20S(OH)D3   中文名称: 20S-（羟基）维生素D3

VDR/vitamin D receptor

化学合成的20S-（OH）D3具有生物活性[1]
在人类正常表皮角质形成细胞、永生化HaCaT角质形成细胞和仓鼠AbC1黑色素瘤细胞中测量的20S-（OH）D3的生物活性显示出与P450scc产生的化合物相似的效果。

首先，我们测试了对角质形成细胞增殖的影响，发现与对照（载体处理）细胞相比，用浓度为10-8M和10-6M的220S-（OH）D3和1,25（OH）2D3处理正常人表皮角质形成细胞24小时，可抑制细胞增殖（图5）。同样，20S-（OH）D3以剂量依赖的方式抑制HaCaT永生化表皮角质形成细胞的增殖（通过3H-胸苷掺入DNA来测量）（图6）。抑制作用与1,25（OH）2D3相似，在低至10-11M的浓度下可见（图6）。有趣的是，前体化合物20S-（OH）-7DHC也抑制了DNA合成，尽管浓度更高（≥10-10M）

接下来，我们测试了20S-（OH）D3对维生素D受体（VDR）、外皮蛋白和CYP24基因表达的影响，发现20S-（OH）D3在与1,25（OH）2D3相当的水平上刺激了VDR的表达（图7A）。然而，与1,25（OH）2D3相比，它在刺激外皮蛋白（2倍对5倍）和CYP24（60倍对10000倍）表达方面的效果较差（图7B和7C）。1,25（OH）2D3和20S-（OH）D3在诱导CYP 24表达方面的差异超过100倍，这与之前报道的生化合成的20S-（羟基）D3相似[18,19,33]，这表明20S-（OH）D3对维生素D3活性形式的失活（24羟基化）影响很小。

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20S-（OH）D3抑制癌症在单层和软琼脂中的菌落形成[2]
通过测量20S-(OH)D3和1，25（OH）2D3抑制癌症细胞集落形成的能力，将它们的抗肿瘤活性进行比较。首先，我们测试了这些化合物对HepG2肝细胞癌集落生长的影响，发现20S-(OH)D3和1,25（OH）2D3以浓度依赖的方式抑制HepG2增殖<与对照（载体处理）细胞相比，浓度为100 nM的strong>20S-（OH）D3和1,25（OH）2D3有效地减少了大于0.2 mm（分别为54±14%和50±6%）和大于0.5 mm（分别是70±20%和92±6%）的集落的CFU（图1A和B）。用较低浓度（0.1和10 nM）的维生素D3衍生物治疗对大于0.2 mm的菌落总数没有显著影响（图1A），但大大阻止了大于0.5 mm的菌落的形成（图1B）。在所有三种测试浓度下，1,25（OH）2D3比20（OH）D3更能抑制大菌落（尺寸>0.5mm）的形成。

接下来，我们测试了20S-(OH)D3对MDA-MB-453人乳腺癌细胞在软琼脂中生长的影响。与对照（载体处理）细胞相比，浓度为100 nM的20（OH）D3和25（OH）D2有效抑制了大于0.2 mm的MDA-MB-453集落的形成（分别为CFU的54±4%和43±7%）（图2A和B）。在两个100nM治疗组中均未检测到大于1.5mm的菌落。在10 nM的浓度下，20（OH）D3抑制了大于1.5 mm的MDA-MB-453集落的形成（图2B）85±11%，而25（OH）D2的作用没有统计学意义。

最后，我们在软琼脂上测试了20S-（OH）D3对MCF7乳腺癌症细胞集落形成的影响。由于该细胞系的集落形成缓慢，我们将测试期延长至26天，以研究其抗增殖作用。浓度为100 nM的20（OH）D3抑制了大于0.2 mm的菌落形成78±28%（图3A）。然而，1α，25（OH）2D3在该模型中表现出相对较强的作用，在低至0.1 nM的浓度下抑制了62±6%的集落形成（图3B）。

高剂量的20S-（OH）D3在治疗3周后对小鼠没有钙血症影响[2]
文献报道，在大鼠模型中，连续7天以3μg/kg的剂量服用20S-(OH)D3对全身钙水平没有影响。在目前的研究中，我们通过给C57BL/6小鼠服用不同剂量的20（OH）D3来评估其潜在毒性，最高剂量为30μg/kg。作为阳性对照，我们使用了25（OH）D2和1,25（OH）2D3，因为据报道，它们在小鼠或大鼠体内浓度≥1μg/kg时会产生高钙血症。如图4所示，即使在3周腹腔注射治疗的最高测试剂量（30μg/kg）下，20（OH）D3也没有显示出高钙血症活性（钙=9.7±0.69，而赋形剂对照组为9.4±0.25mg/dl）。相比之下，剂量仅为2μg/kg的1,25（OH）2D3导致钙的预期急剧上升，高达14.6±0.48mg/dl。剂量为2μg/kg的25（OH）D3显示出轻微的高钙血症作用，将血清钙水平提高到11.5±0.48mg/dl，超过了正常血清钙的10.5mg/dl上限。

细胞色素P450scc和CYP27B1代谢20S-（OH）D3 [1]
如前所述，将20S-（OH）D3掺入磷脂囊泡，并通过P450scc测量其代谢。孵育混合物由510μM磷脂囊泡组成，其中含有20S-（OH）D3，与磷脂的比例为0.1mol/mol，2μM牛细胞色素P450scc，15μM肾上腺素毒素，0.4μM肾上腺毒素还原酶，2 mM葡萄糖6-磷酸，2 U/ml葡萄糖6-磷酸脱氢酶和50μM NADPH。样品预孵育8分钟，通过加入NADPH开始反应，在37°C下振荡孵育6分钟。用二氯甲烷提取后，使用C18柱（Grace 15 cm×4.6 mm，粒径7μm）通过HPLC分析样品。通过与上述类似的程序测量CYP27B1（25-羟基维生素D3 1α-羟化酶）对20S-（OH）D3的代谢。

集落形成试验[2]
该测定遵循如前所述的标准方法。简而言之，将细胞以20个细胞/9.6 cm2的密度铺在24孔板中，培养基含有5%木炭处理的FBS、1%抗生素溶液和分级浓度的20S-（OH）D3、1,25（OH）2D3或25（OH）D3，或乙醇（载体对照）。细胞在37°C下培养7天，每3天更换一次培养基。孵育结束时，用4%多聚甲醛的磷酸盐缓冲溶液（PBS）在4°C下固定菌落过夜，洗涤，用5%结晶紫的PBS染色30分钟，冲洗并风干。使用ARTEK计数器880测量菌落的数量和大小。集落形成单位（CFU）是通过将集落数量除以细胞数量，然后乘以100来计算的。

如前所述，MCF7细胞在软琼脂中生长。HepG2、MCF7和MDA-MB-453细胞的致瘤性是通过它们在软琼脂中形成集落的能力来确定的。细胞在单层中生长，胰蛋白酶消化，并重新悬浮在含有0.4%琼脂糖和10%木炭剥离血清的0.25ml培养基中（1000个细胞/孔）。将细胞悬浮液添加到24孔板中0.8%琼脂层中20S-（OH）D3，25（OH）D3，或1α，25（羟基）2D3从乙醇原料中加入，最终浓度范围为0.1-100nM。试验中包括乙醇对照。两周后，在软琼脂中形成的菌落用MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑）试剂（0.5mg/ml）染色，并在显微镜下评分。单位数由形成的集落数除以接种的细胞数×100计算得出。

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细胞增殖试验[1]
1.MTS测试[1]
将HEKn角质形成细胞铺在96孔板中，10000个细胞/孔。细胞孵育过夜后，向培养基中加入1,25（OH）2D3或化学合成的20S-（OH）D3，最初溶解在乙醇中，然后稀释在含有0.5%BSA的KGM培养基中，使最终浓度达到0.01μM或1μM，而在对照培养中，乙醇载体的最终浓度为0.1%。与这些化合物孵育20小时后，向细胞中加入20μl MTS/PMS溶液。4小时后，使用ELISA平板读数器在490nm处记录吸光度。在六个重复中测量活细胞的数量。

2.DNA合成[1]
如前所述进行DNA合成测试。根据细胞类型，将细胞以5000-25000个细胞/孔的速度接种到24孔板中。在37°C下孵育过夜后，将培养物置于无血清培养基中，使细胞同步处于细胞周期的G0/G1期。24小时后，将20S-(OH)D3加入含有生长补充剂的新鲜培养基中，并再孵育72小时。在规定的时间后，将[3H]-胸苷（比活度88.0 Ci/mmol）加入培养基中至终浓度为0.5μCi/mL。在37°C下孵育4小时后，丢弃培养基，细胞在PBS（磷酸盐缓冲盐水）中的10%TCA中沉淀30分钟，用1 mL PBS洗涤两次，然后在37°C下用1 N NaOH/1%SDS（250μL/孔）孵育30分钟。将提取物收集在闪烁瓶中，并向每个瓶中加入5mL闪烁鸡尾酒。用β计数器测量掺入DNA的3H放射性。

3.集落形成试验[1]
如前所述，该测定遵循我们的标准方法。简而言之，将细胞以192个细胞/孔的密度铺在含有5%ctFBS（木炭处理的胎牛血清）、1%抗生素溶液和分级浓度的20S-（OH）D3或乙醇（载体对照）的培养基中。细胞在37°C下培养7天，每3天更换一次培养基。孵育结束时，将菌落用4%多聚甲醛的PBS溶液在4°C下固定过夜，洗涤，用5%结晶紫的PBS溶液染色30分钟，冲洗并风干。使用ARTEK计数器880测量菌落的数量和大小。集落形成单位是通过将集落数量除以细胞数量，然后乘以100来计算的。

4.ApoTox-Glo三重检测[1]
将黑色素瘤细胞铺在96孔板上，10000个细胞/孔。细胞孵育过夜后，如上所述，将1,25（OH）2D3或20S-（OH）D3加入培养基中。与这些化合物孵育24小时后，向细胞中加入20μl含有GF-AFC底物和双-AAF-R110底物的存活/细胞毒性试剂。在37°C下孵育30分钟后，使用荧光和发光微孔板读数器在400 nm激发/505 nm发射下记录荧光，以检测存活率，在485 nm激发/520 nm发射下检测细胞毒性。将100μl Caspase-Glo 3/7试剂进一步加入细胞中，在室温下孵育30分钟后，记录发光。在四个重复实验中测量了活细胞、细胞毒性细胞和凋亡细胞的数量。 实时荧光定量PCR 使用Absolutely RNA Miniprep试剂盒分离用20S-（OH）D3或1,25（OH）2D3处理的HEKn角质形成细胞的RNA。使用Transcriptor First Strand cDNA合成试剂盒进行逆转录（1μg RNA/反应）。使用在无菌水中稀释5倍的cDNA和TaqMan PCR主混合物进行实时PCR。反应（一式三份）在95°C下进行5分钟，然后进行45个循环，95°C 10秒，60°C 30秒，72°C 30秒钟。引物和探针使用通用探针库设计。数据在罗氏Light Cycler 480上收集。使用比较CT方法将每个基因的扩增产物的量与亲环素B的扩增产物量进行比较。用于RTPCR DNA扩增的引物列表如表2所示。

In vivo toxicity studies [2]
The potential toxicity of 20S-(OH)D3 was evaluated using male C57/BL6 mice. Mice weighed approximately 25–26 g and were seven weeks of age. Two positive control compounds, 1,25(OH)2D3 and 25(OH)D3, were obtained from xx. 20(OH)D3 in autoclaved sesame oil was administered by intraperitoneal (i.p.) injection (50 μl/mouse) once daily for three consecutive weeks to groups of five mice at doses of 5, 10, 20 and 30 μg/kg body weight. In the positive control group, 1,25(OH)2D3 or 25(OH)D3, was given at the dose of 2 μg/kg body weight by i.p. injection following the same pattern. Another group of five mice was injected with autoclaved sesame oil 50 μl /mouse daily, which served as a vehicle control. Clinical signs of toxicity and body weight were assessed twice a week throughout the experimental period. Terminal blood samples (800~1000 μl/mouse) were collected by cardiac puncture at the end of the three-week treatment. The serum (~300 μl/mouse) was immediately separated using BD Microtainer® tubes and stored at −20°C. All animals were sacrificed by cervical dislocation immediately after the blood collection and the main organs (heart, lung, liver, spleen, kidney, adrenal and one piece of skin) of each mouse were collected and stored separately in 10% buffered formalin phosphate solution for subsequent pathological analysis.

[1]. Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity. Steroids. 2010 Dec;75(12):926-35.
[2]. 20-hydroxyvitamin D₃ inhibits proliferation of cancer cells with high efficacy while being non-toxic. Anticancer Res. 2012 Mar;32(3):739-46.
[3]. Design, Synthesis and Biological Activities of Novel Gemini 20S-Hydroxyvitamin D3 Analogs. Anticancer Res. 2016 Mar;36(3):877-86.
[4]. Design, synthesis, and biological action of 20R-hydroxyvitamin D3. J Med Chem. 2012 Apr 12;55(7):3573-7.

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1alpha,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use. [1]

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Aim
To define the potential utility of 20-hydroxyvitamin D3 (20(OH)D3) as a tumorostatic agent, we assessed its in vitro antiproliferative activity and its in vivo toxicity.
Materials and Methods
The antitumor activity of 20(OH)D3 was tested against breast and liver cancer cell lines using colony formation assays. To assess in vivo toxicity, mice were injected with 5–30 μg/kg 20(OH)D3 intraperitoneally each day for 3 weeks. Blood and organ samples were collected for clinical pathology analyses.
Results
20(OH)D3 displays similar tumorostatic activity towards MDA-MB-453 and MCF7 breast carcinomas, and HepG2 hepatocarcinoma, in a dose-dependent manner. This compound is not hypercalcemic, does not cause detectable toxicities in liver, kidney, or blood chemistry in mice at a dose as high as 30 μg/kg. In contrast, both 25(OH)D3 and 1,25(OH)2D3 caused severe hypercalcemia at a dose of 2 μg/kg.
Conclusion
20(OH)D3 possesses high efficacy for inhibiting cancer cell proliferation in vitro and is non-toxic in vivo, supporting its further development as a potential anticancer therapeutic agent.[2]

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Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.[3]

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The non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated by semipreparative HPLC, and its structure is characterized. A comparison of 20R isomer to its 20S counterpart in biological evaluation demonstrates that they have different behaviors in antiproliferative and metabolic studies.[4]

C27H44O2

400.637068748474

400.334

651734-12-2

651734-12-2

162367340

Typically exists as white solid at room temperature

6.2

40.5

2

2

6

29

657

4

CC(C)CCC[C@@](C)([C@H]1CCC\2[C@@]1(CCC/C2=C\C=C/3\C[C@H](CCC3=C)O)C)O

IQEQEOBGZMEDBQ-LWVSKBGXSA-N

InChI=1S/C27H44O2/c1-19(2)8-6-17-27(5,29)25-15-14-24-21(9-7-16-26(24,25)4)11-12-22-18-23(28)13-10-20(22)3/h11-12,19,23-25,28-29H,3,6-10,13-18H2,1-2,4-5H3/b21-11+,22-12-/t23-,24?,25-,26-,27-/m0/s1

(1S,3Z)-3-[(2E)-2-[(1S,7aS)-1-[(2S)-2-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol

20(OH)D3; 20-Hydroxyvitamin D3; 20S(OH)D3; (S,Z)-3-((E)-2-((1S,3aS,7aS)-1-((S)-2-Hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexanol; 20-Hydroxyvitamin D3; 651734-12-2; (1S,3Z)-3-[(2E)-2-[(1S,7aS)-1-[(2S)-2-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。