Irosustat   中文名称: 艾洛司他

Steroid sulfatase (IC50 = 8 nM)

Irosustat (667 COUMATE) 的 IC50 为 8 nM，是类固醇硫酸酯酶的强抑制剂 [1]。 10 μM 的 isosustat (667 COUMATE) 对 MCF-7 细胞形状或增殖没有影响，但它会抑制这些细胞中类固醇硫酸酯酶 (STS) 的活性，IC50 为 0.2 nM [2]。

---

体外抑制STS活性[2]
通过使用胎盘微粒体和MCF-7细胞测定其IC50值，比较了所有化合物抑制STS活性的能力（表1）。在胎盘微粒体中，底物浓度为20μM时，667 COUMATE是最有效的抑制剂，IC50值为8 nM，其次是EMATE和2-MeOE2bisMATE，IC50分别为20和39 nM。在MCF-7细胞中，使用生理浓度的E1S（2-3nM）进行检测，总体IC50值低于使用胎盘微粒体获得的值。然而，667 COUMATE仍然是最有效的抑制剂，IC50为0.2 nM。EMATE和2-MeOE2bisMATE具有相似的抑制效力，IC50分别为0.6和0.5 nM。2-MeOE2缺乏氨基磺酸盐基团，在10μM下测试时，在两种测定中对STS活性都没有任何抑制作用[2]。

isosustat 可有效抑制大鼠肝脏； 1 mg/kg 时，可观察到 >90% 的抑制作用。在卵巢切除大鼠中，irosustat（口服 2 mg/kg，持续 5 天）可抑制硫酸雌酮 (E1S) 的子宫发育。此外，在切除卵巢的大鼠中，Irosustat（2、10 mg/kg，口服）与 E1S 联用可剂量依赖性地抑制 NMU 诱导的乳腺肿瘤的生长 [1]。当给予 Irosustat（667 COUMATE；10 mg/kg，口服）时，大鼠肝脏中的类固醇硫酸酯酶（STS）活性被抑制 97.9 ± 0.06% [2]。

---

体内抑制STS活性[2]
在证明EMATE的修饰衍生物2-MeOE2bisMATE在体外是STS活性的强效抑制剂后，对其进行了体内测试，并与EMATE和667 COUMATE进行了比较。动物接受10mg/kg的单次口服抑制剂剂量，24小时后收集肝脏样本进行STS活性测定。在仅接受载体的动物中，肝脏STS活性为32.56±8.1 nmol/h/mg蛋白。所有测试的化合物都非常有效，对肝脏STS活性的抑制率超过95%

---

体内STS活性的恢复[2]
先前观察到，单次口服667 COUMATE后，STS活性在一周内完全恢复，而EMATE此时几乎没有恢复。在本研究中，我们比较了EMATE、667 COUMATE和2-MeOE2bisMATE在体内抑制STS活性的持续时间。动物接受10mg/kg的单次口服剂量，对于接受667 COUMATE的动物，在第1、3和7天收集肝脏样本，对于接受EMATE或2-MeOE2bisMATE的动物，则在第1，5，10和15天收集肝脏样品。如图4所示，接受667 COUMATE的动物在第3天恢复了10%的抑制STS活性，到第7天，活性部分恢复到对照组的55%。在接受EMATE或2-MeOE2bisMATE的动物中，STS活性在5天内仍受到抑制。到第10天，2-MeOE2bisMATE治疗的动物恢复了80%的STS活性，而EMATE治疗的动物此时仅恢复了35%。然而，在第15天，两组动物的STS活性均已完全恢复。

类固醇硫酸酯酶测定[2]
在胎盘微粒体中[2]
按照Duncan等人的描述纯化胎盘微粒体。使用[6,7-3H]E1S（4×10~5 dpm）进行测定，用未标记的E1S调节至20μM。将酶（125μg蛋白质）与不同浓度的底物±抑制剂一起孵育，通过分配到甲苯中来测定形成的产物。

---

在MCF-7细胞中[2]
MCF-7乳腺癌症细胞维持在含有10%FCS和其他必需营养物的MEM培养基中。在实验中，细胞以每瓶1×105个细胞的数量接种，并在上述培养基中培养至80%融合。STS测定是在无血清培养基中使用[3H]E1S（2 nM）进行的，有或没有抑制剂（1 pM-10μM）。孵育20小时后，通过测量形成的产物来测定培养基中的STS活性。使用库尔特细胞计数器测定每个烧瓶中的细胞数量。

---

在肝组织中[2]
该测定方法与用于胎盘微粒体的方法基本相同，除了使用匀浆大鼠肝脏样品的上清液作为STS的来源来测试酶抑制的程度。

细胞增殖试验[2]
MCF-7细胞在生长培养基（含有酚红、10%胎牛血清（FCS）和必需营养素的最低必需培养基（MEM））中培养。当细胞达到60%融合时，在生长培养基中用药物（0.001-10μM）处理。孵育72小时后，在正常光照条件下拍摄照片，并使用库尔特细胞计数器测定每个烧瓶中附着的细胞数量。

Female Wistar rats (200–250 g) were used. Groups of rats, with three rats in each group for each experiment, were treated p.o. with vehicle (propylene glycol) or the drug (10 mg/kg), with animals receiving a single dose. The animals receiving Irosustat (667 COUMATE) were sacrificed by an approved method on days 1, 3 and 7 and on days 1, 5, 10 and 15 for those receiving EMATE or 2-MeOE2bisMATE, to assess the duration of STS inhibition. Samples of liver tissues were removed and immediately frozen on solid carbon dioxide and stored at −20 °C until assayed. Tissues were homogenized in ice cold PBS-sucrose and, after centrifugation to remove the cell debris, aliquots of supernatants were used for STS assay

[1]. In vivo inhibition of estrone sulfatase activity and growth of nitrosomethylurea-induced mammary tumors by 667 COUMATE. Cancer Res. 2000 Jul 1;60(13):3394-6.

[2]. Inhibition of MCF-7 breast cancer cell proliferation and in vivo steroid sulphatase activity by 2-methoxyoestradiol-bis-sulphamate. J Steroid Biochem Mol Biol. 2003 Feb;84(2-3):351-8.

Irosustat has been investigated for the treatment of Metastatic Breast Cancer and Locally Advanced Breast Cancer.
Irosustat is steroid sulfatase inhibitor BN 83495 selectively binds to and inhibits steroid sulfatase (STS), which may inhibit the production of locally active estrogens and so inhibit estrogen-dependent cell growth in tumor cells, such as those of the breast, ovary, and endometrium. STS is a cytoplasmic enzyme responsible for the conversion of circulating inactive estrone sulfate and estradiol sulfate to biologically active unconjugated estrone and estradiol, respectively.

---

The development of potent steroid sulfatase inhibitors is an important new therapeutic strategy for the treatment of postmenopausal women with breast cancer. A series of tricyclic coumarin sulfamates were synthesized, and their inhibitory properties were examined in vitro and in vivo. In a placental microsomal assay system, 667 COUMATE emerged as the most potent inhibitor with an IC50 of 8 nM. Administration of a single dose (10 mg/kg, p.o.) of 667 COUMATE inhibited rat liver estrone sulfatase activity by 93%. 667 COUMATE was devoid of estrogenicity, as indicated by its failure to stimulate the growth of uteri in ovariectomized rats. In vivo, estrone sulfate-stimulated growth of uteri in ovariectomized rats was inhibited by 667 COUMATE. Using the nitrosomethylurea-induced mammary tumor model, we found that 667 COUMATE caused regression of estrone sulfate-stimulated tumor growth in a dose-dependent manner. The identification of 667 COUMATE as a potent steroid sulfatase inhibitor will enable the therapeutic potential of this type of therapy to be evaluated.[1]

---

The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) inhibits the growth of breast cancer cells and is also a potent anti-angiogenic agent. We have previously shown that the 3-sulphamoylated derivatives of 2-methoxyoestrogens are more potent than the non-sulphamoylated compounds. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-MeOE2 to inhibit the growth of MCF-7 breast cancer cells. Both compounds inhibited cell growth with the IC(50) for 2-MeOE2bisMATE (0.4 microM) being six-fold lower than that for 2-MeOE2 (2.5 microM). Oestrogen sulphamates are potent inhibitors of steroid sulphatase (STS) activity. 2-MeOE2bisMATE was found to retain its STS inhibitory activity and in a placental microsome assay system it was equipotent with oestrone-3-O-sulphamate (EMATE). An in vivo study was also carried out to compare the potency of 2-MeOE2bisMATE with that of EMATE and the non-steroidal STS inhibitor, 667 coumarin sulphamate (667 COUMATE). After a single oral dose (10mg/kg) some recovery of STS activity was detected by day 3 (10%) with activity partially restored (55%) by day 7 after administration of 667 COUMATE. For the other two steroidal compounds, STS activity remained almost completely inactivated for up to 5 days with complete restoration of activity occurring by day 15. The anti-proliferative and STS inhibitory properties of 2-MeOE2bisMATE suggest that it has considerable potential for development as a novel anti-cancer drug.[2]

C14H15NO5S

309.3376

309.067

C, 54.36; H, 4.89; N, 4.53; O, 25.86; S, 10.37

288628-05-7

5287541

White to off-white solid powder

2.3

104

1

6

2

21

562

0

O=C1OC2=CC(OS(=O)(N)=O)=CC=C2C3=C1CCCCC3

DSLPMJSGSBLWRE-UHFFFAOYSA-N

InChI=1S/C14H15NO5S/c15-21(17,18)20-9-6-7-11-10-4-2-1-3-5-12(10)14(16)19-13(11)8-9/h6-8H,1-5H2,(H2,15,17,18)

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.08 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (8.08 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (8.08 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。