Diana Ayan

Impact of estradiol structural modifications (18-methyl and/or 17-hydroxy inversion of configuration) on the in vitro and in vivo estrogenic activity

It is well recognized that the majority of breast cancers are initially hormone-dependent and that 17β-estradiol (17β-E2) plays a crucial role in their development and progression. For this reason, using a compound able to block a specific enzyme involved in the last steps of the biosynthesis of 17β-E2 remains a rational way to treat estrogen-dependent diseases such as breast cancer. The present study describes the biological in vitro and in vivo evaluation of a structural modification (inversion of C18-methyl group at position 13 from β to α face) of 17β-E2 (1) and 17α-estradiol (17α-E2; 2). The two epimers 18-epi-17β-E2 (3) and 18-epi-17α-E2 (4) were obtained in two chemical steps by inversion of the C18-methyl of estrone using 1,2-phenylendiamine in refluxing acetic acid and reduction of ketone at position C17 with LiAlH(4). The new E2 isomers were tested on estrogen-sensitive cell lines (MCF-7 and T-47D), on estrogen-sensitive tissues (uterus and vagina of mice) and on estrogen receptor (ER) to determine their estrogenic potency relatively to natural estrogen 17β-E2 (1). The results show that 18-epi-17β-E2 (3) possesses the lower affinity for ER (RBA = 1.2%), the lower estrogenicity on estrogen-sensitive cells (1000 folds less estrogenic than 17β-E2 in MCF-7) and no uterotrophic (estrogenic) activity when tested on mice. In fact, we observed the following order of estrogenicity: 18-epi-17β-E2 (3)<18-epi-17α-E2 (4) << 17α-E2 (2)17β-E2 (1). These results suggest that the inversion of C18-methyl of natural 17β-E2 scaffold could be a useful strategy to decrease the estrogenicity of E2 derivatives used as enzyme inhibitors in the context of a treatment of estrogen-dependent diseases.

A New Nonestrogenic Steroidal Inhibitor of 17β-Hydroxysteroid Dehydrogenase Type I Blocks the Estrogen-Dependent Breast Cancer Tumor Growth Induced by Estrone

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) converts estrone (E1) into estradiol (E2) and is expressed in many steroidogenic tissues and breast cancer cell lines. Because the potent estrogen E2 stimulates the growth and development of hormone-dependent diseases, inhibition of the final step of E2 synthesis is considered a promising strategy for the treatment of breast cancer. On the basis of our previous study identifying 16β-(m-carbamoylbenzyl)-E2 (CC-156) as a lead compound for the inhibition of 17β-HSD1, we conducted a number of structural modifications to reduce its undesired residual estrogenic activity. The steroid derivative PBRM [3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene] emerged as a potent inhibitor of 17β-HSD1 with an IC50 value of 68 nmol/L for the transformation of E1 into E2. When tested in the estrogen-sensitive breast cancer cell line T-47D and in mice, PBRM showed no estrogenic activity in the range of concentrations tested. Furthermore, with the purpose of evaluating the bioavailability of PBRM and CC-156 injected subcutaneously (2.3 mg/kg), we measured their plasmatic concentrations as a function of time, calculated the area under the curve (AUC0–12h) and showed a significant improvement for PBRM (772 ng*h/mL) compared with CC-156 (445 ng*h/mL). We next tested the in vivo efficiency of PBRM on the T-47D xenograft tumor model in female ovariectomized athymic nude mice. After a treatment with PBRM, tumor sizes in mice stimulated with exogenous E1 were completely reduced at the control group level (without E1 treatment). As a conclusion, PBRM is a promising nonestrogenic inhibitor of 17β-HSD1 for the treatment of estrogen-dependent diseases such as breast cancer. Mol Cancer Ther; 11(10); 2096–104. ©2012 AACR.

Discovery of a non-estrogenic irreversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 from 3-substituted-16β-(m-carbamoylbenzyl)-estradiol derivatives.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is thought to play a pivotal role in the progression of estrogen-sensitive breast cancer by transforming estrone (E1) into estradiol (E2). We designed three successive series of E2-derivatives at position C3 of the potent inhibitor 16β-(m-carbamoylbenzyl)-E2 to remove its unwanted estrogenic activity. We report the chemical synthesis and characterization of 20 new E2-derivatives, their evaluation as 17β-HSD1 inhibitors, and their proliferative (estrogenic) activity on estrogen-sensitive cells. The structure-activity relationship study provided a new potent and steroidal nonestrogenic inhibitor of 17β-HSD1 named 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestra-1(10),2,4-trien-16-yl]methyl}benzamide (23b). In fact, this compound inhibited the transformation of E1 into E2 by 17β-HSD1 in T-47D cells (IC50 = 83 nM), did not inhibit 17β-HSD2, 17β-HSD7, 17β-HSD12, and CYP3A4, and did not stimulate the proliferation of estrogen-sensitive MCF-7 cells. We also discussed the results of kinetic and molecular modeling (docking) experiments, suggesting that compound 23b is a competitive and irreversible inhibitor of 17β-HSD1.

Crucial Role of 3-Bromoethyl in Removing the Estrogenic Activity of 17β-HSD1 Inhibitor 16β-(m-Carbamoylbenzyl)estradiol

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) represents a promising therapeutic target for breast cancer treatment. To reduce the undesirable estrogenic activity of potent 17β-HSD1 inhibitor 16β-(m-carbamoylbenzyl)estradiol (1) (IC50 = 27 nM), a series of analogues with a small functionalized side chain at position 3 were synthesized and tested. The 3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-estra-1,3,5(10)-trien-17β-ol (5) was found to be a potent inhibitor (IC50 = 68 nM) for the transformation of estrone (E1) into estradiol (E2) and, most importantly, did not stimulate the proliferation of estrogen-sensitive MCF-7 cells, suggesting no estrogenic activity. From these results, the crucial role of a bromoalkyl side chain at carbon 3 was identified for the first time. Thus, this new inhibitor represents a good candidate with an interesting profile suitable for further studies including pharmacokinetic and in vivo studies.

Chemical synthesis, cytotoxicity, selectivity and bioavailability of 5α-androstane-3α,17β-diol derivatives

Aminosteroid derivatives represent a new family of compounds with promising antiproliferative activity over different cancer cell lines. Among all the aminosteroid derivatives synthesised in our laboratory, we have identified E-37P as one of the more potent when tested in vitro. Unfortunately, the pharmacokinetic properties of E-37P decrease its effectiveness when tested in vivo. To improve the bioavailability and increase the efficiency of aminosteroid E-37P, two series of analog compounds were synthesised by classic chemical synthesis, they were then characterized, and the concentration that inhibits 50% of cell proliferation (IC50) was determined on different cell lines. RM-133, a 5α-androstane-3α,17β-diol derivative with a quinoline nucleus at the end of the piperazine-proline side-chain at position 2β and an ethinyl at position 17α, showed very good antiproliferative activity among the five cancer cell lines studied (IC50=0.1, 0.1, 0.1, 2.0 and 1.1 μM for HL-60, MCF-7, T-47D, LNCaP and WEHI-3, respectively). Moreover, the plasmatic concentration of RM-133 at 3h, when injected subcutaneously in rats, was 2.3-fold higher than that of E-37P (151 vs 64.8 ng/mL). Furthermore, RM-133 weakly inhibited the two representative liver enzymes, CYP3A4 and CYP2D6, indicating a very low risk of drug-drug interactions. The cytotoxicity of RM-133 against normal cells was tested on peripheral blood lymphocytes (PBL) obtained from different donors and previously activated with phytohemagglutinin-L. PBL responded differently to treatment with RM-133, we observed a stimulation of cell proliferation and/or cytotoxicity in a dose-dependent manner. Based on these results, additional studies are currently underway to evaluate the selectivity of our lead compound against normal cell lines in a more detailed fashion.

Identification of a 17β‐Hydroxysteroid Dehydrogenase Type 10 Steroidal Inhibitor: A Tool to Investigate the Role of Type 10 in Alzheimer’s Disease and Prostate Cancer

Human 17b-hydroxysteroid dehydrogenase type 10 (17bHSD10) is known for its multiple functions and could be involved in the development and progression of many diseases. 17b-HSD10 is a homotetrameric mitochondrial protein expressed in the liver and several other tissues, including brain and gonad. It plays a role in the metabolism of steroid hormones through its 17b-HSD and 3a-HSD activities (Scheme 1),

The Aminosteroid Derivative RM-133 Shows In Vitro and In Vivo Antitumor Activity in Human Ovarian and Pancreatic Cancers

Ovarian and pancreatic cancers are two of the most aggressive and lethal cancers, whose management faces only limited therapeutic options. Typically, these tumors spread insidiously accompanied first with atypical symptoms, and usually shift to a drug resistance phenotype with the current pharmaceutical armamentarium. Thus, the development of new drugs acting via a different mechanism of action represents a clear priority. Herein, we are reporting for the first time that the aminosteroid derivative RM-133, developed in our laboratory, displays promising activity on two models of aggressive cancers, namely ovarian (OVCAR-3) and pancreatic (PANC-1) cancers. The IC50 value of RM-133 was 0.8 μM and 0.3 μM for OVCAR-3 and PANC-1 cell lines in culture, respectively. Based on pharmacokinetic studies on RM-133 using 11 different vehicles, we selected two main vehicles: aqueous 0.4% methylcellulose:ethanol (92:8) and sunflower oil:ethanol (92:8) for in vivo studies. Using subcutaneous injection of RM-133 with the methylcellulose-based vehicle, growth of PANC-1 tumors xenografted to nude mice was inhibited by 63%. Quite interestingly, RM-133 injected subcutaneously with the methylcellulose-based or sunflower-based vehicles reduced OVCAR-3 xenograft growth by 122% and 100%, respectively. After the end of RM-133 treatment using the methylcellulose-based vehicle, OVCAR-3 tumor growth inhibition was maintained for ≥ 1 week. RM-133 was also well tolerated in the whole animal, no apparent sign of toxicity having been detected in the xenograft studies.

Synthesis and preliminary evaluation of a modified estradiol-core bearing a fused γ-lactone as non-estrogenic inhibitor of 17β-hydroxysteroid dehydrogenase type 1

A non-estrogenic inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) was designed based on a modified 3-hydroxy-estra-1,3,5(10)-triene core having an additional five-member lactone ring and a benzamide group. The inhibitor was synthesized, fully characterized and tested for its ability to inhibit the enzyme activity. Estrogenicity was also investigated and tested on estrogen-dependent T-47D cell line. Interestingly, this steroid derivative showed inhibitory potency towards 17β-HSD1 and did not present residual unwanted estrogenic activity.

Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1

ABSTRACT 17&bgr;‐Hydroxysteroid dehydrogenase type 1 (17&bgr;‐HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen‐dependent diseases. A steroidal derivative, 3‐{[(16&bgr;,17&bgr;)‐3‐(2‐bromoethyl)‐17‐hydroxyestra‐1,3,5(10)‐trien‐16‐yl]methyl} benzamide (PBRM), has recently been described as a non‐estrogenic, irreversible inhibitor of 17&bgr;‐HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off‐targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki = 368 nM) and the enzyme inactivation rate (kinact = 0.087 min−1) values for PBRM were determined with purified 17&bgr;‐HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3–5 days) is required to recover 17&bgr;‐HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17&bgr;‐HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non‐estrogenic and steroidal covalent irreversible inhibitor of 17&bgr;‐HSD1 warrant its further development as a potential drug candidate for estrogen‐dependent disorders.

Pharmacokinetic profile of PBRM in rodents, a first selective covalent inhibitor of 17β-HSD1 for breast cancer and endometriosis treatments

The development of a covalent inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a promising approach for the treatment of hormone-dependent breast cancer and endometriosis. After reporting the steroid derivative PBRM as a first potent covalent inhibitor of 17β-HSD1 without estrogenic activity, we are now interested in studying its pharmaceutical behavior. The metabolism study in a human liver microsomal preparation showed a gradual transformation of PBRM into PBRM-O, an oxidized ketonic form of PBRM at position C17. Interestingly, PBRM-O also inhibits 17β-HSD1 and is not estrogenic in estrogen-sensitive T-47D cells. However, when PBRM was injected subcutaneously (sc) in mice, a very small proportion of PBRM-O was measured in a 24 h-time course experiment. A pharmacokinetic study in mice revealed suitable values for half-life (T1/2 = 3.4 h), clearance (CL = 2088 mL/h kg), distribution volume (Vz = 10.3 L/kg) and absolute bioavailability (F = 65%) when PBRM was injected sc at 14.7 mg/kg. A good F value of 33% was also obtained when PBRM was given orally. A tritiated version of PBRM, 3H-PBRM, was synthesized and used for an in vivo biodistribution study that showed its gradual accumulation in various mouse tissues (peak at 6 h) followed by elimination until complete disappearance after 72 h. Elimination was found to occur in feces (93%) and urine (7%) as revealed by a mass balance experiment. PBRM was also evaluated for its toxicity in mice and it was found to be very well tolerated after weekly sc administration (30-405 mg/kg for 8 weeks) or by po administration (300-900 mg/kg for 4 weeks). Overall, these experiments represent important steps in the preclinical characterization of the pharmaceutical behavior of PBRM, as well as for its translation to clinical trials.