH-1 mice were randomized to one placebo group and four chemoprevention groups (diclofenac plus difluoromethylornithine; diclofenac plus calcipotriol; difluoromethylornithine plus calcitriol; and diclofenac plus difluoromethylornithine plus calcipotriol). The mice received UVB radiation for 20 weeks followed by 17 weeks with topical application of chemoprevention. The number of mice with tumors, number of tumors per group and tumor area size were compared using a linear regression model. Results: Chemoprevention with diclonefac plus calcipotriol and diclonefac plus difluoromethylornithine had a significant inhibiting effect on the number of tumors per group and the area of tumors. Moreover, diclonefac plus difluoromethylornithine had a significant inhibiting effect on the number of mice with tumors. Conclusion: Potentially, non-melanoma skin cancer in humans may be prevented with these agents with few adverse effects. Therefore, clinical studies are needed to determine their therapeutic/preventive effect and possible adverse effects.[3]

Absorption
Clinical studies of radiolabeled ointments have shown that when the ointment is applied topically to psoriatic plaques, approximately 6% (±3%, standard deviation) of the calcipotriol dose is absorbed systemically; when applied to normal skin, 5% (±2.6%, standard deviation) is absorbed systemically.
Elimination Pathway
Active vitamin 1,25-dihydroxyvitamin D3 (calcipotriol) is known to circulate in the liver and be excreted in bile. There is evidence that maternal 1,25-dihydroxyvitamin D3 (calcipotriol) may enter fetal circulation, but it is unclear whether it is secreted into human breast milk.
Metabolism/Metabolites
Hepatic metabolism. After systemic absorption, calcipotriol is rapidly metabolized, following a metabolic pathway similar to that of natural hormones. The potency of the major metabolites is much lower than that of the parent compound.
Hepatic metabolism. After systemic absorption, calcipotriol is rapidly metabolized, following a metabolic pathway similar to that of natural hormones. The potency of the major metabolites is much lower than that of the parent compound. Excretion pathway: The active form of vitamin D3 (calcitriol) is known to be recycled in the liver and excreted via bile. There is evidence that maternal 1,25-dihydroxyvitamin D3 (calcitriol) may enter fetal circulation, but it is unclear whether it is secreted into human breast milk.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Currently, there is no information regarding the use of calcipotriol during lactation. Due to poor absorption after topical application, the risk to breastfeeding infants may be low, and its use during lactation is generally considered acceptable. However, some sources recommend avoiding application to the nipple area. Avoid applying combination preparations containing betamethasone (Enstaira) to the breasts. Only water-soluble creams or gels should be applied to the breasts, as ointments may expose the infant to high concentrations of mineral oil through licking.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

[1]. Effects of vitamin D3, calcipotriol and FTY720 on the expression of surface molecules and cytolytic activities of human natural killer cells and dendritic cells. Toxins (Basel). 2013 Oct 28;5(11):1932-47.

[2]. Calcipotriol Increases hCAP18 mRNA Expression but Inhibits Extracellular LL37 Peptide Production in IL-17/IL-22-stimulated Normal Human Epidermal Keratinocytes. Acta Derm Venereol. 2014 Sep;94(5):512-6.

[3]. Combination chemoprevention with diclofenac, calcipotriol and difluoromethylornithine inhibits development of non-melanoma skin cancer in mice. Anticancer Res. 2013 Aug;33(8):3033-9.

Calcipotriol hydrate is the monohydrate form of calcipotriol. It is used in combination with the corticosteroid betamethasone dipropionate for the topical treatment of adult plaque psoriasis. It is an anti-psoriatic drug. It contains calcipotriol. Calcipotriene is a synthetic vitamin D derivative, usually formulated as a topical dermatological medication, and is an anti-psoriatic drug. Calcipotriene (calcipotriol) competes with active 1,25-hydroxy-2D3 (natural vitamin D) for the 1,25-hydroxy-2D3 receptor, thereby regulating cell proliferation and differentiation. It induces keratinocyte differentiation and inhibits their proliferation, reversing the abnormal keratinocyte changes in psoriasis and normalizing epidermal growth. (NCI04) See also: Calcipotriene (with active moiety); Betamethasone dipropionate; Calcipotriol hydrate (component).

C27H42O4

430.6200

430.308

C, 75.31; H, 9.83; O, 14.86

147657-22-5

Calcipotriol;112965-21-6

49800068

White to off-white solid powder

5.026

4

4

5

31

743

7

O([H])[C@]([H])(/C(/[H])=C(\[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])(C([H])([H])[C@@]([H])(C\3([H])[H])O[H])O[H])/C([H])([H])C([H])([H])C([H])([H])[C@]12C([H])([H])[H])C1([H])C([H])([H])C1([H])[H].O([H])[H]

XBKHACNRWFKJNC-MANNPBRJSA-N

InChI=1S/C27H40O3.H2O/c1-17(6-13-25(29)20-8-9-20)23-11-12-24-19(5-4-14-27(23,24)3)7-10-21-15-22(28)16-26(30)18(21)2;/h6-7,10,13,17,20,22-26,28-30H,2,4-5,8-9,11-12,14-16H2,1,3H3;1H2/b13-6+,19-7+,21-10-;/t17-,22-,23-,24+,25-,26+,27-;/m1./s1

(1R,3S,5Z)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(E,2R,5S)-5-cyclopropyl-5-hydroxypent-3-en-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol;hydrate

MC-903; PRI 2201; Psorcutan; Calcipotriol monohydrate; CALCIPOTRIENE HYDRATE; 147657-22-5; Calcipotriol (monohydrate); calcipotriene; UNII-S7499TYY6G; Calcipotriene monohydrate; S7499TYY6G; Sorilux; MC903; Calcitrene; CCRIS 7700; Daivonex; Dovonex; MC 903; Calcipotriene; Calcipotriol Monohydrate

2934.99.03.00

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)

DMSO: ~100 mg/mL (~232.2 mM)
Ethanol: ~100 mg/mL (~232.2 mM)

Solubility in Formulation 1: 5 mg/mL (11.61 mM) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH 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: ≥ 5 mg/mL (11.61 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH 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: ≥ 5 mg/mL (11.61 mM) (saturation unknown) in 10% EtOH + 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 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.81 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 5: ≥ 2.5 mg/mL (5.81 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 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 6: ≥ 2.5 mg/mL (5.81 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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Solubility in Formulation 7: 2.5 mg/mL (5.81 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 8: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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 (Please use freshly prepared in vivo formulations for optimal results.)