WO2001026651A2

WO2001026651A2 - Selective estrogen receptor modulators in the treatment or reduction of the risk of acquiring hypertension, cardiovascular diseases, and insulin resistance

Info

Publication number: WO2001026651A2
Application number: PCT/CA2000/001222
Authority: WO
Inventors: Fernand Labrie; Andre Marette
Original assignee: Endorecherche, Inc.
Priority date: 1999-10-14
Filing date: 2000-10-13
Publication date: 2001-04-19
Also published as: AU7896100A; WO2001026651A3

Abstract

Novel methods for the medical treatment and/or inhibition of the development of hypertension, cardiovascular diseases, insulin resistance and diabetes in susceptible warm-blooded animals including humans involving administration of a selective estrogen receptor modulator, particularly compounds having general structure (I).

Description

SELECTΓVE ESTROGEN RECEPTOR MODULATORS IN THE TREATMENT OR REDUCTION OF THE RISK OF ACQUIRING HYPERTENSION, CARDIOVASCULAR DISEASES, AND INSULIN RESISTANCE.

HELD OF THE INVENTION

The present invention relates to a method for treating or reducing the risk of acquiring hypertension, cardiovascular diseases, insulin resistance and diabetes, in susceptible warm-blooded animals, including humans of both sexes which consists in adrninistering a selective estrogen receptor modulator (SERM), particularly, adrninistering a benzopyran derivative.

BACKGROUND OF THE RELATED ART Hypertension is a common vascular disease. It is particularly seen, but not exclusively, in obese persons, postmenopausal women and the elderly and is frequently accompanied by insulin resistance. There is a wide variety of treatments using even nonpharmacologic intervention (Orbach and Lowenthal, 1998) or pharmacologic therapies. Among the last, it can be noted the use of beta-blockers and angiotensin analogues.

The beneficial effects of estrogens on the risk for developing coronary heart disease (CHD) and insulin resistance is well documented. Estrogen replacement therapy in postmenopausal women reduces the clinical manifestations of CHD (Stampfer et al., 1991). These observations have led to the hypothesis that estrogen possesses cardioprotective effects

(Mendelson and Karas, 1999). Moreover, it is well documented that led to the hypothesis that estrogen possesses cardioprotective effects

(Mendelson and Karas, 1999). Moreover, it is well documented that estrogen replacement therapy also improves insulin resistance in postmenopausal women with or without diabetes (Brussaard et al. Diabetologia 40: 843-9, ; Lindheim et al / Soc Gynecol Investig 1: 150-4, 1994 ; Lindheim et al. Fertil Steril 60: 664-7, 1993). Estrogens, however, are recognized to play a predominant role in breast cancer development and growth (McGuire et al., 1975). Moreover, estrogen replacement therapy as commonly used in non-hysterectomized women requires the addition of progestins to counteract the endometrial proliferation and the risk of endometrial cancer induced by estrogens. In addition, since both estrogens and progestins are thought to increase the risk of breast cancer (Bardon et al., 1985; Colditz et al., 1995), the use of estrogen-progestin replacement therapy is accepted by a limited number of women and, usually, for too short periods of time.

Selective estrogen receptor modulators (SERMs) are used for breast cancer prevention and treatment. Tamoxifen is a mixed estrogen agonist/antagonist that is widely used for breast cancer prevention and therapy. We recently showed that the SERM EM-800 in addition to being a pure antiestrogen in the mammary gland and uterus, was more potent than tamoxifen and ICI 182780 in inhibiting estrone-stimulated uterine weight in ovariectomized mice (Luo et al., 1997; Couillard et al., 1998; Martel et al., 1998).

Considerable evidence now suggests that ovarian hormones have an important influence on blood flow distribution (Magness et al., 1993; Magness et al., 1998) and vascular reactivity (Barber and Miller, 1997;

Skarsgard et al., 1997; Huang et al., 1998). Moreover in EP 792641 and EP 659426, lasofoxifene and some 2-phenyl-3-azoylbenzothiophenes were disclosed for treating pulmonary hypertension, a disease which is however, different from general hypertension, object of the present invention. To our knowledge, very little is known about the effects of estrogen and/or estrogen-like drugs on the hemodynamic actions of acetylcholine, bradykinin and insulin.

Dehydroepiandrosterone (DHEA) is shown to have beneficial effects on different diseases like diabetes, obesity, cancer, menopausal symptoms, hypertension etc.. Particularly, it has been reported that DHEA prevents dexamathasone-induced hypertension in rats (Shafagoj et al. 1992) and DHEA has been disclosed as agents for preventing, controling, or reversing hypertension in GB 2240472 (J.C. Masterson).

In addition to its well known actions on carbohydrates and lipid metabolism, insulin exerts hemodynamic influence on different vascular beds promoting glucose metabolism in different peripheral tissues (Anderson and Mark, 1993; Baron, 1993; Baron, 1994). A regional non- uniformity in insulin hemodynamic responses has been described (Pitre et al., Am J Physiol 271: E658-68, 1996). A vasodilator effect of insulin was observed in the kidney and hindquarter beds but not the superior mesenteric bed of normotensive. The overall change in blood pressure is thus dependent on the sum of the local effects of insulin on these vascular beds. This regional variation in hemodynamic regulation has also been documented for other vasodilators such as acetylcholine and bradykinin (Gregg et al. J Vase Res 32: 106-11, 1995 ; Honda et al. Physiol Behav 63: 55-8, 1997). It is therefore important to remember that the measurement of vascular effects of any vasodilator in one vascular bed is not indicative of its hemodynamic action in other beds. Furthermore, it is not possible to predict changes in blood pressure simply based on the hemodynamic effect of any given compound on one or more vascular beds. One must directly measure the effect of the compound on mean arterial blood pressure.

SUMMARY OF THE INVENTION

It is accordingly an object of the present invention to provide effective methods of treatment for hypertension, cardiovascular diseases, insulin resistance and diabetes, comprising adrninistering to an animal, particularly an human of both sexes, that needs such treatment, a therapeutic compound chosen among the class of Selective Estrogen

Receptor Modulators (SERMs).

More particularly, this invention claims the beneficial effects of SERMs on hypertension and insulin resistance, particularly on the hemodynamic actions of insulin, acetylcholine, and bradykinin, as well as on the metabolic action of insulin on glucose disposal in peripheral tissues.

As used herein, a selective estrogen receptor modulator (SERM) is a compound that either directly or through its active metabolite functions as an estrogen receptor antagonist ("antiestrogen") in breast tissue, yet provides an estrogen-like effect on bone tissue and on serum cholesterol levels (i.e. by reducing serum cholesterol or other tissues). Non-steroidal compounds that function as estrogen receptor antagonists in vitro, in human breast cancer cell lines or in in vivo models of human breast cancer (especially if the compound acts as an antiestrogen in human breast cancer cells growing as xenografts in nude mice) is likely to function as a SERM. Conversely, steroidal antiestrogens tend not to function as SERMs because they tend not to display any beneficial effect on serum cholesterol.

It is preferred that the selective estrogen receptor modulator of the invention has a molecular formula with the following features :

a) two aromatic rings spaced by 1 to 2 intervening carbon atoms, both aromatic rings being either unsubstituted or substituted by a hydroxyl group or a group converted in vivo to hydroxyl;

b) a side chain possessing an aromatic ring and a tertiary amine function or salt thereof.

It is also accordingly another object of the present invention to provide effective methods of reducing the risk for hypertension, insulin resistance, and diabetes, and related complications such as insulin resistance and diabetes comprising aciministering to an animal, particularly an human, who needs this reduction a therapeutic amount of a Selective Estrogen Receptor Modulator (SERM). Experiments in our laboratory indicate that EM-652.HC1 is unlikely to exert negative effects in the breast and endometrium, effects that can be problematic in some prior art treatments. Benzopyrans of this type are likely to provide particularly useful methods of treatment and prevention.

Because of correlations between undesirable insulin resistance and the onset or continuation of insulin resistance-related diabetes, the methods of the invention are expected to effectively treat or reduce the risk of such diabetes. Because of correlations between hypertension and hypertension-related cardiovascular disease, the methods of the invention are expected to effectively treat or reduce the risk of such cardiovascular disease.

Applicants utilize methods believed to effect hypertension beyond pulmonary hypertension.

Applicants believe that the combination of DHEA and a SERM has more beneficial effect that DHEA alone in preventing or treating hypertension or insulin resistance.

This combination permits aclministery lower doses of DHEA and thus avoiding typical side effects due to high doses of DHEA like virilism and endometrium stimulation in women. At the same time, SERM plays an important role in reducing the risk of acquiring breast, ovarian and endometrial cancer osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, obesity etc. Moreover, the applicant believes that the administration of DHEA to SERM permits a more complete effect than a SERM or DHEA alone. In another aspect, the invention provides therapeutic and prophylactic methods as described herein which includes the foregoing combination of active ingredients, wherein DHEA may be, optionally replaced or supplemented with other sex steroid precursors as described herein.

A patient in need of treatment or of reducing the risk of onset of a given disease is one who has either been diagnosed with such disease or one who is susceptible to acquiring such disease.

DESCRIPTION OF THE DRAWINGS

Figure 1 describes schematically the modulation of the cardiovascular function by acetylcholine (Ach), bradykinin, and insulin.

Figure 2 describes schematically a possible mechanism of action of SERMs on the cardiovascular system.

Figure 3 describes the effects of the treatments on acetylcholine-mediated reductions in blood pressure: intact female rats; ovariectomized female rats; ovariectomized female rats with 0.5 cm estradiol implants; ovariectomized female rats receiving EM-652.HC1 (0.5 mg, per os, ID). Figure 3 indicates that the EM-652.HC1 treatment reverses the effect due to the ovariectomy.

Figure 4 describes the effects of the treatments on acetylcholine-induced reductions in vascular reactivity in the aorta and l mdlirnb muscle vascular beds. Figure 4 indicates that the EM-652.HC1 treatment reverses the effect due to the ovariectomy.

Figure 5 describes the effects of the treatments on bradykinin-mediated changes in blood pressure.

Figure 6 describes the effects of the treatments on insulin-mediated changes in blood pressure. Figure 6 indicates that the EM-652.HC1 treatment potentiates insulin-mediated reduction in blood pressure in ovariectomized animals. Figure 7 describes the effects of the treatment on insulin-mediated changes in glucose utilization in peripheral tissues. This data shows that the statistically significant difference of whole-body insulin-mediated glucose disposal between EM-652.HC1 ovariectomized rats and untreated ovariectomized rats proves the effectiveness of EM-652.HC1 against hypertension.

Figure 8 describes the basal perfusion pressures in mesenteric vascular bed following Methoxamine (MTX, 50 μM) infusion. Intact control (TNT); OVX control (OVX); OVX + EM-652.HC1 (0.5 mg/rat) (OVX-EM); OVX + DHEA (100 mg/rat) (OVX-DHEA); OVX + EM-652.HC1 + DHEA (OVX-EM- DHEA). Figure 8 indicates that in absence of vasodialatory factors, the blood pressured was not modified by different treatments.

Figure 9 describes the acetylcholine (ACh)-induced vasodilatory responses in MTX-preconstricted mesenteric vascular bed. Intact control (TNT); OVX control (OVX); OVX + EM-652.HC1 (0.5 mg/rat) (OVX-EM); OVX + DHEA (100 mg/rat) (OVX-DHEA); OVX + EM-652.HC1 + DHEA (OVX-EM-

DHEA). *: P < 0.05 versus OVX; . P < 0.05 versus INTACT (ANOVA followed by Fischer post hoc test). Figure 9 indicates that blood pressure of intact control rats and EM-652.HC1, DHEA, or EM-652.HC1 + DHEA- treated ovariectomized rats was decreased versus ovariectomized untreated rats. This data shows that the statistically significant difference of blood pressure between EM-652.HC1 ovariectomized rats and untreated ovariectomized rats proves the effectiveness of EM-652.HC1 against hypertension.

Figure 10 describes the adenosine diphosphate (ATP)-induced vasodilatory responses in MTX-preconstricted mesenteric vascular bed. Intact control

(TNT); OVX control (OVX); OVX + EM-652.HC1 (0.5 mg/rat) (OVX-EM); OVX + DHEA (100 mg/rat) (OVX-DHEA); OVX + EM-652.HC1 + DHEA (OVX-EM-DHEA). *: P < 0.05 versus OVX. (ANOVA followed by Fischer post hoc test). Figure 10 indicates that blood pressure of ovariectomized rats treated with the combination of EM-652.HC1 + DHEA is decreased versus ovariectomized untreated rats.

Figure 11 describes the sodium nitroprusside (SNP)-induced vasodilatory responses in MTX-preconstricted mesenteric vascular bed. Intact control (TNT); OVX control (OVX); OVX + EM-652.HC1 (0.5 mg/rat) (OVX-EM);

OVX + DHEA (100 mg/rat) (OVX-DHEA); OVX + EM-652.HC1 + DHEA (OVX-EM-DHEA).

Figure 12 describes the effect of EM-652.HC1 and estradiol on whole- body insulin-mediated glucose disposal in ovariectomized rats. Rats were ovariectomized and treated with either EM-652.HC1 (EM), or estradiol (E2) or left untreated (OVX) for 3 weeks. A groups of intact (CTRL) animals was also included. All OVX rats were paired-fed to the CTLR group. Data are mean ± SE of 8-10 rats. ** p<0.01 vs intact group. This data shows that the statistically significant difference of whole-body insulin-mediated glucose disposal between EM-652.HC1 ovariectomized rats and untreated rats proves the effectiveness of EM-652.HC1 against hypertension.

Figure 13 describes the effect of EM-652.HC1 and estradiol on insulin- induced PI 3-kinase activity in skeletal muscle of ovariectomized rats. Rats were ovariectomized and treated with either EM-652.HC1 (EM), or estradiol (E2) or left untreated (OVX) for 3 weeks. A groups of intact (CTRL) animals was also included. All OVX rats were paired-fed to the CTLR group. Data are mean ± SE of 5-6 rats. ** p<0.01 vs insulin values in intact and OVX groups. DETAIL DESCRIPTION OF THE INVENTION

It has been demonstrated that the cardiovascular function is modulated by insulin (Townsend et al., 1992; Steinberg et al., 1994; Wu et al., 1994; Chen and Messina, 1996; Pitre et al., 1996), acetylcholine (Gryglewski, 1995; Drexler and Hornig, 1999) and bradykinin (Lin and Frishman, 1996; Scholkens, 1996). Although insulin is a potent vasodilator in some vascular beds (ex. skeletal muscle, kidney), it can also cause vasoconstriction of other vascular beds (ex. mesenteric), and the net influence of this hormone on blood pressure therefore depends on the sum of these opposite changes in different vessels. The vasodilatory effects of acetylcholine, bradykinin, and insulin is mediated, at least in part, via the release of nitric oxide (NO) by the endothelial cells of the blood vessels. Nitric oxide activates the production of cGMP in smooth muscle cells which leads to vasodilatation of the vessels (reduced vascular reactivity) as shown in figure 1.

The inventors have shown that SERMs of the invention exert their beneficial actions on blood pressure by potentiating the hemodynamic responses of several well-known vasodilatory factors. A possible mechanism of action of SERMs on the cardiovascular system is described in figure 2. By increasing blood flow in different vascular beds, SERMs lower blood pressure and therefore improve the cardiovascular profile.

Furthermore, the increased blood flow facilitates uptake and utilization of lipids and glucose, thereby contributing to improve the metabolic profile as well. Thus, in agreement with the present invention, blood pressure is lowered in intact control rats (non-OVX), by treatment with increasing doses of acetylcholine (A -Ch), but this lowering effect is impaired in OVX animals and estrogen replacement therapy failed to restore this defect. The compound EM-652.HC1, a preferred SERM of the invention, not only restores this defect but also tends to potentiate the beneficial lowering effect of acetylcholine on blood pressure as shown in figure 3. Acetylcholine induces also a lowering effect on vascular resistance of both aortic and muscle vessels which is impaired by ovariectomy an effect that estrogen replacement failed to restore. SERMs of the invention, moreover, restored the acetylcholine action in OVX rats as shown in figure 4.

A single dose of bradykinin ( 10 μg/kg) induced reduction in blood pressure (figure 5). The lack of estrogen (OVX) or its replacement (OVX- E₂) appears to have little effect on this parameter while EM-652.HC1 potentiates bradykirtin-induced reduction in blood pressure.

Although a tendency to insulin-induced hypertension was observed in intact rats (Figure 5), it failed to reach statistical significance. Insulin (10 mU/kg/min) reduces blood pressure in ovariectomized rats and estrogen replacement prevents this effect. In contrast, SERMs of the invention potentiate insulin-mediated reduction in blood pressure in ovariectomized animals as shown in figure 6.

Insulin-stimulated glucose disposal rate (GDR) was reduced in ovariectomized rats (Figure 7), confirming that estrogen lack causes insulin resistance in peripheral tissues. This impairment was partially corrected by estrogen replacement. However, SERMs of the invention totally reversed the insulin resistance induced by ovariectomy.

Results of a similar experiment in which all OVX rats were paired-fed to the CTLR group were reported in Figure 12. The effects of EM-652.HC1 and estradiol on insulin sensitivity were measured by the hyperinsulinemic-euglycemic clamp technique. Insulin was infused at a rate of lCtoU/kg/min to rats for 2 hours and glucose disposal rates were measured as described in Roy et al. (1998). Results show that EM- 652.HC1, but not estradiol, increases insulin-mediated glucose disposal in rats.

Figure 13 shows the effect of EM-652.HC1 and estradiol on insulin- stimulated phosphatidylinositol (PI) 3-kinase activity. After treatment with either EM-652.HC1, or estradiol, a maximal dose of insulin or saline (basal) was injected i.v. and muscles were rapidly sampled (4 min). PI 3- kinase activity was markedly increased in all groups. However, EM-652- treated rats exhibited increased activation of enzymatic activity by insulin. Since PI 3-kinase activation is a pivotal and essential step in the insulin signaling cascade leading to increased glucose transport in muscle, these data strongly suggest that EM-652.HC1 increases insulin sensitivity at least in part by potentiating insulin action on PI 3-kinase.

Applicants believe that adrninistration of SERMs of the invention has utility in the reduction of the development of hypertension, insulin, resistance, and diabetes. Preferred SERMs discussed herein relate to: (1) all diseases stated to be susceptible to the invention; (2) both therapeutic and prophylactic applications; and (3) preferred pharmaceutical compositions and kits.

Preferred SERMs of the invention are benzopyran derivatives having a following molecular formula:

wherein Ri and R∑ are independently selected from the group consisting of hydroxyl and a moiety convertible in vivo to hydroxyl;

wherein R3 is a species selected from the group consisting of saturated, unsaturated or substituted pyrrolidinyl, saturated, unsaturated or substituted piperidino, saturated, unsaturated or substituted piperidinyl, saturated, unsaturated or substituted morpholino, nitrogen-containing cyclic moiety, nitrogen-containing polycyclic moiety, and NRaRb (Ra and Rb being independently hydrogen, straight or branched Ci-Cβ alkyl, straight or branched C_∑-Cδ alkenyl, and straight or branched C2-C6 alkynyl). or salt thereof.

wherein R4 is selected from the groups consisting of hydrogen and methyl.

One preferred benzopyran derivative of the invention is EM-800 reported in WO 96/26201. The molecular structure of EM-800 is:

Another preferred benzopyran derivative of the invention is EM-652.HC1 (EM-01538) :

Benzopyran derivatives administered in accordance with the invention are preferably administered in a dosage range between 0.01 to 10 mg/kg of body weight per day (preferably 0.05 to 1.0 mg/kg), with 60 mg per day, especially 20 mg per day, for a person of average body weight when orally administered, or in a dosage range between 0.003 to 3.0 mg/kg of body weight per day (preferably 0.015 to 0.3 mg/kg), with 20 mg per day, especially 10 mg per day, for a person of average body weight when parentally a«iministered (i.e. intramuscular, subcutaneous or percutaneous administration). Preferably the benzopyran derivatives are aclministered together with a pharmaceutically acceptable diluent or carrier as described below.

Other preferred SERMs of the invention include Tamoxifen ((Z)-2-[4-(l^Z- c ^henyl-l-butenyl)phenoxy]-N,N-dimethyle ananrιine) (available from Zeneca, UK), Toremifene (available from Orion-Farmos Pharmaceutical,

Finland, or Schering-Plough), Droloxifene and CP-336,156 (ris-lR-[4 - pyrroUdino-ethoxyphenyl]-2S-phenyl-6-hydroxy-l,2 4,- terrahydronapthalene D-(-)-tartrate salt) (Pfizer Inc., USA described in US 5,889,042) (also called Lasofoxifene), Raloxifene (Eli Lilly and Co., USA), LY 335563 and LY 353381 (EH Lilly and Co., USA described in WO

98/45287, WO 98/45288, and WO 98/45286), Idoxifene (SmithKline Beecham, USA), Levormeloxifene (3,4-trarts-2,2-dimethyl-3-phenyl-4-[4- (2-(2-(pyrrohdin-l-yl)ethoxy)phenyl]-7-methoxychroman) (Novo

Nordisk, A/S, Denmark) which is disclosed in Shalmi et al. WO 97/25034, WO 97/25035, WO 97/25037,WO 97/25038 ; and Korsgaard et al. WO 97/25036), GW5638 (described by Willson at al., Endocrinology, 138(9), 3901-3911, 1997) and indole derivatives (disclosed by Miller et al. EP 0802183A1), TSE 424, and ERA 923 developed by Wyeth Ayerst (USA) and disclosed in JP10036347 (American home products corporation) and nonsteroidal estrogen derivatives described in WO 97/32837. Other SERMs of the invention are disclosed in: WO 99/07377; WO 98/48806; EP

0823437A2; EP 0838464A1; EP 0835867A1,

EP 0835868A1; EP 0792641 Al; EP 0873992A1 and ER 0895989A1.

Any SERM used as required for efficacy against breast cancer or osteoporosis, as recommended by the manufacturer, can be used. Appropriate dosages are known in the art. Any other non steroidal antiestrogen commercially available can be used according to the invention. Any compound having activity similar to SERMs (example: Raloxifene can be used).

Except when otherwise noted or where apparent from context, dosages herein refer to weight of active compounds unaffected by pharmaceutical excipients, diluents, carriers or other ingredients, although such additional ingredients are desirably included, as shown in the examples herein. Any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry is appropriate for use herein, and the terms "exapient", "diluent", or "carrier" include such nonactive ingredients as are typically included, together with active ingredients in such dosage forms in the industry. For example, typical capsules, pills, enteric coatings, solid or liquid diluents or excipients, flavorants, preservatives, or the like may be included.

The active ingredients of the invention may be formulated and adrrtinistered in a variety of manners.

The SERMs of the invention can also be administered, by the oral route, and may be formulated with conventional pharmaceutical excipients, e.g. spray dried lactose, microcrystalline cellulose, and magnesium stearate into tablets or capsules for oral administration.

Except where otherwise stated, the preferred dosage of the each active compounds is the same regardless of the disease whose likelihood of onset is being reduced.

AU of the active ingredients used in any of the methods discussed herein may be formulated in pharmaceutical compositions which also include one or more of the other active ingredients. In some preferred embodiments of the invention, for example, one or more active ingredients are to be formulated in a single pharmaceutical composition.

The active substance can be worked into tablets or dragee cores by being mixed with solid, pulverulent carrier substances, such as sodium citrate, calcium carbonate or dicalcium phosphate, and binders such as polyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly by adding also lubricants such as magnesium stearate, sodium lauryl sulfate, "Carbowax" or polyethylene glycol. Of course, taste-improving substances can be added in the case of oral administration forms.

As further forms, one can use plug capsules, e.g. of hard gelatin, as well as closed solf-gelatin capsules comprising a softner or plasticizer, e.g. glycerine. The plug capsules contain the active substance preferably in the form of granulate, e.g. in mixture with fillers, such as lactose, saccharose, mannitol, starches, such as potato starch or amylopectin, cellulose derivatives or highly dispersed silicic acids. In solf-gelatin capsules, the active substance is preferably dissolved or suspended in suitable liquids, such as vegetable oils or liquid polyethylene glycols.

The lotion, ointment, gel or cream should be thoroughly rubbed into the skin so that no excess is plainly visible, and the skin should not be washed in that region until most of the transdermal penetration has occurred preferably at least 4 hours and, more preferably, at least 6 hours.

A number of transdermal drug delivery systems that have been developed, and are in use, are suitable for delivering the active ingredient of the present invention. The rate of release is typically controlled by a matrix diffusion, or by passage of the active ingredient through a controlling membrane.

Mechanical aspects of transdermal devices are well known in the art, and are explained, for example, in United States Patents 5,162,037, 5,154,922, 5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444, 4,568,343, 5,064,654, 5,071,644, 5,071,657, the disclosures of which are incorporated herein by reference. Additional background is provided by European Patent 0279982 and British Patent Application 2185187.

The device may be any of the general types known in the art including adhesive matrix and reservoir-type transdermal delivery devices. The device may include drug-containing matrixes incorporating fibers which absorb the active ingredient and /or carrier. In a reservoir-type device, the reservoir may be defined by a polymer membrane impermeable to the carrier and to the active ingredient. In a transdermal device, the device itself maintains active ingredient in contact with the desired localized skin surface. In such a device, the viscosity of the carrier for active ingredient is of less concern than with a cream or gel. A solvent system for a transdermal device may include, for example, oleic acid, linear alcohol lactate and dipropylene glycol, or other solvent systems known in the art. The active ingredient may be dissolved or suspended in the carrier.

For attachment to the skin, a transdermal patch may be mounted on a surgical adhesive tape having a hole punched in the middle. The adhesive is preferably covered by a release liner to protect it prior to use. Typical material suitable for release includes polyethylene and polyethylene-coated paper, and preferably silicone-coated for ease of removal. For applying the device, the release liner is simply peeled away and the adhesive attached to the patient's skin. In United States Patent 5,135,480, the disclosure of which is incorporated by reference, Bannon et al. describe an alternative device having a non-adhesive means for securing the device to the skin.

With respect to all of the dosages recommended herein, the attending clinician should monitor individual patient response and adjust dosage accordingly. EXAMPLES OF EFFECTIVENESS

MEASUREMENT OF HEMODYMANIC PARAMETERS

Example 1

All surgical procedures and parameters recording were performed on isofluorane anaesthetised animals. A cannula was inserted into the left carotid artery for the measurement of arterial blood pressure (BP, mmHg) by a low volume pressure transducer (transpac Abbott, Transonic, N.Y., USA). Another cannula was inserted in the left jugular vein for the i.v. infusion of drugs.

For blood flow (ml/min) measurement, a midline laparatomy was performed, and a Transonic flow probe was implanted around the upper abdominal aorta (to monitor abdominal aortic blood flow). Another probe was implanted around the distal abdominal aorta, just before the iliac bifurcation (to monitor hindquarters blood flow). Blood flow was determined by the ultrasonic transit time shift technique with the use of a small animal flowmeter (model T206; Transonic, NY, U.S.A.) and a 1 mm ultrasonic flow probe (model 1RB; Transonic, NY, U.S.A.). The flow probes were connected to the flowmeter, which in turn was interfaced with a Power Macintosh compatible computer that acquires data for abdominal aortic blood flow, hindquarters blood flow, carotid BP and heart rate, using BIOPAC data acquisition software (BIOPAC, CA,

U.S.A.). When drugs were tested, graded doses were administered and maximal hemodynamic effects were monitored for each dose. For most drugs (except insulin) the maximal effects were observed within 1-2 minutes. For insulin, the parameters were measured when a steady-state metabolic and hemodynamic action was reached (between 60-120 rnin). Vascular resistance was calculated by dividing Mean Arterial Blood Pressure (MABP) by arterial blood flow.

Example 2 URMA-r-31-00

Animals and treatment

Eight to ten week-old female Sprague-Dawley rats (Crl:CD(SD)Br) (Charles River Laboratory, St-Constant, Canada) weighing approximately 200-250g at start of treatment were used. The animals were acclimatized to the environmental conditions (temperature: 22 ±

3°C; humidity: 50 ± 20%; 12-h light-12-h dark cycles, lights on at 07:15h) for 1 week before starting the experiment. Animals were housed individually and were allowed free access to tap water and certified rodent feed (Lab Diet # 5002, pellets) during the acclimation period. During the study period, the rats received an enriched carbohydrate diet ad libitum. The enriched diet (diet #3) was composed of (g/lOOg): Com starch, 31.2; Dextrose, 31.2; Casein, 20.0; Corn oil, 6.4; dl-Methionine, 0.3; Vitamin mix, 1.0; ATN-76 mineral mix, 4.9; Fiber, 5.O.. The experiment was conducted in a Canadian Council on Animal Care approved facility in accordance with the CCAC Guide for Care and Use of Experimental

Animals. Sixty rats were randomly distributed between 5 groups of 12 animals each as follows: 1) Intact control; 2) OVX control; 3) OVX + EM-652.HC1 (0.5 mg/rat); 4) OVX + DHEA (100 mg/rat); 5) OVX + EM-652.HC1 + DHEA. On day 0 of the study, the animals of the appropriate groups were bilaterally ovariectomized (OVX) under isoflurane anesthesia. EM- 652.HC1 and DHEA were administered orally in 0.4% methylcellulose for

20 days (Study Day 1 to 20). Animals of control groups received the vehicle alone. Approximately 24 hours after the last dosing, non-fasted animals were killed by exsanguination at the abdominal aorta under isoflurane anesthesia.

Immediately following exsanguination, the superior mesenteric artery was cannulated with a PE-90 tubing and the gut removed. The MAB was perfused at 5 ml rnin'l and superfused at 0.2 ml min^~l with modified Krebs' bicarbonate solution (mM: NaCl 118, KCI 4.7, MgCl2-6H2θ 1.2, aH2Pθ41.0, CaCl2-2H2θ 2.6, NaHCOβ 25, glucose 11.1; 37°C; pH 7.35 - 7.45), oxygenated with a 95% oxygen and 5% carbon dioxide gas mixture. The Krebs' solution routinely contained 1 μM indomethacin to block cyclo-oxygenase pathway. A bubble trap system, with a flow rate of 0.2 ml min^~l, removed any air bubbles in the perfusate. Arteriolar vasoconstriction produced an increase in perfusion pressure (PP, mmHg) which was measured with a strain gauge transducer (Beckman, Palo Alto, CA, USA), placed in the perfusion circuit just before the mesenteric arterial bed. Mean PP was recorded after electronic integration of the pulsatile pressure signal. All PP values given were corrected by subtraction of the pressure generated by the tubing of the perfusion system. Twenty minutes following noradrenaline (NA; 10^~9 to 3 X 10"? mol) dose-response curves (DRC), MAB was preconstricted with methoxamine (MTX, 30 - 50 μM) for 30 min at the end of which time an acethylcholine (Ach) bolus was injected into the perfusate. Bolus injections was repeated with stepwise increases in the concentration at 5 min intervals of ACh (10"12 to 10^~9 mol), adenosine triphosphate (ATP;

10"¹⁰ to lO^-7 mol) and sodium nitroprusside (SNP; 10"⁹ to 10^-6 mol). A 20 min recovery period was allowed between each drug.

MEASUREMENT OF INSULIN ACTION ON PERIPHERAL GLUCOSE DISPOSAL

Example 3

Three days before the experiment, rats were anesthetized with a Ketamine/Xylazine solution i.p. at 80 mg/kg and 10 mg/kg, respectively, for catheterization of the carotid artery and the jugular vein. The right carotid artery was isolated and PE-50 (Polyethylene tubing, 0.40 mm ID, 0.80 mm OD) catheter was inserted 2.5 cm down the vessel while the cephalic end of the artery was ligated. Thereafter, the left jugular vein was isolated and three PE-10 tubings (Polyethylene tubing, 0.28 mm ID, 0.61 mm OD) were inserted 3 cm into the vessel and sutured in situ. All the catheters were passed subcutaneously to exit dorsally at the midscapular region, which was covered with a stainless steel extension spring. The spring was secured to the rat by a rigid polyethylene kneck (90° angle) that was sutured, subcutaneously at the exit incision site. The spring and catheters were then suspended from a contort holder directly above the cage ensuring the rat 360° of free and unrestricted motion.

Heparinized saline (50 IU/ml) was infused into the catheters to prevent coagulation.

Three days after canulation, the overnight fasted rats were weighed and transferred to a quiet isolated room. The extension spring and catheters were suspended directly above the cage (providing free and unrestricted movement) with the end of the jugular catheters attached to a syringe Pump (Razel, CT) with a 5 cc syringe for glucose and insulin infusions. Heparinized saline was taken out of each catheter and replaced by fresh saline prior to the start of the experiment. The carotid artery catheter was occluded with a 23G needle and sterile tuberculin syringe. Once installed, the rat was allowed 60 minutes to acclimatize to its new surroundings. Once the animal was acclimatized, a first blood sample was collected via the carotid artery catheter for measurement of basal levels of glucose and insulin and care was taken not to disturb the rat during all interventions.

Whole-body insulin action was determined by the hyperinsulinemic euglycemic clamp as previously described (Roy et al. Am J Physiol 274: E692-E699, 1998). After the acclimatation period, insulin (Hu ulin, Eli Lilly, IN) infusion (10 mU/kg/min) was started and blood glucose was monitored at 5-minute intervals using a One Touch TJ Hospital in order to prevent hypoglycemia. D-glucose (50% wt/vol) infusion was normally started 5-10 minutes after starting insulin infusion according to blood glucose levels, and the rate of glucose infusion was then adjusted to maintain normoglycemia throughout the study. A 300 μl sample of blood was collected at 20-min intervals for later measurements of insulin levels and red blood cells were re-injected following each sampling to prevent a fall in the hematocrit and minimize stress. The animals were infused for a pre-study period of 60 min which allowed to attain steady-state insulin action. Steady state glucose disposal rate was determined during the last 60 minutes of the clamp. Example 4

Effect of EM-652.HC1, TSE 424, Lasofoxifene, LY 353381 and Raloxifene plasma insulin level on ovariertomized female rats.

URMA-r-45-00

We have running experiments showing the effectiveness of the following SERMs (EM-652.HC1, TSE 424, Lasofoxifene, LY 353381 and Raloxifene) on the plasma insulin level. For this purpose, tested compounds were a iministered by oral gavage for 20 days (0.5 mg/rat for each compound; 0.5 ml/rat) in 0.4% methylcellulose to ovariectomized female rats. Intact control, ovariectomized (OVX) control and OVX rats treated with 17β-estradiol (E₂) were used as reference.

TEST ANIMAL:

Species : Rattus norvegicus

Strain : Sprague-Dawley Rat (Crl:CD® (SD) BR VAF/Plus™)

Sex : Female

Body Weight : At onset of dosing, body weights were approximately 200-

225g. 1

- 28 -

Housing and maintenance

a) Housin :

The rats were housed individually in stainless steel cages of conventional design during the acclimation and study periods.

b) Temperature: Environmental conditions (temperature, humidity) in the rat room were recorded continuously using a computerized automated system. The targeted conditions were of 22 ± 3°C and 50 ± 20 % relative humidity.

c) Light-Dark Cycle:

The photoperiod were 10 hours of light and 14 hours of darkness. Lights were opened at 07:15, and closed at 17hl5.

d) Diet:

During acclimation period, rats received a certified rodent diet (Lab Diet # 5002, pellet) and tap water ad libitum while during study period, they received a high carbonhydrate diet (diet # 3) and tap water ad libitum. The diet were composed of (g/lOOg): Corn starch, 31.2; Dextrose, 31.2;

Casein, 20.0; corn oil, 6.4; dl-Methionine, 0.3; Vitamine mix, 1.0; ATN-76 mineral mix, 4.9; fiber, 5.0. Rats were fasted (with access to water only) around 07h00 the morning of their necropsy.

Randomization :

Rats were assigned to each group in order to have equivalent mean body weights. 6651

- 29

METHODS AND EXPERIMENTAL DESIGN

Test groups:

Seventy-seven rats were assigned to 8 groups of 9-10 rats for conduct of the study outlined below.

51

- 30 -

Animal preparation:

On day 0 of the study, rats from group 2 to 8 were ovariectomized (by bilateral flank incision) under Isoflurane anesthesia. Rats from group 1 were sham-operated. .

Body Weights

Rats were weighed on day 0 (surgery) and then, every 2 days during study period as well as on the day of necropsy.

Food Consumption

Food consumption were evaluated every 2 days.

Blood Samples

Insulin was measured on serum sample using the Linco RIA kit.

Res lts

Table 1

Claims

WHAT IS CLAIMED IS:

1. A method of treating or reducing the risk of acquiring hypertension other than pulmonary hypertension in warm-blooded animals in need a such treatment or reduction comprising administering a therapeutically effective amount of a selective estrogen receptor modulator.

2. A method of treating or reducing the risk of acquiring hypertension-related cardiovascular diseases in warm-blooded animals in need a such treatment or reduction comprising acinrinistering a therapeutically effective amount of a selective estrogen receptor modulator.

3. A method of treating or reducing the risk of acquiring insulin resistance in warm-blooded animals in need of such treatment or reduction comprising administering a therapeutically effective amount of a selective estrogen receptor modulator.

4. A method of treating or reducing the risk of acquiring insulin resistance-related diabetes in warm-blooded animals in need a such treatment or reduction comprising administering a therapeutically effective amount of a selective estrogen receptor modulator.

5. The method of any of claims 1 to 4 wherein said selective estrogen receptor modulator is administered to a human.

6. The method of any of the foregoing Claims 1 to 4 wherein the selective estrogen receptor modulator has a molecular formula with the following features :

b) a side^' chain possessing an aromatic ring and a tertiary arnine function or salt thereof.

7. A method of Claim 6 wherein the side chain is selected from the group consisting of :

and

8. The method of Claim 6 wherein the selective estrogen receptor modulator is selected from the group consisting of a benzothiophene derivatives, triphenylethylene derivatives, indole derivatives, benzopyran derivatives, and centchroman derivatives.

9. The method of Claim 6 wherein the selective estrogen receptor modulator is a benzothiophene derivative compound of the following formula:

wherein Ri and R2 are independently selected from the group consisting of : hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl ;

wherein Rs and R4 are either independently selected from the group consisting of : C1-C4 alkyl, or wherein Rj, Rj and the nitrogen to which they are bound, together are any structure selected from the group consisting of pyrrolidino, dimethyl-1- pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino and morpholino; wherein A is selected from the group consisting of -CO-, -CHOH, and -CH2-;

wherein B is selected from the group consisting of phenylene, pyridylidene, and -cydoC4H2N2-.

10. The method of Claim 6 wherein the selective estrogen receptor modulator is selected from the group consisting of Raloxifene, LY 353381 and LY 335563.

11. The method of Claim 6 wherein the selective estrogen receptor modulator is a triphenylethylene derivative compound of the following formula :

wherein D is -OCH2CH₂N(R₃)R4 or -CH=CH-COOH ( Rs and R either being independently selected from the group consisting of C1-C4 alkyl, or R3, R4, and the nitrogen atom to which they are bound, together 1

36 -

being a ring structure selected from the group consisting of pyrrolidino, dimethyl-1- pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexame yleneimino and morpholino);

wherein E and K are independently hydrogen or hydroxyl ; wherein J is hydrogen or halogen.

12; The method of Claim 11 wherein selective estrogen receptor modulator is Tamoxifen, OH-tamoxifen, Droloxifene, Toremifene, Iodoxifene, and GW5638.

13. The method of Claim 6 wherein the selective estrogen receptor modulator is an indole derivative compound of the following formula:

wherein D is selected from the groups consisting of -OCH2CH₂N(R₇)R8 , -CH=CH-CO N(R₇)Rs , -CC-(CH₂)n-N(R )R8 (R₇ and Re either being independently selected from the group consisting of Ci-Cβ alkyl, or R₇, Re and the nitrogen atom to which they are bound, together being a ring structure selected from the group consisting of pyrrolidino, dimethyl-1- pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholino);

wherein X is selected from the group consisting of : hydrogen, and

C1-C6 alkyl;

wherein Ri, R₂ R3, R4, Rs, and Re are independently selected from the group consisting of : hydrogen, hydroxyl, G-Cό alkyl, and a moiety converted in vivo in hydroxyl.

14. The method of Claim 6 wherein the selective estrogen receptor modulator is a centchroman derivative compound of the following formula :

1

- 38 -

wherein Ri and R2 are independently selected from the group consisting of : hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl;

wherein R5 and Re are independently hydrogen or Ci-Cβ alkyl ;

wherein D is -OCH₂CH2N(R3)P4 ( R3 and R4 either being independently selected from the group consisting of Ci-Q alkyl, or R3, R₄ and the nitrogen atom to which they are bound, together being a ring structure selected from the group consisting of pyrrolidino, dimethyl-1- pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholino).

15. The method of Claim 14 wherein the centchroman derivative is Levormeloxifene (3,4-trans-2,2-dimethyl-3-phenyl-4-[4-(2-(2- (pyrrolidin-l-yl)ethoxy)phenylJr7-methoxychroman).

16. The method of ^Qaim 6 wherein the selective estrogen receptor modulator is a compound, or salt thereof having the molecular formula :

wherein Ri and R2 are independently hydrogen, hydroxyl or a moiety which is converted to hydroxyl in vivo ;

wherein Z is selected from the group consisting of -O-, -NH-, -S-, and -CH2-.;

wherein theRlOO is a bivalent moiety which distances L from the B-ring by 4-10 intervening atoms ;

wherein L is a bivalent or rrivalent polar moiety selected from the group of -SO-, -CON-, -N<, and -SON< ;

wherein Gi is selected from the group consisting of hydrogen, a Ci to C5 hydrocarbon or a bivalent moiety which joins G₂ and L to form a 5- to 7- membered heterocydic ring, and halo or unsaturated derivatives of the foregoing.

wherein G is either absent or selerted from the group consisting of hydrogen, a Ci to C5 hydrocarbon or a bivalent moiety which joins Gi and L to form a 5-to 7- membered heterocydic ring, and halo or unsaturated derivatives of the foregoing.

wherein G3 is selected from the group consisting of hydrogen, methyl and ethyl.

17. The method of Claim 16, wherein compound or salt is a benzopyran derivative that is optically active due to a majority of its enantiomer having an absolute configuration S on carbon 2 said enantiomer of S configuration having the molecular structure:

5^!

wherein Ri and R2 are independently selected from the group consisting of hydroxyl and a moiety convertible in vivo to hydroxyl;

wherein R3 is a spedes selerted from the group consisting of saturated, unsaturated or substituted pyrrolidinyl, saturated, unsaturated or substituted piperidino, saturated, unsaturated or substituted piperidinyl, saturated, unsaturated or substituted morpholino, nitrogen-containing cyclic moiety, mtrogen-containing polycyclic moiety, and NRaRb (Ra and Rb being independently hydrogen, straight or branched C1-C6 alkyl, straight or branched C2-C6 alkenyl, and straight or branched C2-C6 alkynyl);

wherein R4 is selected from the group consisting of hydrogen and methyl.

18. The method of Claim 17 wherein said compound or salt substantially lacks (ZR)-enantiomer.

51

42

19. The method of Claim 18 where said selective estrogen receptor modulator is selerted from the group consisting of:

20. The method of Claim 18 wherein the benzopyran derivative is a salt of an add selerted from the group consisting of acetic add, adipic add, benzenesulfonic add, benzoic add, camphorsulfonic add, dtric add, fumaric add, hydroiodic add, hydrobromic add, hydrochloric add, hydrochlorothiazide add, hydroxy-naphthoic add, lactic add, maleic add, methanesulfonic add, methylsulfuric add, 1,5- naphthalenedisulfonic add, nitric add, palmitic add, pivalic add, phosphoric add, propionic add, sucdnic add, sulfuric add, tartaric add, terephthalic add, p-toluenesulfonic add, and valeric add.

21. The method of Claim 20 wherein the add is hydrochloric add.

22. The method of any of the foregoing Claims 1 to 4 wherein said selective estrogen receptor modulator is:

23. The method of daim 1, further comprising administering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,

4-androstene-3,17-dione and any prodrug that is converted in vivo any into the foregoing precursors.

24. The method of daim 2, further comprising administering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,

25. The method of daim 3, further comprising administering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,

26. The method of daim 4, further comprising aciministering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,

27. A method of treating or reducing the risk of acquiring hypertension in warm-blooded animals in need of such treatment or reduction comprising administering a therapeutically effective amount of a benzopyran derivative that is optically active due to a majority of its enantiomer having an absolute configuration S on carbon 2, said enantiomer of S configuration having the molecular structure:

wherein Ri and R2 are independently selerted from the group consisting of hydroxyl and a moiety convertible in vivo to hydroxyl;

wherein R3 is a moiety selerted from the group consisting of saturated, unsaturated or substituted pyrrolidinyl, saturated, unsaturated or substituted piperidino, saturated, unsaturated or substituted piperidinyl, saturated, unsaturated or substituted morpholino, nitrogen-containing cydic moiety, nitrogen-containing polycydic moiety, and NRaRb (Ra and Rb being independently hydrogen, straight or branched Ci-Cβ alkyl, straight or branched C2-C6 alkenyl, and straight or branched C2-C6 alkynyl); 1

- 46 -

wherein R is selected from the group consisting of hydrogen and methyl.

28. The method of daim 27 wherein said hypertension is not pulmonary hypertension.

29. The method of daim 27 wherein said benzoplyras is EM 652.HC1.

30. The method of daim 27 further comprising aclministering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,

31. A pharmaceutical composition comprising both a SERM and administering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,l 7β-diol, 4-androstene-3,17-dione and any prodrug that is converted in vivo any into the foregoing precursors.

32. A kit comprising both a SERM and administering a therapeutically effective amount of one sex steroid precursor selerted from the group consisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,