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].

---

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].

---

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

---

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.

---

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.

---

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 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

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.

---

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.

---

View More

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.

---

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