Introduction

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Compounds that can bind to and activate the estrogen receptors (ERs) but cause differential estrogenic or antiestrogenic responses in specific tissue are currently being investigated as alternatives to estrogens for the prevention and treatment of chronic postmenopausal pathologies.

Estrogen replacement therapy has been used primarily to prevent perimenopausal symptoms in addition to preventing and treating osteoporosis (Kiel et al., 1987). Although many other beneficial activities of estrogens have been described, including improvements in cognitive functions and decreases in the risk of coronary disease through their effect on lipids profile (Cumming, 1991; Stampfer and Colditz, 1991), there are several undesirable side effects associated with chronic estrogen therapy that create difficulties in compliance.

In particular, the return of withdrawal bleeding is one of the major reasons for a woman stopping estrogen therapy. In addition, estrogens when administered without progestin, substantially increase the incidence but not the mortality of endometrial cancer (Ziel and Finkle, 1975; Vesey, 1984); furthermore, concerns about the increased risk of breast cancer associated with estrogen replacement therapy have been raised (Cauley et al., 1999; Jacobs, 2000).

These adverse effects of estrogens have led to an increased interest in drugs thought to maintain estrogen protective effects on bone and serum lipid profile, whereas being characterized by antagonist activity or no activity in reproductive tissues. In addition to an improved side effect profile, compared with estrogens, a tissue-selective estrogen agonist/antagonist may have potential utility in the treatment and prevention of estrogen-dependent cancer, because of its antiestrogen effects on reproductive tissues (Eppenberger et al., 1991).

The well known antiestrogen tamoxifen maintains bone mass and lower blood lipids in rats (Jordan et al., 1987) and postmenopausal women (Love et al., 1994). On the other hand, the estrogenic effects in the endometrium in women can result in an increased incidence of endometrial cancer coincident with prolonged tamoxifen therapy (Kedar et al., 1994), preventing its use to mimic some of the helpful actions of estrogens after the menopause. Subsequent work has seen the first approval of a selective estrogen receptor modulator (SERM) for the prevention and treatment of osteoporosis (raloxifene; Black et al., 1994) and the emergence of several new compounds with this possible spectrum of activities (Sato et al., 1999; Miller et al., 2001). However, the development of many of these compounds as drugs has failed because of their unacceptable uterine changes (Sato et al., 1996; Grese et al., 1997). Moreover, although in humans raloxifene increased bone mineral density, showing a significant reduction in vertebral fractures, it is less effective than other inhibitors of bone resorption at the skeleton (Khovidhunkit and Shoback, 1999). In fact, raloxifene increases bone mineral density at the spine by 1.5 to 2% (Delmas et al., 1997) compared with 4 to 5% for estrogen replacement therapy and 7 to 9% for bisphosphonates (Liberman et al., 1995).

There is increasing evidence that some compounds with a benzopyran structure can initiate estrogen agonist/antagonist actions when applied to biological systems (Sato et al., 1999). We demonstrated that some benzopyran derivatives (i.e., 3-phenyl-4-piperidinyl-ethoxy-benzyl-benzopyrans and, in particular, 3-phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-7-ol, CHF 4056) are SERMs that produce beneficial effects on bone and cholesterol levels similar to those previously reported for raloxifene, whereas they maintain antagonist effects on the uterus (Galbiati et al., 1999, 2002). These compounds diverged dramatically from EE2 and from others SERMs characterized by a benzopyran structure such as levormeloxifene, in its lack of significant estrogenic effects on uterine tissue.

In the present study we characterized the pharmacological properties of a novel benzopyran derivative CHF 4227 (4-methoxy derivative of CHF 4056, Fig. 1), which was found in our search for tissue-selective estrogen characterized by an improved estrogen agonist/antagonist activity compared with raloxifene in bone, in serum lipids, and in uterine tissue. A molecule characterized by this pharmacological profile may have therapeutic advantages over the marketed raloxifene for the treatment of postmenopausal pathologies, such as osteoporosis and estrogen-dependent cancer.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structure for CHF 4227. CHF 4227 is a new benzopyran derivative that is classified as a selective estrogen receptor modulator based on its in vivo pharmacological profile.

We investigated the effect of CHF 4227 for the following activities: 1) binding affinity to human ER- and ; 2) antiestrogenic and estrogenic effect on uterine growth in an immature female rat model (Eppenberger et al., 1991); 3) estrogenic effects on bone, serum cholesterol, and uterus in an OVX rat model of postmenopausal bone loss (Kalu, 1991); and 4) prevention of development of dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma in rats (Huggins at al., 1961).

Herein, the pharmacological properties of CHF 4227 observed in the above-mentioned experimental models are reported. In addition, its in vivo effects were compared with those of EE2 and raloxifene.

	Materials and Methods

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All animals studies were performed in strict accordance with the Decreto Legislativo sulla Sperimentazione Animale (Italian law on rules for animal experimentation, Decree 116, January 27, 1992) and the "European Directive for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" (European Union Directive #86/606/CEE).

Chemicals. 3-(4-Methoxy)phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (mol.wt. = 508.06; CHF 4227) and [6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]-methanone hydrochloride (mol.wt. = 510.06; raloxifene) were synthesized by Chiesi Farmaceutici S.p.A. (Parma, Italy). EE2 (mol.wt. = 296.4) and diethylstilbestrol (mol.wt. = 268.4) were obtained from Sigma-Aldrich (St. Louis, MO). Kits for radioimmunoassay of osteocalcin were supplied by Biomedical Technologies (Stoughton, MA). All other reagents were purchased from Sigma-Aldrich.

Human ER- and ER- Binding. ER- and - binding analysis was performed as described previously (Obourn et al., 1993). Briefly, the standard assay was performed in a volume of 100 µl, containing a final concentration of 0.5 nM [³H]estradiol (PerkinElmer Life Sciences, Boston, MA), increasing concentration of unlabeled CHF 4227 (0.01-100 nM), 5 µl of diluted (1:100 in binding buffer) human recombinant ER- or - (insect Sf9 cells), and 95 µl of binding buffer (10 mM Tris pH 7.5, 10% glycerol, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin). The incubation was carried out at room temperature for 3 h. After incubation, 100 µl of 50% hydroxylapatite slurry (equilibrated in 50 mM Tris pH 7.4 and 1 mM EDTA) was added to each tube and vortexed three times over 15 min. One milliliter of wash buffer (40 mM Tris pH 7.4, 1 mM EGTA, 1 mM EDTA, and 100 mM KCl) was added to each reaction and was centrifuged at 10,000g for 5 min, and the supernatant was aspirated. The wash step was repeated two more times and then the hydroxyapatite pellet was resuspended in 400 µl of ethanol, transferred to a scintillation vial, and counted. Nonspecific binding was defined as that which occurred in the presence of 1 µM diethylstilbestrol and represented 10 to 15% of the total binding. K_i values were calculated using the equation of Cheng and Prusoff (1973) using the observed half-maximal inhibition concentration (IC₅₀) of the tested compound, the concentration of radioligand used in the assay, and the dissociation constant value of the ligand. The data were also fitted by an iterative program (RECEPT) for nonlinear regression analysis (Benfenati and Guardabasso, 1984) both to a one-site and to a two-site model. The one-site model was then chosen when it yielded the best correlation coefficient and when the improvement of goodness-of-fit for the two-site model was not statistically significant (P < 0.05) according to the F test on the sums of squared errors.

Immature Female Rat Study. Female Sprague-Dawley rats (21 days old), weighing approximately 40 to 50 g (Charles River Italica, Calco, Italy) were treated by oral gavage with either vehicle (0.5% methylcellulose, 3 ml/kg), CHF 4227 (0.001-10 mg/kg · day), raloxifene (0.01-10 mg/kg · day), or EE2 at 0.05 mg/kg · day for 3 days. The compounds under investigation were also administered 15 min before the EE2 gavage, used as estrogenic stimulus to increase uterine weight. Nonestrogenic controls were given vehicle alone.

Four-Week OVX Rat Study. Virgin Sprague-Dawley rats (9-10 month old), weighing approximately 280 to 300 g (Harlan Nossan, Correzzana, Italy) were used in this study. The animals were acclimatized to the local vivarium conditions (22 ± 2°C; 12-h light/dark cycle) for 2 weeks and housed individually during the experimental period.

Bone Densitometry. Bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DEXA) using a Hologic QDR-1000 plus instrument equipped with dedicated software for small animal measurements. An ultra high-resolution mode (line spacing 0.0254 cm and resolution 0.0127 cm) was used with a collimator of 0.63-mm diameter. This technique provides an integrated measure of both cortical and trabecular bone.

Uterine Histology. Formalin-fixed uteri were processed for conventional paraffin embedding. Sections of about 5 µm in thickness were obtained from each block. Slides were stained with hematoxylin and eosin before undergoing image analysis for the measurement of endometrium epithelia and myometrial thickness. The measurements were performed using an Ibas20 computerized imaging system run on a 386 personal computer (Kontron Instruments, Watford, Herts, UK). The images were acquired with a black-and-white camera (JVC, Yokohama, Japan) fitted with a 50-mm macro lens (for myometrial thickness) or an Axioscope microscope (for endometrium epithelia). A black-and-white camera was used because it is more sensitive than a color one.

Uterine Eosinophil Peroxidase Activity. The test protocol was based on the method described by White at al. (1991). The assay is based on the oxidation of o-phenylenediamine by uterine eosinophil peroxidase in the presence of H₂O₂. In brief, after the removal of uterus and recording of whole uterine weight, the uterine horns were bisected. One horn from each animal was weighed and homogenized (Polytron Kinematica, Luzern, Switzerland) on ice in 50 mM Tris buffer, pH 8.0 (200 µl/mg of tissue), containing 0.05% (v/v) Triton X-100. Samples were centrifuged at 3000 rpm for 10 min at 4°C in a centrifuge (J2-MI; Beckman Coulter, Inc., Fullerton, CA). The resulting supernatant was filtered through a 45-µm filter. Duplicate 200-µl aliquots of the filtered supernatant (equivalent to 1 mg of tissue) were added to a spectrophotometric cuvette. The reaction was initiated with the addition of 800 µl of substrate solution containing 3.5 mM o-phenylenediamine 2HCl and 0.0005% H₂O₂ in 50 mM Tris buffer, pH 8. The apparent maximal velocity (mOD/min) was determined by continuous recording of the absorbance at 490 nM at room temperature.

Prevention of Development of DMBA-Induced Mammary Tumors. Mammary carcinomas were induced in rats by a single intragastric administration of 20 mg of DMBA (Sigma-Aldrich) in 1 ml of corn oil at 50 days of age. Forty days later, tumor measurement (the two largest perpendicular diameters of each tumor) was performed with calipers biweekly. The animals were treated for 6 months with 0.5% methylcellulose (n = 40) or CHF 4227 2 mg/kg · day (n = 19), commencing 3 days before the oral administration of DMBA. Some of the control animals died or were killed by cervical dislocation under anesthesia before the end of the experiment because their tumors grew too large. The tumors size and number in these rats at death together with those measured at later time from the surviving animals were used for the analysis of the incidence of tumors, average tumor number per rat, and average tumor size per tumor-bearing animal. All rats were killed 6 months after DMBA administration.

Statistical Analysis. Results are expressed as mean ± S.E.M. Significance was determined by analysis of variance and when analysis of variance was significant, by the Newman-Keuls test for post hoc multiple comparisons. Probability values of <0.05 were considered to be statistically significant. Analysis of the incidence of development of mammary tumors was performed using Fisher's exact test. Analysis of the tumor number per rat was carried out using the Mann-Whitney U test.

	Results

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Human ER- and ER- Binding Effects. CHF 4227 binds with high affinity to purified recombinant human ER- (K_i = 0.017 ± 0.002 nM) and ER- (K_i = 0.099 ± 0.005 nM). Compared with the well known SERM raloxifene (K_i for ER- = 1.62 ± 0.348 and for ER- = 0.071 ± 0.008 nM), the affinity for ER- and ER- was 4- and 16-fold higher, respectively. This competitive binding assay showed that CHF 4227 competes for a single binding site on both ER- and ER-.

Immature Female Rat Assay. In immature female rats, treatment with EE2 at 0.05 mg/kg p.o. for 3 days significantly increased uterine wet weight (~170%) compared with vehicle-treated controls. This concentration of EE2 was the lowest producing near-maximal effect and was chosen on the basis of preliminary dose-response experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   A, effect of CHF 4227 () and raloxifene () on EE2-induced uterine weight increase in immature rats. B, effect of CHF 4227 (), raloxifene (), and EE2 () on uterine weight in immature rats. The estrogenic and antiestrogenic activity was expressed as uterine weight/body weight ratios. Data are expressed as mean ± S.E.M (n = 8). Statistical significance relative either to EE2-treated control (A) or vehicle-treated control (B) is denoted by *, P < 0.05; **, P < 0.01.

OVX Rat Assay. CHF 4227 effects on a number of estrogen target tissues (bone, uterus, and serum cholesterol) were evaluated in 9- to 10-month-old OVX rats that were dosed for 4 weeks postsurgery and compared with OVX and sham controls.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of CHF 4227 (), raloxifene (), and EE2 () on bone mineral density of lumbar vertebrae L1 to L4 in OVX rats treated for 4 weeks. Bone mineral density values are reported as percentage of change in BMD from the baseline. Each point is the mean ± S.E.M. of two to four experiments. Statistical significance relative to vehicle-treated OVX rats is denoted by *, P < 0.01. For each experiment, n = 7 to 8.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of CHF 4227 (), raloxifene (), and EE2 () on serum cholesterol levels in OVX rats treated for 4 weeks. Each point is the mean ± S.E.M. of two to four experiments. Statistical significance relative to vehicle-treated OVX rats is denoted by *, P < 0.01. For each experiment, n = 7 to 8.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of CHF 4227 (), raloxifene (), and EE2 () on uterine weight (uterine weight/body weight ratio) (A) and uterine peroxidase activity (B) in OVX rats treated for 4 weeks. Uterine peroxidase activity is reported as the mean Vmax. Each point is the mean ± S.E.M. of two to four experiments. Statistical significance relative to vehicle-treated OVX rats is denoted by *, P < 0.05; **, P < 0.01. For each experiment, n = 7 to 8.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of CHF 4227 (), raloxifene (), and EE2 () on endometrium epithelia thickness in OVX rats treated for 4 weeks. Data are expressed as pixel ± S.E.M (n = 7 to 8). Statistical significance relative to vehicle-treated OVX rats is denoted by *, P < 0.01.

Prevention of Development of DMBA-Induced Mammary Tumors. Mammary carcinoma induced by DMBA in rats is a widely used model to study the factors that control hormone-sensitive breast cancer in women. In fact, the development and growth of these tumors are particularly sensitive to the stimulatory action of estrogens and prolactin (Asselin et al., 1977).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of daily oral administration of 2 mg/kg CHF 4227 () or vehicle () on the number of animals who developed palpable mammary carcinoma induced by DMBA throughout the 180-day observation period. Data are expressed as percentage of the total number of animals in each group. Each point is the mean ± S.E.M. of one experiment. The inset represents the effect on average tumor number per animal (mean ± S.E.M). Statistical significance relative to vehicle-treated rats is denoted by *, P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Long-term use of estrogens is effective in reducing the risks associated with the decreased production of ovarian steroid after the climacteric (Kiel et al., 1987). Unfortunately, concerns regarding the increased incidence of breast and endometrial cancer and some other undesirable side effects, including breakthrough bleeding, are considerable drawbacks to the initiation and long-term use of estrogens (Vesey, 1984; Jacobs, 2000). Accordingly, a therapeutic agent that can mimic the protective effects of estrogen on nonreproductive tissues without inducing significant proliferative effect on the uterus and breast, would be highly desirable for postmenopausal women.

CHF 4227 is a novel orally active nonsteroidal estrogen agonist/antagonist. In vitro binding studies have demonstrated that CHF 4227 displays high affinity to the human ER- and ER- (K_i values of 0.017 and 0.099 nM, respectively). Differential tissue selectivities of CHF 4227 have been studied in OVX rats treated for 4 weeks with endpoints of body weight, serum cholesterol lowering, bone tissue, and uterine stimulation.

Bilateral ovariectomy in the rat decreases circulating serum estrogen levels, which results in increased bone turnover. Bone loss in the OVX rat reflects the skeletal changes observed in postmenopausal women: rapid decrease in bone mass, preferential loss of trabecular bone, and responsiveness to estrogen replacement therapy (Kalu, 1991). In our experimental conditions, ovariectomy resulted in significant osteopenic responses after 4 weeks in the lumbar spine L1 to L4 as measured by DEXA densitometric techniques. CHF 4227 prevented the ovariectomy-induced reduction in BMD of L1 to L4 lumbar vertebrae, with an ED₅₀ of about 0.003 mg/kg · day. Total protection against OVX-induced bone loss was observed between 0.1 and 1 mg/kg · day; moreover, at the same doses CHF 4227 fully prevented also the rise in serum osteocalcin levels induced by castration. In line with previously reported data (Black et al., 1994), raloxifene only partially prevented the OVX effects on bone tissue (maximal effect = 60% protection) seeming less effective and about 100 times less potent than CHF 4227. Thus, our data suggest that CHF 4227 might exert in clinical studies an improved benefit in the bone tissue compared with raloxifene, which does not seem to be as efficacious as estrogens and bisphosphonates (Liberman et al., 1995). Although the results of these studies show that CHF 4227 will provide protection against OVX-induced bone loss after 4 weeks, a longer term study is in progress to show that these effects will be maintained and that CHF 4227 are not simply delaying the eventual loss of BMD due to estrogen deficiency.

EE2 produced a marked hypocholesterolemic effect in OVX rats; this effect is attributed to up-regulation of hepatic low-density lipoprotein receptors, resulting in enhanced clearance of circulating low-density lipoprotein (Brown and Goldstein, 1980). Under the same experimental conditions, CHF 4227 significantly decreased total serum cholesterol in a dose-dependent manner with an ED₅₀ of about 0.007 mg/kg · day and maximal cholesterol lowering effect observed at 0.1 mg/kg · day. As in the bone tissue, again CHF 4227 is significantly more potent (about 50 times) than raloxifene in lowering plasma cholesterol levels.

Although the mechanisms for the increased potency of CHF 4227 in vivo, relative to raloxifene, are not fully clear at the moment, the higher binding affinity to the estrogen receptors (4 times for ER- and 16 times for ER-) and the higher oral bioavailability of CHF 4227 may be relevant factors. In particular, the oral bioavailability of CHF 4227 in rats is 64% (data not shown), which is a significant improvement over raloxifene (5%; Rosati et al., 1998). The insertion of the 4'-methoxy group seems to be responsible for the higher oral bioavailability of CHF 4227; in line with this hypothesis, the demethoxylated derivative CHF 4056 (Galbiati et al., 2002) is characterized by an oral bioavailability similar to the one of raloxifene. In parallel, also the in vivo potency and efficacy of CHF 4056 and raloxifene are superimposable (Galbiati et al., 2002), underlying the fact that the bioavailability may be an important feature to justify the improved SERM profile of CHF 4227.

Presently, we are investigating the in vivo metabolism of CHF 4227, relative to CHF 4056, to clarify possible biotransformation mechanisms that could account for the higher bioavailability of CHF 4227. In this respect, preliminary data suggest that a minimization of glucuronidation process in position 7 (on phenolic group of the benzopyran moiety) of CHF 4227 might be an important point.

Estrogens play a predominant role in breast cancer development and growth (McGuire et al., 1975). Because the first step in the action of estrogens in target tissue is binding to the estrogen receptor, a logical approach for the prevention and treatment of estrogen-sensitive breast cancer is the use of compounds that block the interaction of estrogens with their specific receptor. Consequently, the use of the antiestrogen tamoxifen is the standard therapy for breast cancer, at all stages of the disease; in addition, raloxifene therapy is associated with a potential decreased risk of developing breast cancer (Clemett and Spencer, 2000). CHF 4227, being characterized by marked estrogen antagonist activity on reproductive tissue, might be of interest in the prevention and treatment of estrogen dependent tumors. To test this hypothesis, we studied the effect of CHF 4227 on the development of DMBA-induced mammary carcinoma in rats, a model widely used to study the factors that control hormone-sensitive breast cancer in women (Asselin et al., 1977). In this assay, CHF 4227 significantly prevents the development of DMBA-induced mammary tumors, the incidence being reduced from 87.5 to 26.3% 6 months after DMBA administration. Moreover, our data clearly show that CHF 4227 not only significantly reduces the percentage of rats bearing DMBA-induced tumors but also decreased tumor number per animal that have developed tumors during treatment with CHF 4227. Thus, these results show that CHF 4227 may be of interest in the prevention of estrogen-dependent tumors. A treatment study will be performed to test also the efficacy of CHF 4227 on the growth of established mammary tumors. Whether being used as a selective estrogen for prevention and treatment of osteoporosis or as an estrogen antagonist for breast cancer, it is critical that CHF 4227 does not induce a significant estrogen agonist effects on uterine tissue.

In line with the results observed in the immature rats assay, also in aged OVX rats CHF 4227 has minimal stimulatory effects on the uterus. In fact, after 4 weeks of treatment, histological analysis of the uterine tissue showed that CHF 4227, like raloxifene, has nonsignificant effects on endometrium epithelia thickness. In contrast, EE2 increased epithelial thickness, demonstrating significant uterine hypertrophic effects. This trend was reproduced in analysis of uterine eosinophil peroxidase activity to show that CHF 4227 is much less stimulatory in the uterus than EE2. CHF 4227 differed substantially also from others benzopyran estrogens agonist/antagonist such as levormeloxifene, which, as detailed previously (Galbiati et al., 2002), significantly increased uterine epithelial thickness and uterine eosinophil peroxidase in OVX rats. It is worth noting that the clinical development of levormeloxifene was discontinued after reports of endometrial thickening side effects in postmenopausal women (Mitlak and Cohen, 1999).

After 4 weeks of treatment, CHF 4227 caused a statistically significant increase in uterine weight relative to the OVX controls, although it was much less pronounced than that observed in EE2-treated animals. However, this marginal effect on uterine weight was coupled with the lack of a stimulatory activity on endometrium epithelia and uterine peroxidase, indicating that it may not be clinically relevant. In favor of this consideration, CHF 4227's profile on uterine tissue in OVX rats is superimposable to the one previously observed for raloxifene (Black et al., 1994), which, in clinical studies with postmenopausal women, did not show stimulatory effects on the uterus (Delmas et al., 1997). Thus, CHF 4227 is expected to have a tissue selectivity profile in postmenopausal women similar to the one of raloxifene and strictly different compared with those of others SERMs such as tamoxifen and levormeloxifene. The full estrogen antagonist effect of CHF 4227 in the uterine tissue of immature rats indicates that its tissue selectivity is not simply due to a selective tissue distribution.

In conclusion, the new benzopyran derivative CHF 4227 is a selective estrogen receptor modulator that compares favorably in efficacy and potency with raloxifene in preventing bone loss, lowering serum cholesterol levels, and antagonizing EE2 stimulation of the uterus. Moreover, the results observed in the DMBA-induced mammary tumors model indicate that CHF 4227 has significant chemopreventive effects. This profile along with the minimal uterine stimulation suggests a therapeutic advantage to CHF 4227 over estrogens, raloxifene, or tamoxifen for the prevention and/or treatment of osteoporosis and estrogen-dependent tumors. In particular, the lack of a significant agonist activity on uterine tissue may avoid some of the potential estrogen-related clinical side effects that may arise with the use of long-term estrogens and tamoxifen therapy, whereas the higher efficacy and potency of CHF 4227 on bone tissue indicate that this compound may be superior to raloxifene as antiosteoporotic drug. Clinical studies with CHF 4227 will be performed to confirm its preclinical profile in postmenopausal women.