VDR/vitamin D receptor.

In the absence of IL-17A or IL-22 stimulation, calcipotriol either had no effect (2–20 nM) or only slightly increased (0.2 nM) the expression of IL-8 mRNA in NHEK cells. Our earlier research was validated by the addition of IL-17A and IL-22, which greatly raised the mRNA expression of IL-8. In a dose-dependent manner, 2, 20, and 40 nM calcipotriol can suppress this increased IL-8 mRNA expression [1]. The expression of NK cytotoxic receptors, or KIRs, is modulated when medicines are administered to natural killer (NK) cells. For four hours, calcipotriol, FTY720, or 100, 10, or 1 ng/mL of 1,25(OH)2D3 were applied as a pretreatment to human NK cells. Following a 4-hour incubation period, the expression of NKp30 on the surface of NK cells was considerably elevated by all three doses of 1,25(OH)2D3, calcipotriol, and FTY720 [2].

Basic Protocol: TOPICAL APPLICATION OF MC903 INDUCES AD-LIKE SKIN INFLAMMATION[6]
Materials
Mice (C57BL/6J wild type, 6-10 weeks old)
Isoflurane (Baxter, cat. no. 100190360400)
MC903, calcipotriol, hydrate
Ethyl alcohol (EtOH, ethanol) , pure, 190 proof
37°C mouse heating pad
Precision-dial thickness gauge
High-precision laboratory scale
Additional reagents and equipment for Support Protocols 1–5.
Days 0-14: Induce dermatitis
1. Anesthetize mice using 4% isoflurane in 2 L/min of oxygen for induction and 2% isoflurane in 0.4 L/min of oxygen for maintenance.
2. Weigh mice using a high-precision laboratory scale and place animals on a 37°C mouse heating pad to maintain body temperature. Record weights over the course of treatment.
3. Measure both left and right ear thickness using a precision-dial thickness gauge under light anesthesia.
4. Topically, apply 1 nM MC903 in 100% absolute ethyl alcohol (EtOH) using a 2-20 µl pipette to both sides of each ear (10 μl on dorsal and ventral ear sides for a total volume of 20 μl per ear) on every treatment day according to the treatment regimen. Control mice are treated in parallel with the same volume of EtOH (as a vehicle control) only.
5. After the treatment, keep animals on a 37°C mouse heating pad to maintain constant body temperature during recovery, then return to cages.
6. Over the next 2 weeks (see treatment regimen), repeat measurements before topical application of MC903 or EtOH (steps 2-5). A total of 7 applications of MC903 or EtOH will be applied, with the final topical application on day 14.
Day 14: Itch assessment
7. On day 14, 24 hr before the experimental endpoint, record a video of the mice and quantify itch events via time-lapse videography. Determine and quantify itch events for 30 min.
Day 15: Experimental endpoint
8. At the experimental endpoint (day 15), euthanize mice by CO2 asphyxiation, repeat measurements (step 3), and harvest ear skin tissues using sterile sharp scissors and forceps (Alam et al., 2020, Mac-Daniel, Buckwalter, Gueirard, & Ménard, 2016). For ear draining lymph nodes, carefully cut the skin at the corresponding neck region and dissect the auricular lymph nodes (Alam et al., 2020, Mac-Daniel et al., 2016)

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The diclofenac plus DFMO plus calcipotriol group resulted in 1 out of every 32 animals dying, whereas all other animals in this group survived. Every group had a similar survival rate. Diclofenac plus calcipotriol (p=0.018) and diclofenac plus DFMO plus calcipotriol (p=0.002) therapy groups showed considerably less weight gain when compared to the placebo (linear regression model) [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).

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.[1]

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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.[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]

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Calcipotriol (MC903) can be used to induce Atopic Dermatitis (AD) mice model
Calcipotriol (MC903) was used to establish AD-like mice model. A murine model of AD-like disease was established as in a prior study. Briefly, mice were administered a daily topical dose of a 15 µL 0.005% Calcipotriol (MC903) scalp solution (MC903), which was applied to the dorsal and ventral sides of each ear for 12 consecutive days. Control animals received 15 µL of ethanol instead. Mice in the Calcipotriol (MC903) + poly (I:C) group were then treated with poly (I:C) in a concentration of 5 µg/g bodyweight. The impact of poly (I:C) treatment on these animals was assessed based upon changes in lesions, bodyweight, ear thickness, and histopathological findings. In addition, serum interleukin 4 (IL-4), interferon-γ (IFN-γ), immunoglobulin E (IgE), IL-13, and TSLP levels were measured using enzyme-linked immunosorbent assay (ELISA), while tissue IL-13 and TSLP levels were assessed using ELISA, Western blotting, and immunohistochemical staining, and mast cell infiltration was assessed through toluidine blue (TBO) staining.[from: Ann Transl Med. 2022 Feb;10(4):209. ]

Absorption, Distribution and Excretion
Clinical studies of radiolabeled ointments have shown that when the ointment is applied topically to psoriatic plaques, approximately 6% (±3%, SD) of the calcipotriol dose is absorbed systemically; when applied to normal skin, approximately 5% (±2.6%, SD) is absorbed systemically. The active form of vitamin 1,25-dihydroxyvitamin D3 (calcipotriol) is known to be recycled in the liver and excreted via 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: Calcipotriol is rapidly metabolized after systemic absorption, following a pathway similar to that of natural hormones. The potency of primary 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]. 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.

[2]. 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.

[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.

[4]. Induction of Antigen-Specific Tolerance by Peripheral Phagocytosis. US 20150283231 A1.

[5]. Nanoemulsion loaded gel for topical co-delivery of clobitasol propionate and calcipotriol in psoriasis. Nanomedicine. 2017 May;13(4):1473-1482.

[6]. A Mouse Model of MC903-Induced Atopic Dermatitis. Atopic Dermatitis. Curr Protoc. 2023 Mar;3(3):e695.

Calcipotriol is an open-ring cholesterane compound with the chemical name 26,27-cyclo-9,10-open-ring cholester-5,7,10,22-tetraene, containing hydroxyl substituents at positions 1, 3, and 24. It is used (in hydrate form) in combination with the corticosteroid betamethasone dipropionate for the topical treatment of adult plaque psoriasis. It acts as both a drug allergen and an anti-psoriatic agent. It belongs to the cyclopropane class of compounds and is a secondary alcohol, triol, hydroxy-open-ring steroid, and open-ring cholesterane. Calcipotriol (INN) or calcipotriene (USAN) is a synthetic derivative of calcitriol or vitamin D. Calcipotriene is a vitamin D analog. Calcipotriene is a synthetic vitamin D derivative, usually formulated for topical dermatological treatment with anti-psoriatic activity. Calcipotriol (Calcipotriol) competes with active 1,25-dihydroxy-2D3 (the natural form of vitamin D) for the 1,25-dihydroxy-2D3 receptor, thereby regulating cell proliferation and differentiation. It induces keratinocyte differentiation and inhibits their proliferation, reverses the abnormal keratinocyte changes in psoriasis, and restores normal epidermal growth. (NCI04)
Calcipotriol is only found in individuals who have used or taken this drug. It is a synthetic derivative of calcitriol or vitamin D. The exact mechanism by which calcipotriol alleviates psoriasis is not fully understood. However, studies have shown that it has a similar affinity for the vitamin D receptor to calcitriol, but its activity in regulating calcium metabolism is less than 1% that of calcitriol. The vitamin D receptor (VDR) belongs to the steroid/thyroid receptor superfamily and is present on various tissue cells, including those in the thyroid, bone, kidneys, and T cells of the immune system. T cells are known to play a role in psoriasis, and it is thought that the binding of calcipotriol to VDR may regulate the transcription of genes related to T cell differentiation and proliferation. See also: Calcipotriol hydrate (active ingredient); Betamethasone dipropionate; Calcipotriol (ingredient); Calcipotriol; Nicotinamide (ingredient)... See more...

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Drug Indications
For the treatment of moderate plaque psoriasis in adults.
Treatment of Psoriasis
Mechanism of Action
The exact mechanism by which calcipotriol alleviates psoriasis is not fully understood, but studies have shown that it has a similar affinity for vitamin D receptors to calcitriol, while its activity in regulating calcium metabolism is less than 1% of that of calcitriol. Vitamin D receptors (VDRs) belong to the steroid/thyroid receptor superfamily and are present on various tissue cells, including those in the thyroid, bone, kidneys, and T cells of the immune system. T cells are known to play a role in psoriasis, and it is believed that calcipotriol, upon binding to VDRs, regulates gene expression in T cells. This regulation is thought to affect gene products associated with cell differentiation and proliferation.

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Calcipotriol (MC903) Can Be Used to Induce Mouse Models of Atopic Dermatitis (AD)
Atopic dermatitis (AD) is a chronic, relapsing, and extremely itchy inflammatory skin disease, particularly affecting children. The pathogenesis of AD is not fully understood, and there is currently no cure. Therefore, various gene- or chemically induced AD mouse models have been developed. These preclinical mouse models are indispensable research tools for studying the pathogenesis of AD and evaluating the efficacy of novel candidate AD therapies. A commonly used AD mouse model is established by inducing an AD-like inflammatory phenotype very similar to human AD through topical application of the hypocalcemic vitamin D3 analog MC903. Furthermore, this model has minimal impact on systemic calcium metabolism compared to vitamin D3-induced AD models. Therefore, an increasing number of studies are using MC903-induced AD models to study the in vivo pathobiology of AD and to test new candidate small molecule and monoclonal antibody therapies.
This protocol details the functional measurements, including skin thickness measurement (skin thickness is a surrogate indicator of ear skin inflammation), pruritus assessment, histological assessment (for assessing structural changes associated with AD skin inflammation), and preparation of single-cell suspensions of ear skin and draining lymph nodes for flow cytometry assessment of inflammatory leukocyte subsets in these tissues. © 2023 Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Topical application of MC903 induces AD-like skin inflammation. Ancillary Protocol 1: Measurement of ear skin thickness. Ancillary Protocol 2: Pruritus assessment. Ancillary Protocol 3: Dissection of ear skin and ear draining lymph nodes. Ancillary Protocol 4: Histological assessment and quantification. Ancillary Protocol 5: Preparation of single-cell suspensions of ear skin and draining lymph nodes for flow cytometry assessment of inflammatory immune cell infiltration. [Reference: Mouse model of atopic dermatitis induced by MC903. Curr Protoc. Mar 2023; 3(3):e695.] doi: 10.1002/cpz1.695. ]

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Compared to the vector control group, poly(I:C) administration significantly exacerbated calcipotriol-induced AD-like skin lesions in mice. In animals treated with poly(I:C), serum IL-4, IL-13, and TSLP levels were significantly elevated, while IFN-γ levels remained unchanged. Compared to control mice, poly(I:C) treatment also increased IL-13 and TSLP levels in the skin lesions and increased dermal mast cell infiltration and IgE production. Conclusion: These data suggest that poly(I:C) treatment and the exogenous activation of TLR3 exacerbate calcipotriol-induced atopic dermatitis-like skin lesions in mice by increasing the production of TSLP and other T helper cell 2 (Th2)-related cytokines. [Reference: Polyinosinic acid-polycytidylic acid exacerbates calcipotriol-induced atopic dermatitis-like skin lesions in mice by increasing the expression of thymic stromal lymphopoietin. Ann Transl Med.] Feb 2022; 10(4):209.

C27H40O3

412.61

412.297

, 78.60; H, 9.77; O, 11.63

112965-21-6

Calcipotriol monohydrate;147657-22-5;Impurity F of Calcipotriol;112875-61-3

5288783

White to off-white solid powder

1.1±0.1 g/cm3

582.0±50.0 °C at 760 mmHg

166-168ºC

250.6±24.7 °C

0.0±3.7 mmHg at 25°C

1.580

5.43

3

3

5

30

743

7

C[C@H](/C=C/[C@H](C1CC1)O)[C@H]2CC[C@@H]\3[C@@]2(CCC/C3=C\C=C/4\C[C@H](C[C@@H](C4=C)O)O)C

LWQQLNNNIPYSNX-JQWURIRRSA-N

InChI=1S/C27H40O3/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/b13-6+,19-7+,21-10-/t17-,22+,23-,24+,25-,26-,27-/m1/s1

(5Z,7E,22E,24S)-24-Cyclopropyl-9,10-secochola-5,7,10(19),22-tetraene-1alpha,3beta,24-triol

Calcipotriene; calcipotriol; MC-903PRI; 2201MC903PRI-2201; Calcitrene ; CCRIS 7700; Daivonex; Dovonex; MC 903; Psorcutan Sorilux.

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)

DMSO : ~100 mg/mL (~242.37 mM)
Ethanol : ~50 mg/mL (~121.18 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.06 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 (6.06 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 3: ≥ 2.5 mg/mL (6.06 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 4: ≥ 2.5 mg/mL (6.06 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.)