Steroid sulfatase (IC50 = 8 nM)

With an IC50 of 8 nM, Irosustat (667 COUMATE) is a strong inhibitor of steroid sulfatase [1]. At 10 μM, isosustat (667 COUMATE) has no effect on MCF-7 cell shape or proliferation, but it inhibits the activity of steroid sulfatase (STS) in these cells with an IC50 of 0.2 nM [2].

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Inhibition of STS activity in vitro [2]
The ability of all the compounds to inhibit STS activity was compared by determining their IC50 values using placental microsomes and MCF-7 cells (Table 1). In placental microsomes, with a 20 μM substrate concentration, 667 COUMATE was the most potent inhibitor with an IC50 value of 8 nM followed by EMATE and 2-MeOE2bisMATE with IC50s of 20 and 39 nM, respectively. In MCF-7 cells, where the assays were carried out using a physiological concentration of E1S (2–3 nM), the overall IC50 values were lower than those obtained using placental microsomes. However, 667 COUMATE still remained the most potent inhibitor with an IC50 of 0.2 nM. EMATE and 2-MeOE2bisMATE had similar inhibitory potencies with IC50s of 0.6 and 0.5 nM, respectively. 2-MeOE2, which lacks a sulphamate group, did not have any inhibitory effect on STS activity in both the assays when tested at 10 μM [2].

Rat liver is efficiently inhibited by isosustat; at 1 mg/kg, >90% inhibition is seen. In ovariectomized rats, irosustat (2 mg/kg orally for 5 days) inhibits the development of the uterus in response to estrone sulfate (E1S). Moreover, in ovariectomized rats, the growth of NMU-induced mammary tumors was dose-dependently inhibited by Irosustat (2, 10 mg/kg, oral) in combination with E1S [1]. Steroid sulfatase (STS) activity in rat liver was inhibited by 97.9 ± 0.06% when Irosustat (667 COUMATE; 10 mg/kg, oral) was administered [2].

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In vivo inhibition of STS activity [2]
Having shown that 2-MeOE2bisMATE, a modified derivative of EMATE, was a potent inhibitor of STS activity in vitro, it was tested in vivo and compared with EMATE and 667 COUMATE. Animals received a single oral dose of the inhibitor at 10 mg/kg, with liver samples being collected 24 h later for the assay of STS activity. In animals receiving vehicle only, liver STS activity was 32.56±8.1 nmol/h/mg protein. All the compounds tested were very effective and produced more than 95% inhibition of liver STS activity

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Recovery of STS activity in vivo [2]
It was previously observed that, after administration of a single oral dose of 667 COUMATE, STS activity was completely restored within a week in contrast to EMATE when little recovery had occurred by this time. In the present study, we have compared the duration of inhibition of STS activity in vivo by EMATE, 667 COUMATE and 2-MeOE2bisMATE. The animals received a single oral dose at 10 mg/kg and the liver samples were collected on days 1, 3 and 7 for animals receiving 667 COUMATE and on days 1, 5, 10 and 15 for those receiving EMATE or 2-MeOE2bisMATE. As shown in Fig. 4, 10% of inhibited STS activity was recovered by day 3 in animals receiving 667 COUMATE, with activity partially restored to 55% of the controls by day 7. In animals receiving EMATE or 2-MeOE2bisMATE, STS activity remained inhibited up to 5 days. By day 10, 80% of STS activity was restored in 2-MeOE2bisMATE treated animals, while those with EMATE showed only 35% recovery by this time. However, complete recovery of the STS activity was observed by day 15 in both groups of animals.

Steroid sulphatase enzyme assay [2]
In placental microsomes [2]
Placental microsomes were purified as described by Duncan et al. The assay was carried out using [6,7-3H]E1S (4×10~5 dpm) adjusted to 20 μM with unlabelled E1S. The enzyme (125 μg of protein) was incubated with the substrate ± inhibitor at different concentrations and the product formed was determined by partitioning into toluene.

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In MCF-7 cells [2]
MCF-7 breast cancer cells were maintained in MEM medium with 10% FCS and other essential nutrients. For experiments, the cells were seeded at 1×105 cells per flask and cultured in the above medium until 80% confluent. The STS assay was carried out using [3H]E1S (2 nM) in serum free medium, with or without the inhibitor (1 pM–10 μM). After 20 h of incubation, STS activity was assayed in the medium by measuring the product formed. The number of cells in each flask was determined using a Coulter cell counter.

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In liver tissue [2]
The assay methodology was essentially the same as that used for placental microsomes except that supernatants from the homogenized rat liver samples were used as a source of STS to test the extent of enzyme inhibition.

Cell proliferation assay [2]
MCF-7 cells were cultured in growth medium (minimum essential medium (MEM) containing, phenol red, 10% foetal calf serum (FCS) and essential nutrients). When the cells reached 60% confluency, they were treated with the drugs (0.001–10 μM) in growth medium. After 72 h of incubation, photographs were taken under normal conditions of light and the number of attached cells in each flask was determined using a Coulter cell counter.

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 and locally advanced breast cancer. Irosustat is a steroidal sulfatase inhibitor; its compound, BN 83495, selectively binds to and inhibits steroidal sulfatase (STS), thereby inhibiting the production of locally active estrogen and subsequently suppressing estrogen-dependent cell growth in tumor cells such as breast, ovarian, and endometrial cells. STS is a cytoplasmic enzyme responsible for converting inactive circulating estrone sulfate and estradiol sulfate into biologically active unbound estrone and estradiol, respectively. Developing potent steroidal sulfatase inhibitors is an important new therapeutic strategy for postmenopausal breast cancer patients. We synthesized a series of tricyclic coumarin sulfonamide compounds and investigated their inhibitory activity in vitro and in vivo. In the placental microsomal activity assay system, 667 COUMATE exhibited the strongest inhibitory activity, with an IC50 value of 8 nM. A single oral dose (10 mg/kg) of 667 COUMATE inhibited estrone sulfatase activity in rat liver by up to 93%. 667 COUMATE is not estrogenic because it does not stimulate uterine growth in ovariectomized rats. In vivo experiments showed that 667 COUMATE inhibits estrone sulfate-stimulated uterine growth in ovariectomized rats. Using a nitrosomethylurea-induced breast tumor model, we found that 667 COUMATE inhibits estrone sulfate-stimulated tumor growth in a dose-dependent manner. 667 COUMATE has been identified as a potent steroid sulfatase inhibitor, which will help evaluate the therapeutic potential of such therapies. [1]

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The endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2) inhibits the growth of breast cancer cells and is also a potent anti-angiogenic agent. We have previously demonstrated that 3-sulfonated derivatives of 2-methoxyestradiol are more effective than non-sulfonated compounds. In this study, we compared the ability of 2-methoxyestradiol disulfonate (2-MeOE2bisMATE) and 2-MeOE2 to inhibit the growth of MCF-7 breast cancer cells. Both compounds inhibited cell growth, with 2-MeOE2bisMATE showing a six-fold lower IC50 value (0.4 μM) than 2-MeOE2 (2.5 μM). Estrogen sulfamate is a potent steroid sulfatase (STS) inhibitor. Studies have shown that 2-MeOE2bisMATE retains its STS inhibitory activity and, in a placental microsomal assay system, its potency is comparable to that of estrone-3-O-sulfamate (EMATE). Furthermore, an in vivo study compared the potency of 2-MeOE2bisMATE with EMATE and the nonsteroidal STS inhibitor 667 coumarin sulfamate (667 COUMATE). Following a single oral dose of 667 COUMATE (10 mg/kg), partial recovery of STS activity was detected on day 3 (10%) and on day 7 (55%). For the other two steroid compounds, STS activity was almost completely inactivated over a period of up to 5 days, and fully recovered by day 15. The antiproliferative and STS-inhibiting properties of 2-MeOE2bisMATE suggest that it has great potential for development as a novel anticancer 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.08 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 (8.08 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 (8.08 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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 (Please use freshly prepared in vivo formulations for optimal results.)