GB2374412A

GB2374412A - Hypertension treatment and assay

Info

Publication number: GB2374412A
Application number: GB0109091A
Authority: GB
Inventors: Jan-Ike Gustafsson; Zhao Bian
Original assignee: Karo Bio AB
Current assignee: Karo Pharma AB
Priority date: 2001-04-11
Filing date: 2001-04-11
Publication date: 2002-10-16
Also published as: GB0109091D0

Abstract

Methods of assaying compounds for blood-pressure modulating activity comprising the step of determining the ability of the compound to affect estrogen receptor b (ER b ) activity. It also relates to the use of ER b modulating compounds for modulating blood-pressure and in particular for treating hypertension.

Description

Hypertension Treatment and Assay The invention relates to the treatment of hypertension with estrogen receptor P (ERss) ligands and to assays for drugs for treating hypertension. These are based on the identification by the inventors that estrogen receptor ss is involved in modulating hypertension.

Human essential hypertension is a complex disorder with multiple genetic and environmental determinants (1). In pre-menopausal women, hypertension is less common and severe than in men of similar age (2). Differences between women and men with regard to cardiovascular diseases, including hypertension, tend to diminish after women reach menopause, suggesting that endogenous estrogen and/or progesterone may play a role in the development of hypertension (3). Hormone replacement therapy (HRT) has been shown to decrease blood pressure (BP) in healthy postmenopausal women as well as in postmenopausal women with mild to moderate hypertension (4,5), possibly through blocking of calcium channels (6), increasing circulating bradykinin (7), and inducing eNOS activity and secretion of nitric oxide (NO) (8,9). In addition, estrogen has recently been shown to play an important role in controlling BP by regulating the vasoconstrictor-vasodilator balance of renin-angiotensin system (RAS) (10) as well as ATI receptor expression (11).

Estrogen influences cell growth, differentiation and functioning in many target tissues, following activation of its receptors. To date, two estrogen receptors, namely ERa and ERss, have been identified (12,13). Human ERP shows approximately 88% identity to mouse Ergs, and 47% identity to human ERa (14). Principally, the estrogen receptors are ligand-activated transcription factors, switching expression of target genes on or off (15). However, recent knowledge suggests that the biological action of estrogen/ERs is far more complicated than we previously thought (16,17). It has been shown that ERss is expressed in rat heart (18) and in human vascular smooth muscle (19). The inventors believe that ER, plays an important role in the cardiovascular system, mediating the protective effects of estrogen against cardiovascular disease, including hypertension. Importantly, it has been recently

demonstrated that the vasoprotective effect of estrogen on vascular injury in rat is ERs-dependent (20). The inventors have performed a study designed to evaluate the effects of dysfunction of ERss on cardiovascular parameters in mice with targeted disruption of ERss gene (21).

Ligands which bind ERss are known. WO 98/56812, for example, discloses methods for designing ligands which bind to ERss and ligands identified by such methods. Such ligands include raloxifene and its analogues as shown in WO 98/56812. Such ERss ligands are

expected to modulate ERss activity and to modulate hypertensive activity which is under ERss control.

The inventors have realised that ERss is important in the modulation of blood-pressure. Accordingly, they have identified that compounds having blood-pressure modulating activity may be identified by screening the compound for its effect on estrogen receptor ss activity.

A first aspect of the invention provides a method of assaying compounds for blood-pressure modulating activity'comprising the step of determining the ability of the compound to affect estrogen receptor ss activity.

By"activity"we mean the physiological activity of the estrogen receptor ss. A review of the structure and functioning of the estrogen receptor is provided in an article by Katzenellenbogen J., et al., Steroids (1997), 62 (3), 268-303.

ER-ss activity may be assessed in a number of ways: ER-ss affinity may be assessed by a number of assays known in the art: (i) Radioligand displacement (filter) assay, as shown in, for example, WO 98/56812; (ii) Scintistrip ER binding assay. This uses ERss protein bound to plastic, the binding of a radioligand causes the plastic to fluoresce. This is disclosed in WO 98/56812, Haggblad J. et aL (Biotechniques, Vol. 18 (1995), pages 1460151), Barkem T. et al. (J. Steroid.

Biochem. Molec. Biol., Vol. 38, pages 667-75) and Salononsson M. et al. (J. Steroid Biochem. Molec. Biol, Vol 50 (1994), pages 313-318).

(iii) By measuring displacement of a fluorescent ligand, see for example: Hwang K. J., Carlson K. E. , Anstead G. M., Katzenellenbogen J. A.: Donor-acceptor tetrahydrochrysenes, inherently fluorescent, high-affinity ligands for the estrogen receptor: binding and fluorescence characteristics and fluorometric assay of receptor. Biochemistry. 1992 Nov 24; 31 (46): 11536-45.

(iv) Using a fluorescence polarization assay, see for example: Bolger R. , Wiese T. E., Ervin K. , Nestich S. , Checovich W.: Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environ. Health Perspect. 1998 Sep; 106 (9): 551-7.

(v) Using binding assays with or without co-factors or serum. See for example: Gee A. C. , Carlson K. E. , Marini P. G., Katzenellenbogen B. S. , Katzenellenbogen J. A.: Coactivator peptides have a differential stabilizing effect on the binding of estrogens and antiestrogens with the estrogen receptor. Mol Endocrinol. 1999 Nov; 13 (11) : 1912-23.

(vi) Digestion assays, measure the ability of a ligand to change the conformation of ER so that its various sites are more prone or less prone to enzymatic cleavage. See, for example, digestion assay: Kraichely D. M. , Sun J. , Katzenellenbogen J. A. , Katzenellenbogen B. S.: Conformational changes and coactivator recruitment by novel ligands for estrogen receptor-alpha and estrogen receptor-beta: correlations with biological character and distinct differences among SRC coactivator family members. Endocrinology. 2000 Oct; 141 (10): 3534-45.

The confirmation of ERss with binding compounds can be measured by, e. g.

BioKey/Molecular Braille type assays, as described in: Wijayaratne A. L. , Nagel S. C. , Paige L. A. , Christensen D. J., Norris J. D. , Fowlkes D. M., McDonnell D. P.: Comparative analyses of mechanistic differences among antiestrogens.

Endocrinology. 1999 Dec; 140 (12): 5828-40.

Chang C. Y., Norris J. D., Gron H., Paige L. A., Hamilton P. T., Kenan D. J., Fowlkes D., McDonnell D. P. : Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta. Mol. Cell. Biol. 1999 Dec; 19 (12): 8226-39.

Norris J. D. , Paige L. A. , Christensen D. J., Chang C. Y. , Huacani M. R. , Fan D. , Hamilton P. T. , Fowlkes D. M., McDonnell D. P.: Peptide antagonists of the human estrogen receptor.

Science. 1999 Jul 30; 285 (5428): 744-6.

Paige L. A.. Christensen D. J., Gron H. , Norris J. D. , Gottlin E. B. , Padilla K. M. , Chang C. Y., Ballas L. M. , Hamilton P. T., McDonnell D. P. , Fowlkes D. M.: Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta. Proc.

Natl. Acad. Sci. U. S. A. 1999 Mar 30 ; 96 (7): 3999-4004.

The effect of compounds on the stability of ERss may be assessed using NMR based assays.

See for example: Sensing conformational changes in nuclear receptor structure upon ligand binding by NMR: Chen S. , Johnson B. A. , Li Y. , Aster S. , McKeever B. , Mosley R. , Moller D. E. , Zhou G. Both coactivator LXXLL motif-dependent and-independent interactions are required for peroxisome proliferator-activated receptor gamma (PPARgamma) function. J. Biol. Chem.

2000 Feb 11 ; 275 (6): 3733-6.

Using NMR to monitor drug-receptor interactions reviewed in : Keifer P. A.: NMR spectroscopy in drug discovery: tools for combinatorial chemistry, natural products, and metabolism research. Prog. Drug Res. 2000; 55: 137-211.

Cell reporter assays may be used to determine the effect of binding of compounds on ER-p, for example as shown in WO 98/56812.

Variations of cell assays using the trans-activation by ER (including ERss) of AP-1, may also be-used-see for example: Kushner P. J., Agard D. A. , Greene G. L. , Scanlan T. S. , Shiau A. K. , Uht R. M. , Webb P.: Estrogen receptor pathways to AP-1. J. Steroid Biochem. Mol. Biol. 2000 Nov. 30; 74 (5): 311-317.

Weatherman R. V. , Scanlan T. S.: Unique Protein Determinants of the Subtype-selective Ligand Responses of the Estrogen Receptors (ERalpha and ERbeta) at AP-l Sites. J. Biol.

Chem. 2001 Feb. 9; 276 (6): 3827-3832.

Paech K. , Webb P. , Kuiper G. G. , Nilsson S. , Gustafsson J. , Kushner P. J. , Scanlan T. S.: Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP-1 sites.

Science. 1997 Sep. 5; 277 (5331): 1508-10.

The effects on gene or protein expression especially of marker genes relevant to blood pressure (in cells expressing predominately ERss or with ERss selective compounds), may be tested using quantitative PCR. Branched DNA assay, and the use of fluorescently tagged antibodies to proteins regulated by ERss, may also be used.

Furthermore, ex vivo effects on relevant tissues, such as blood vessels or in tissues expressing predominately ERss or with ER, selective compounds, and assays for measuring tension of blood vessel strips and nitric oxide production, known in the art, may also be used to assay ERss activity.

Methods for assaying ERss modulating compounds for hypertensive activity may be carried out using a number of assays including using ex vivo techniques to measure the effects of compounds on nitric oxide production. Nitric oxide reduces blood pressure. An example of such an assay is shown in the paper by Prevot V., et aI., Endocrinology, Vol. 140 (2) (1999), pages 652-9.

The compound used in the invention may have an antagonist or an agonist effect on blood-pressure. In particular, it is preferable that the blood-pressure modulating activity is anti-hypertensive activity.

Preferably, the method comprises the step of contacting the compound with ERss and determining the effect of the compound of the activity of the ERss. The method may comprise contacting the compound with ERss and determining the binding affinity of the compound with ERss. Methods of determining ERR binding ability are known in the art as discussed above.

The inventors have also recognised that ERss modulating compounds may be used to modulate blood-pressure in mammals, such as humans.

A further aspect of the invention provides the use of an ERss modulating compound for the manufacture of a medicament to modulate blood-pressure. Preferably, the use is for the treatment of hypertension.

A further aspect of the invention provides a method of modulating blood-pressure comprising administering to a patient an ERss modulating compound. The invention also includes within its scope a method of treating hypertension comprising administering an ERss modulating compound.

Oral dosages of the ERss modulating compounds, when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day, preferably 0. 01 mg per kg of body weight per day (mg/kg/day) to 10 mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day. For oral administration, the compounds are preferably provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from about 1 mg to about 100 mg of active ingredient.

The ERss modulating compounds may be agonists or antagonists.

Preferably, the ERss modulating compound used in the invention is an ERss ligand. Such ligands are known in the art. Such compounds are discussed, for example, in WO 98/56812. Preferably, the ERss modulating compound is selected from raloxifene, centchroman, coumestrol, diethylstilbestrol, esculin, tamoxifen, zearalenone and zindoxifen. Preferably, the ERss modulating compound has a general formula Z:

Preferably, modifications to the steroid nucleus may be made at a positions at the 7-, 9-, 12-, 14-, 16-and 17-positions, and ss-substitutions at the 8-, 11-, 15-and 18-positions.

Preferably, those substituents are hydrophobic substituents, such as methyl, ethyl or isopropyl, or halogens such as chlorine, bromine or iodine.

Preferably, the ERss modulating compound is a 2'-, 3'-, 5'-and/or 6'-substituted 2-aryl benzothiophene. Such a compound may be substituted at one or more of the 2', 3', 5'and 6' positions.

Estradiol and estradiol metabolites such as 2-methoxyestradiol, 4-hydroxyestradiol, 16ahydroxyestradiol and 16a-hydroxyestrone may also be used. Genistein and modrefen, and compounds disclosed in WO 00019994 and WO 00007996 may also be suitable.

Other compounds of use include those shown in Figure 3.

Pharmaceutically acceptable salts of the compounds may be used.

The compounds may be used in the form of pharmaceutical compositions.

Pharmaceutical compositions comprising ERss modulating compounds, or pharmaceutically acceptable salts thereof, may comprise any pharmaceutically acceptable carrier. adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to. ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers. polyethylene glycol and wool fat.

The pharmaceutical compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.

The pharmaceutical compositions may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and

solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but. are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation.

Topically-transdermal patches are also included in this invention.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

Definitions "ERP agonism" : An ERss agonist is a compound that displays > 50% of the activity of the natural estrogen 17ss-estradiol (E2) or the synthetic estrogen moxestrol, activity defined as e. g the increased expression of a gene product that is transcriptionally controlled by an

estrogen-response-element (ERE)-promoter-gene construct (ERE-reporter vector) in the presence of an ERss.

"ERss antagonism" : An ERss antagonist is a compound that displays 5% or no agonist activity compared to the activity displayed by the natural estrogen 17ss-estradiol (E2) or the synthetic estrogen moxestrol, or a compound that decrease the activity of E2 or the synthetic estrogen moxestrol down to < 5% of the activity displayed by E3 or the synthetic estrogen moxestrol alone, activity defined as e. g the increased expression of a gene product that is transcriptionally controlled by an estrogen-response-element (ERE)-promoter-gene construct (ERE-reporter vector) in the presence of an ER.

The invention will now be described by example only, with reference to the following figures: Figure 1 shows the kinetics of the mean arterial blood-pressure (MAP), measured in conscious freely-moving mice. Each symbol in the diagram is a mean +/-SEM from 1 hour recording (30 times) of 9 to 10 animals.

Figure 2 shows variations of the mean arterial blood-pressure (MAP) in mice. Values are expressed as mean +/-SEM (mmHg) of 24 hours recording (twice per minute) from 9 to 10 animals in each group.

Figure 3 shows examples of suitable ER, ligands which may be used to regulate blood-pressure.

Methods All mice used in the study were bred from the heterozygous parents (ERR +/-), since female BERKOs are subfertile (21), and were fed with a standard diet ad libitum. Genotype was confirmed by PCR, using primers published previously (21). Nineteen male mice weighing 26-36 g and twenty female mice weighing 22-28 g were included in this study. The animal experiment was approved by the regional Animal Ethical Committee. Mice were anaesthetized with 2% isofluran (Abbott Lab Ltd, Kent, England). The right carotid artery

was cannulated with a PE-10 catheter, which was tunnelled subcutaneously to exit at the back, and connected to a swivel tether system (custom made). Sterile saline was infused continuously via the cannula (2-3 ul/min). All mice recovered completely from anaesthesia within a few minutes after the termination of isofluran. The mean arterial BP (MAP) in

conscious, freely-moving mice was started to be recorded (Grass Polygraph model 7) at 1 h post-operation, and it lasted for 24 h (twice per min), consisting of 6 h light cycle, 12 h dark cycle and 6 h light cycle.

To evaluate cardiac mass, animals were sacrificed by exsanguination under isofluran anaesthesia. The heart was perfused from the apex of the left ventricle with cold BPS for 1 min under a constant pressure (100 mmHg), followed by triphenyl tetrazolium chlorid (TTC) perfusion. The hearts were removed and weighted, the indices were calculated as ratio of cardiac mass wet weight (mg) to body weight (g). Finally, the hearts were fixed with PBS/4% formaldehyde for 4-6 h for other studies.

All data were presented as meanSEM. Statistical analysis was done by the two-tailed unpaired Student's t-test and one-way analysis of ANOVA, respectively.

Results The inventors first monitored the changes of BP in adult BERKOs and their wt littermates at two months of age, using non-invasive tail-cuff method (RTBP1001, Kent Scientific Corporation, Litchfield, CT) or intra-arterial measurement. However, no dramatic elevation of BP was detected in BERKOs until they were 5 months old, when BP was affected in 50% of screened BERKOs (n=20). We therefore decided to perform the experiment with a group of 6-7 months old animals.

Figure 1 illustrates the kinetics of MAP in conscious, freely-moving mice. There was a clear elevation of MAP in both male and female BERKOs throughout the 24 h observation, as compared to their wt littermates. Within the first 1-3 h of a light cycle, the MAP was slightly above the baseline when mice were adapting to the experimental conditions. After entering the dark cycle, most of the experimental mice became gradually more active, with additional

elevation of MAP recorded during this period (12 h dark cycle). All the mice became less active during the following 6 h light cycle, reflected in some decline of MAP back to the baseline. Figure 2 represents the statistical analysis of MAP from total 24 h recordings (mean : l : SEM). MAP in male BERKOs (1374. 6 mmHg, n=9) was significantly higher (P < 0. 0001) than in wt males (1092. 7 mmHg, n=10), and in female BERKOs (1234. 9 mmHg, n=10), respectively. Furthermore, MAP in female BERKOs was significantly elevated (P < 0. 0001) compared to wt females (1062. 9 mmHg, n=10). Unexpectedly, a significant difference (P < 0.001) of MAP was also seen between wt male and wt female animals. However, when the data collected from 12 h light cycle recordings only were

analyzed, no differences (P =0. 11) in MAP between wt males (1082. 8 mmHg) and wt females (1063. 2 mmHg) were observed. MAP in heterozygous mice at 6-7 months of age tended to be higher than those of wt counterparts, but these differences did not reach statistical significance (data not shown).

The cardiac mass study is summarized in the Table. The size of the intact heart and the transverse section from a BERKO were similar to that from a littermate control. The ratio of the heart wet weight against body weight was not significantly different (P > 0.05) between BERKOs (n=7) and their wt littermates (n=6). In addition, using H & M and/or Masson's trichrome staining, histological examination of organs, including heart, lungs and kidneys obtained from both male and female 6-7 months old BERKOs did not reveal any obvious pathological alterations, such as hypertrophy or fibrosis (data not shown).

Table. Comparison of cardiac mass in BERKO and WT

WT BERKO nr HW/BW ratio (%) HW/BW ratio (%) 1 212/36. 3 0.058 148/24.9 0.059 2 147/27.6 0.053 146/23.4 0.062 3 163/25.2 0.065 166/30.9 0.054 4 144/26.3 0.055 147/24.6 0.060 5 143/26.2 0.055 208/34.4 0.061 6 142/23.4 0.061 198/34.1 0.058 7 152/28.7 0.053 mean 0.0578 0.0581 s. d. 0.004 0.003

HW: heart wet weight (mg), BW: body weight (g).

Discussion Estrogen has been shown to interact with different systems in the regulation of BP. Among those systems, the RAS has been considered to be a key regulator of BP and volume homeostasis (22,23). All the components of RAS as well as angiotensin II (Ang II) receptors (ATI and AT2) have been identified in cardiac tissue. The RAS has also been shown to control BP in the brain, by regulation of angiotensinogen production through angiotensinogen gene-activating elements (24). It has recently been shown that AT2 has an opposite effect to ATI in the regulation of BP and sodium secretion (25).

Estrogen in man and in experimental animals can affect RAS by suppression of renin (26), reduction of angiotensin-converting enzyme (ACE) activity (27), and decreasing ATI

receptor gene expression and density (28). However, the precise mechanisms of this regulation are largely unknown.

The NOS system is a well-documented regulatory pathway which plays an important role in keeping BP within normal limits (29). The function of estrogen in regulating eNOS/iNOS has been well documented both in vitro and in vivo, from animal experiments to studies in man. The chronic presence of estrogen causes a greater release of NO and flow-induced dilatation of skeletal muscle arterioles in female than that in male rats (30). This is further supported by the fact that elevated BP is established in eNOS-deficient mice (31), and that administration of NOS antagonist results in increased peripheral resistance and elevated BP in animals (32) and in man (33). A gender difference of BP in BERKOs was also observed in this study, which is likely due, to a large extent, to the presence of estrogen in female (2, 3, 34) and androgen in male (35-37). Practically, androgen receptor has been found to be upregulated in different tissues in BERKOs (our unpublished observation). In addition, a low level of circulating estrogen is associated with a reduced production of NO, resulting in hypertension in pre-eclampsia (38). This issue may be further elucidated by the use of transgenic mice deficient in aromatase (ARKO). In the study, heterozygotes (ERss +/-) showed a tendency towards an increase in BP over wt, but the difference did not reach statistical significance (data not shown), suggesting that sufficient ERss is produced from a single copy of the gene to maintain normal BP. Whether the heterozygotes might show a different BP from wt in other environmental and genetic backgrounds remains to be determined.

So far, six female and two male patients have been diagnosed to carry mutations in the aromatase gene (39), and one man has been described to carry a point mutation in ERa (40).

However, none of these subjects had hypertension. These seemingly contrasting findings between clinical cases and an animal model may have several explanations. 1) Mouse may be quite different from man regarding estrogenic control of blood pressure. 2) Development of hypertension is an age-dependent phenomenon. All the reported patients are still young (less than 35-year-old) and they may develop hypertension later as compared to age-matched healthy individuals. 3) All the patients have received treatments with different regimens on

the basis of their endocrine disturbances, and such therapy might prevent the development of hypertension.

Although no net beneficial effect of HRT on postmenopausal women with established cardiovascular diseases evidenced by Heart and Estrogen/progestin Replacement Study, the HERS study (41), additional drug trials with sufficient size and length of treatment are needed to further clarify the HRT issue. Comments on the statistical analysis, interpretations as well as limitations about the HERS study have been addressed lately (42,43).

Estrogen modulates G-protein-coupled receptors and exerts long-term effects in most, if not all, cells through the regulation of gene transcription (15). It has become apparent that ER-mediated actions of estrogen are much broader than initially perceived (44). The present study clearly indicates that disturbance in ERB function may lead to the development of hypertension.

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Claims

Claims 1. A method of assaying compounds for blood-pressure modulating activity comprising the step of determining the ability of the compound to affect estrogen receptor

ss (ERss) activity.
2. A method according to claim 1, wherein the blood-pressure modulating activity is anti-hypertensive activity.
3. A method according to claim 1 or claim 2, wherein the method comprises the step of contacting the compound with ERss and determining the effect of the compound on the activity of the ERss.
4. A method according to claim 1 or claim 2, wherein the method comprises contacting the compound with ERss and determining the binding affinity of the compound with ERss.
5. Use of an ERss modulating compound for the manufacture of a medicament to modulate blood-pressure.
6. Use according to claim 5, for the treatment of hypertension.
7. A method of modulating blood-pressure comprising administering to a patient, an ERss modulating compound.
8. A method of treating hypertension comprising administering an ERss modulating compound.
9. A method or use according to any one of claims 5 to 8, wherein the ERss modulating compound is an ERR ligand.

<Desc/Clms Page number 21>
10. A method or use according to claim 5 or claim 6, wherein the ER-ss modulating compound is in an amount of 0. 01 nM to 200 nM.
11. A method or use according to claim 10, wherein the compound is in an amount of 0.1 nM to 10 nM.
12. A method or use according to claim 9, wherein the ERss modulating compound is selected from raloxifene, raloxifene, centchroman, coumestrol, diethylstilbestrol, esculin, tamoxifen, zearalenone, zindoxifen, Serm-3, EM-800, ICI-182780, lasofoxifene, EM-652, Fc-1271a, TSE-424, trans-dihydroraloxifene, LY-357489, LY-326315, NNC-45-0095, enclomiphene, toremifene, tibolone, modrefen, nafoxidene, idoxifene, droloxifene, levormeloxifene, RU-39411, RU-45144, GW-5638, GW-7604, MDL-103323 and miproxifene.
13. A method or use according to claim 9, wherein the ERss modulating compound has a general formula Z :

and has a hydrophobic constituent at one or more of the 9a or 12a positions.