PDE/phosphodiesterase; antipsoriatic anteagent; vitamin D3 analog

Pefcalcitol, an analog of vitamin D3 (VD3), is an anti-psoriatic drug candidate that is designed to achieve much higher pharmacological effects, such as keratinocyte differentiation, than those of VD3, with fewer side effects [2].

SMD-502, a new vitamin D3 (VD3) analog, is an antipsoriatic drug candidate. The compound exhibited fewer side effects than known VD3 analogs in animal models, probably because the compound is rapidly converted into pharmacologically inactive form after permeating into systemic circulation from the application site on skin. This feature of the compound makes it difficult to assess genotoxic risk in vivo with the standard approach of bone marrow or peripheral blood micronucleus assays in rodents because of the low blood concentration level. To evaluate in vivo mutagenicity of the compound in the present study, mutant frequency (MF) in skin and liver of gpt delta transgenic mice was examined with percutaneous administration of SMD-502 for 28 days. In tissues collected 7 days after the end of administration, no significant increase in the MF was observed in either skin or liver. Additionally, when the compound was tested in a GDL1 cell line established from gpt delta mice, GDL1 cells exhibited no significant increase in MFs even under conditions in which they would be exposed to a much higher concentration of the compound than in the in vivo study. The results in this study further supported the consideration that SMD-502 has no mutagenic activity [1].

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Genotoxicity of the compound was evaluated in a rat skin micronucleus (MN) test.[2]
Results: In the rat skin MN test, pefcalcitol showed positive when specimens were stained with Giemsa, whereas neither an in vitro chromosome aberration test in CHL cells nor an in vivo bone marrow MN test in rats indicated clastogenicity. To elucidate the causes of the discrepancy, the MN specimens were re-stained with acridine orange (AO), a fluorescent dye specific to nucleic acid, and the in vivo clastogenicity of the compound in rat skin was re-evaluated. The MN-like granules that had been stained by Giemsa were not stained by AO, and AO-stained specimens indicated that pefcalcitol did not increase the frequency of micronucleated (MNed) cells. Histopathological evaluation suggested that the MN-like granules in the epidermis were keratohyalin granules contained in keratinocytes, which had highly proliferated after treatment with pefcalcitol. Conclusions: Pefcalcitol was concluded to be negative in the rat skin MN test. The present study demonstrated that Giemsa staining gave a misleading positive result in the skin MN test, because Giemsa stained keratohyalin granules.

Bone marrow MN test [2]
Pefcalcitol solution (5 mg/mL) was injected via the tail vein in dose volumes of 0.75, 1.5, 3, and 6 mL/kg to achieve dose levels of 3.75, 7.5, 15, and 30 mg/kg, respectively. These doses were selected because a dose of 30 mg/kg was the maximum tolerated dose for rat single dose i.v. treatment in a preliminary study (data not shown). MMC was administered intraperitoneally at a dose of 2 mg/kg with a dosing volume of 4 mL/kg. Five male rats per group were used. A solution composed of propylene glycol, ethanol, N-methyl-2-pyrrolidinone, and saline (48:6:6:40 by volume) served as a vehicle control and was dosed at 6 mL/kg. Approximately 24 h or 48 h after treatment, bone marrow cells were collected from femurs. Smear specimens of bone marrow from the rats treated with the chemicals or with vehicle were fixed with 99.8% methanol for 5 min and stained with 40 μg/mL AO. MNed erythrocytes among approximately 2000 polychromatic erythrocytes (PCE) were scored at 1000× magnification using fluorescent microscopes equipped with a filter set of blue excitation (460–490 nm) and a barrier filter at 515 nm. To evaluate the bone marrow toxicity, reduction in the ratio of PCE to all erythrocytes, which is the total of PCE plus normochromatic erythrocytes (NCE), was measured by counting approximately 1000 erythrocytes.

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Skin MN test [2]
The skin MN test followed the Nishikawa method, using a slight modification by Reus et al. An ethanol solution of Pefcalcitol prepared at 20 mg/mL was applied onto shaved dorsal skin (3 cm × 4 cm) of rats at a volume of 1 mL/kg, which resulted in a dose of 20 mg/kg. The dose of 20 mg/kg was set because the single dose level was supposed to be sufficient to examine AO stainability of the granules, and the preliminary rat skin MN test (data not shown) showed that pefcalcitol at this dose sufficiently increased the frequency of MN-like granules. The MMC solution at 1 mg/mL was administered in the same manner as pefcalcitol at a volume of 0.2 mL/head to achieve a dose of 0.2 mg/head. Five male rats were used for each dosing group. Skin samples were taken approximately 72 h after treatment. Epidermis cells were harvested through distinct dermo-epidermal separation from the skin samples with cold-digestion in thermolysin (200 μg/mL) followed by isolation with a trypsin solution (phosphate-buffered saline containing 2.5 mg/mL trypsin and 0.4 mg/mL EDTA). The epidermis cells were then swollen with hypotonic 0.075 mol/L KCl solution, fixed with methanol/acetic acid (3:1 by volume), and dropped on to microscope slides after re-suspension in 1% acetic acid methanol solution. The slides were stained with 3% Giemsa in 1/15 mol/L phosphate buffer (pH 6.8) for microscopic analysis, and approximately 2,000 cells per animal were evaluated for the occurrence of MNed cells. The coordinates of each cell with MNi were recorded using a microscope with automated scanning.

[1]. Lack of Mutagenicity of SMD-502, a New Vitamin D3 Analog for Topical Application, in Skin and Liver of gpt delta Transgenic Mice and in GDL1 Cells. Genes and Encironment, 2012, 34 (3), 107-114.
[2]. Giemsa-stained pseudo-micronuclei in rat skin treated with vitamin D3 analog, pefcalcitol. Genes Environ. 2017 Jun 1:39:17.

Pefcalcitol has been investigated for the basic science of Psoriasis.

C26H34F5NO4

519.24

C, 60.11; H, 6.60; F, 18.28; N, 2.70; O, 12.32

381212-03-9

16122802

Typically exists as solid at room temperature

1.3±0.1 g/cm3

614.0±55.0 °C at 760 mmHg

325.1±31.5 °C

0.0±4.0 mmHg at 25°C

1.523

5.42

3

9

7

36

955

5

FC(C(F)(F)F)(CNC(CO[C@@H](C)C1=CC[C@H]2/C(=C/C=C3\C(=C)[C@H](C[C@@H](C\3)O)O)/CCC[C@@]21C)=O)F

SVCSMAZYWOQCBW-NVJMFHFGSA-N

InChI=1S/C26H34F5NO4/c1-15-18(11-19(33)12-22(15)34)7-6-17-5-4-10-24(3)20(8-9-21(17)24)16(2)36-13-23(35)32-14-25(27,28)26(29,30)31/h6-8,16,19,21-22,33-34H,1,4-5,9-14H2,2-3H3,(H,32,35)/b17-6+,18-7-/t16-,19+,21-,22-,24+/m0/s1

2-[(1S)-1-[(3aS,4E,7aS)-4-[(2Z)-2-[(3S,5R)-3,5-dihydroxy-2-methylidenecyclohexylidene]ethylidene]-7a-methyl-3a,5,6,7-tetrahydro-3H-inden-1-yl]ethoxy]-N-(2,2,3,3,3-pentafluoropropyl)acetamide

Pefcalcitol; 381212-03-9; M518101; SMD-502; KT5224XSHW; SMD502; M5181; M-5181; 2-(((1S,3R,5z,7E,20S)-1,3-dihydroxy-9,10-secopregna-5,7,10(19),16-tetraen-20-yl)oxy)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; 2-[(1S)-1-[(3aS,4E,7aS)-4-[(2Z)-2-[(3S,5R)-3,5-dihydroxy-2-methylidenecyclohexylidene]ethylidene]-7a-methyl-3a,5,6,7-tetrahydro-3H-inden-1-yl]ethoxy]-N-(2,2,3,3,3-pentafluoropropyl)acetamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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