Field of the Invention

The present invention relates to therapeutic agents that bind to the estrogen receptor and have been found to be useful in the treatment of osteoporosis.

Background of the Invention

The decrease in estrogen that occurs at the menopause is an important etiological factor in the increased incidence of osteoporotic fractures and cardiovascular disease in postmenopausal women. Although postmenopausal bone loss can be prevented by estrogen replacement therapy (ERT), unopposed ERT increases the risk of endometrial cancer. An ideal therapy would retain the desirable skeletal and cardiovascular effects of estrogen without having the unwanted effects on reproductive tissues. Tamoxifen is an antiestrogen that has been shown to lower cholesterol levels and protect against bone loss in postmenopausal women. Tamoxifen is also effective in the ovariectomized rat model of osteoporosis. However, tamoxifen has been shown to have unwanted side effects, in particular by causing endometrial hypeφlasia and endometrial cancer. See 1. Love RR, Wiebe DA, Newcomb PA, Cameron L, Leventhal H, Jordan VC, Feyzi J, DeMets DL. (19 1). Effects of tamoxifen on cardiovascular risk factors in postmenopausal women. Annals of Internal Medicine, 115, 860-864. 2. Love RR, Mazess RB, Barden HS, Epstein S, Newcomb PA, Jordan VC, Carbone PP, DeMets DL. ( 1992). Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. New England Journal of Medicine, 326, 852,856. 3. Turner RT, Wakely GK, Hannon KS, Bell NH. (188). Tamoxifen inhibits osteoclast-mediated resoφtion of trabecular bone in ovarian hormone-deficient rats. Endocrinology, 122, 1146- 1 150.

Summary of the Invention

This invention provides a method for the prevention and treatment of postmenopausal diseases without having an overt uterotrophic effect. The method comprises administering to a human in need thereof an effective amount of a compound of formula I

wherein X represents 3- or 4- iodo or bromo and the Rl and R^ symbols, which may be the same or different, represent C1.3 alkyl, especially methyl or ethyl, groups or R! represents a hydrogen atom and R^ a Cj_3 alkyl group or R 1 and R^ together with the nitrogen atom to which they are attached represent a saturated heterocyclic group, typically having 5 or 6 ring atoms, especially a pyrrolidino, piperidino, 4- methylpiperidino or moφholino group, and their pharmaceutically acceptable acid addition salts.

Detailed Description of the Invention

The present invention is a therapeutic method for treating postmenopausal diseases with a group of compounds that have been previously prepared and evaluated as effective in the treatment of estrogen receptor-positive breast cancer. These compounds are described in formula I above and in U.S. patent 4,839,155.

The preferred compound for the described method of treatment is

(E)-l-[2-[4-[ l-(4-lodophenyl)-2-phenyl- l-butenyl]phenoxy]pyrrolidine Such compounds are known to bind to the estrogen receptor and to cause either estrogen agonist or antagonist effects depending on the tissue being studied.

The term "postmenopausal diseases" refers to osteoporosis and atherosclerotic cardiovascular diseases such as myocardial infarction and stroke and an increase in plasma cholestrerol. The method of this invention is useful in

preventing bone loss and in producing a plasma lipid profile that is associated with a reduced risk of atherosclerosis.

The ability to prevent bone loss is assessed by studies in an ovariectomized rat model of osteoporosis and by studies in postmenopausal women.

Histomoφhometric studies have shown that when the ovaries are removed from adult female rats, progressive bone loss occurs in the proximal tibial metaphysis. Three months after ovariectomy (OVX), 60-70% of cancellous bone has been removed through an increase in bone turnover in which excessive bone resoφtion predominates. Bone loss also occurs in the lumbar spine although at a slower rate. OVX-induced bone loss in rats (which can be completely prevented by replacement doses of estrogen) forms the basis of the most widely used and best characterized animal model of osteoporosis.

Sprague-Dawley rats were used at the age of 7-8 months. Baseline bone mineral density (BMD) was measured by dual energy x-ray absoφtiometry (DXA) in the lumbar 3-6 region of the spine and the proximal tibial metaphysis. The rats were then apportioned into groups of 8- 10 having approximately the same mean and standard deviation values for lumbar BMD. Groups of rats were bilaterally ovariectomized (OVX) and in each experiment one group was sham operated.

Idoxifene was prepared for oral dosing as a suspension in a 1 % aqueous solution of carboxymethyl cellulose. Rats were dosed by oral gavage once daily. In each experiment one OVX group and the sham group received oral dosing vehicle by gavage once daily. Dosing commenced on the day following surgery.

Plasma cholesterol levels were determined after 2 weeks of treatment.

Lumbar and tibial BMDs were measured at 1 month intervals. The animals were sacrificed, the uteri removed and the wet weight determined. Tibiae were collected post mortem and embedded and sectioned for histomoφhometry (15). Cancellous bone area and perimeter were measured in the secondary spongiosa, 1.2 mm from the growth plate. Cancellous bone area (Cn.B. Ar) was expressed as a percent of the medullary area. The secondary structural parameters, trabecular width, trabecular number and trabecular separation, were calculated from the primary area and perimeter measurements using equations developed by Parfitt et al.

Initial dose-ranging study of the effect of idoxifene on bone loss, plasma cholesterol and uterine weight in the ovariectomized rat model of osteoporosis.

The aim of this study was to determine the optimal dose of idoxifene for the prevention of bone loss in the OVX rat model of osteoporosis.

BMD was measured after 1, 2 and 3 months of treatment. In addition to removing the tibiae for histomoφhometry, the femora and vertebrae (LI and 2) were removed for the ex vivo measurement of BMD (femur only) and mechanical testing. Idoxifene was dosed at 2, 8, 40 and 200 micrograms/kg d.

The no-effect dose of idoxifene was 2 μg/kg according to all measured parameters.

Only the 200 μg/Tcg dose of idoxifene caused a significant prevention of OVX-induced decrease in BMD in the lumbar spine. This dose was effective and caused 100% inhibition of bone loss at 1 month. In the lumbar spine after 3 months of treatment, idoxifene at 200 μg/kg caused about a 50% inhibition of bone loss.

In the proximal tibia at 1 month, doses of idoxifene from 8-200 μg/kg caused about a 50% inhibition of bone loss. This degree of protection had fallen to about 25% at three months which was not significant. This suggested that 200 μg/kg is not the optimal dose of idoxifene at this skeletal site.

Bone mineral density measured ex vivo showed that 200 μg/kg idoxifene maintained proximal femoral BMD at the level of sham controls. This dose also maintained femoral mid-shaft medullary cross-sectional area at the level of sham controls, an indication that idoxifene prevents cortical as well as cancellous bone loss. Idoxifene did not adversely affect the mechanical strength of either the femoral diaphysis in a 3-point bending test or the L2 vertebral body in an axial compression test.

Histomoφhometry revealed a small but non-significant effect of idoxifene on proximal tibial cancellous bone area after 3 months of treatment , which was in agreement with BMD measurements. There were no differences between any of the groups with respect to trabecular width. Despite its lack of effect on tibial cancellous bone area in the long term, activity of idoxifene at doses as low as 40 μg/kg were apparent with respect to trabecular number and separation.

Idoxifene (200 μg/kg) significantly reduced plasma cholesterol (figure 5). After 3 months of treatment, all doses of idoxifene caused a very slight but statistically significant increase in uterine weight.

Dose-refining study of the effect of idoxifene on bone loss, plasma cholesterol and uterine weight in the ovariectomized rat model of osteoporosis.

The aim of this study was to determine the optimal dose of idoxifene for the prevention of bone loss in the OVX rat model of osteoporosis. BMD was measured at 1 month and treatment continued for a further 2 weeks before collection of the uteri and tibiae.

Idoxifene at 200 and 500 μg/kg completely prevented bone loss in the lumbar spine. There was no significant effect of idoxifene at 1000 μg/kg in the lumbar spine. Idoxifene at 200-1000 μg/kg completely prevented bone loss in the proximal tibial metaphysis.

Histomoφhometry revealed that idoxifene optimally prevented OVX- induced loss of cancellous bone at 500 μg/kg. Prevention of OVX-induced reduction in trabecular width occured significantly at 200 and 500 μg/kg idoxifene. Trabecular number was significantly preserved at 500 and 1000 μg/kg idoxifene. Idoxifene at 200- 1000 μg/kg significantly prevented the OVX-induced increase in trabecular separation.

All doses of idoxifene significantly reduced plasma cholesterol levels (figure 1 1). There was no effect of idoxifene on uterine wet weight at any of the doses tested.

The dose 500 μg/kg of idoxifene was consistently identified by all measured parameters as optimal after 6 weeks of treatment in the OVX rat.

Over a 6 week treatment period, the optimal dose of idoxifene was found to be 500 μg/kg. The minimally effective dose for prevention of bone loss in the spine was 200 μg/kg and 100 μg/kg for its cholesterol-lowering effect. In summary, the bone-protective and cholesterol-lowering effects of idoxifene are useful in the prevention of postmenopausal diseases without having an overt uterotrophic effect.

Three different doses of idoxifene (2.5, 5 and lOmg/day) were compared with placebo in a study of three months duration in postmenopausal women. These

women had evidence of low bone mineral density at the beginning of the study. The presence of an effect on bone that would be consistent with a reduction in the rate of bone loss was detected by measuring changes in biochemical markers of bone resoφtion (urinary collagen crosslink excretion, measured as excretion of C- telopeptides, free crosslinks and total crosslinks) and bone formation (serum osteocalcin).

The observed dose related reduction in the levels of these biochemical markers (see below) is representative of a reduction in bone turnover and is consistent with the effects of estrogen on bone turnover in postmenopausal women. All changes are described as percentage change from the baseline value. Statistically significant differences from placebo are designated by the following notation: *=p<0.01, **=p<0.001.

Placebo 2.5 5.0 10.0

C-Telopeptide 9.8 (4.3) -0.2 (4.3) -6.8 (5.2)* -16.0 (6.3)**

Free Crosslinks 4.4 (3.0) 2.6 (3.0) -1.5 (2.4) -6.4 (2.3)*

Total Crosslinks 2.4 (2.9) -4.8 (2.7) -2.4 (4.8) -12.6 (2.6)**

Osteocalcin -1 (0.01) -6 (0.02) -9 (0.02)** -17 (0.02)**

Changes in the levels of different lipid parameters, fibrinogen and other coagulation/fibrinolysis parameters were also measured before and after treatment as an index of the likely effect of idoxifene on the risk of cardiovascular disease. The results of this study are described below.

Placebo 2.5 5.0 10.0

Total chol 0.5 (1.1) -0.9 (1.1) -4.2 (1.2)* -9.8 (1.1)**

LDL-chol 1.3 (1.5) -1.3 (2.0) -4.7 (1.7)* -15.2 (1.6)**

HDL-chol 2.4 (1.4) 2.7 (1.5) -1.4 (1.5) 0.8 (1.7)

HDL/LDL ratio 2.2 (1.6) 7.1 (2.6) 5.2 (2.1) 21.7 (3.1)** lipoprotein (a) 5.0 (3.0) -2.0 (6.0) 1.0 (4.0) -6.0 (4.0) triglycerides 1.3 (3.8) 1.8 (3.9) -3.2 (3.6) 3.8 (3.8)

Fibrinogen 5.8 (3.0) - 1.7 (2.4) -8.3 (3.1)* -16.9 (2.4)**

D-dimer 0.0 (3.1) 4.7 (4.3) -2.1 (2.7) -7.1 (2.7)

Factor VII 5.4 (2.4) - 10.1 (2.4)** -9.6 (2.1 )** - 1 1.1 ( 1.6)**

The compounds of the instant invention and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example, polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or coloring agent.

A composition in the form of a tablet can be prepared using any suitable pharmacuetical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.

The compounds of the instant invention and their pharmaceutically acceptable salts which are active when administered parenterally (i.e. by injection of infusion) can be formulated as solutions or suspensions. A composition for parenteral administration will generally consist of a solution or suspension of the active ingredient in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration. A typical suppository composition comprises a compound of the instant invention or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or coca butter or other low melting vegetable or synthetic waxes or fats. A typical transdermal formulation comprises a conventional aqueous or non- aqueous vehicle, for example, a cream, ointment lotion or paste or in the form of a medicated plaster, patch or membrane.

For topical administration, the pharmaceutical compositions adapted include solutions, suspensions, ointments, and solid inserts. Typical pharmaceutically acceptable carriers are, for example, water, mixtures of water and water-miscible solvents such as lower alkanols or vegetable oils, and water soluble ophthalmologically acceptable non-toxic polymers, for example, cellulose

derivatives such as methyl cellulose. The pharmaceutical preparation may also contain non-toxic auxiliary substances such as emulsifying, preserving, wetting and bodying agents, as for example, polyethylene glycols; antibacterial components such as quaternary ammonium compounds; buffering ingredients such as alkali metal chloride; antioxidants such as sodium metabisulfite; and other onventional . ingredients such as sorbitan monolaurate.

Preferably the composition is in unit dose form. Doses of the compounds of the instant invention in a pharmaceutical dosage unit will be an efficiacious, non- toxic quantity selected from the range of .01 - 200 mg/kg of active compound, preferably .1 - 100 mg/kg. The selected dose is administered to a human patient in need of treatment or prevention of osteoporosis or in the lowering of plasma cholesterol or prevention of cardiovascular disease from 1-6 times daily, orally, rectally, topically, by injection, or continuously by infusion. Oral dosage units for human administration preferably contain from 10 to 500 mg of active compound. Lower dosages are used generally for parenteral administration. Oral administration is used when safe, effective, and convenient for the patient.

No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.

Example \ An oral dosage form for administering orally active Formula (I) compounds is produced by screening, mixing and filling into hard gelatin capsules the ingredients in proportions, for example, as shown below.

Ingredients Amounts (E)- 1 -[2-[4-[ 1 -(4-lodophenyl)-2-phenyl- 1 -butenyl] phenoxy]pyrrolidine 100 mg magnesium stearate 10 mg lactose 100 mg

Exarηpje 2,

The sucrose calcium sulfate dihydrate and orally active Formula (I) compounds are mixed and granulated with a 10% gelating solution. The wet granules are screened, dried, mixed with the starch, talc and stearic acid, screened and compressed into a tablet.

Ingredients Amounts

(E)-l-[2-[4-[ l -(4-lodophenyl)-2-phenyl- l-butenyl]

phenoxyjpyrrolidine 75 mg calcium sulfate dihydrate lOO mg sucrose 15 mg starch 8 mg talc 4 mg stearic acid 2 mg

(E)- 1 -[2-[4-[ 1 -(4-lodophenyl)-2-phenyl- 1 -butenyi ]phenoxy Jpyrrolidine, 50 mg, is dispersedin 25 ml of normal saline to prepare an injectable preparation.

Example 4 This experiment was conducted to compare the mechanism of action of idoxifene and raloxifene in osteoblasts. Using a construct containing an estrogen response element (ERE) upstream of a luciferase reporter gene (described below) it has now been shown that idoxifene, like estrogen, is a pure agonist through the ERE in osteoblasts. The potency of agonist action was similar between the natural steroid hormone estrogen and idoxifene. Raloxifene, at the same concentrations as idoxifene (0.01 to IOμM), gave an extremely weak signal, which was of similar magnitude to the to vehicle control, through the ERE.

Competition experiments were performed to confirm the mechanism of action through this response element. Raloxifene inhibited the agonist activity of both estrogen and idoxifene through the ERE. At doses of ligand (either estrogen or idoxifene ) at lOOnM, there was a maximum agonist response. Co-treatment with 500nM raloxifene reduced the reporter gene activity to vehicle control levels. In contrast, idoxifene at 500nM did not diminish the maximal agonist action of estrogen at lOOnM in osteoblasts. If submaximal concentrations of idoxifene and estrogen were used (< lOOnM) there was additive actions of the two agonists through the ERE in osteoblasts.

Experimental Procedures

Cells were seeded in either 6-well plates at 1.5 x lO^cells/well or in 24-well plates at 1.5 x 10^ cells/well in phenol red-free medium. A DNA construct comprising a mouse mammary tumor virus promoter in which the glucocorticoid response elements have been replaced with five copies of a 33-base pair vitellogenin estrogen response element was employed. This is upstream of the Luciferase reporter gene (MMTV-ERE-Luc) "(Wen, D.X., Y-F. Xu, M. . Goldman and P. McDonnell. 1994. The A and B isoforms of the human progesterone receptor operate through distinct signalling pathways within target cells. Molec. Cell. Biol. 14: 8356-8364)". The renilla-Luciferase vector was used to correct for transfection efficiency using the dual-luciferase detection method (Promega, Madison, WI). DNA was introduced into rat osteosarcoma (Ros 17/2.8) cells by the lipofectin method (Life Technologies, Gaithersburg, MD). Cells were co-transfected with 2 μg per well in 6-well plates and 140ng per well in 24-well plates of MMTV ERE-Luc and 25ng of the control renilla-Luciferase vector (pRL-CMV). Transfection efficiency was corrected for by co-transfection with a renilla-Luciferase vector, which utilizes a different substrate, coelenterazine, for its bioluminescent readout (Promega, Madison WI). Cells were incubated overnight. Transfection medium was then removed and cells were incubated for 48 with or without hormones. Cells were washed in phosphate buffered saline and then lysed with 500μl/well lx passive lysis buffer (PLB) for 15 minutes while roc-king sample on a rocking platform. Lysates were centrifuged for 30secs. at 12,000g and the clear lysate was transferred to a tube prior to reporter enzyme analysis. Samples(20μl) were transfered to a 96 well luminescence detection plate and reacted with lOOμl of each assay reagent (Promega, Madison, WI). Each assay reagent was injected by a microlumat LB96P luminometer (Wallac,

Gaithersburg, MD), which measured luciferase activity. Luciferase activity provides a surrogate of transcriptional activation of estrogen responsive gene that contains the estrogen response element (ERE). Therefore upregulation of luciferase activity is indicative of an agonist effect, whereas down-regulation indicates antagonism through the ERE.

Discussion;

Idoxifene is an agonist through the estrogen response element (ERE) in osteoblasts. In contrast to idoxifene, raloxifene is an antagonist through the ERE in osteoblasts at the doses tested, which suggests a distinct mechanism for the bone sparing effects seen with raloxifene. Thus, raloxifene is able to exert its biological

effect through a non-ERE containing sequence present on the 5'-untranslated region of the human TGFβ3 promoter. In the same cellular system, raloxifene inhibited the ERE -containing vitellogenin promoter expression and exhibited therefore pure estrogen antagonism. The raloxifene response element (Yang, N, N., Venugopalan, M., Hardikar, S., and Glasebrook, A. (1996) Identification of an estrogen response element activated by metabolites of 17beta-estradiol and raloxifene Science 273: 1222-1225) is not present on the same genes as the ERE suggesting modulation through this response element will result in effects on different genes. This distinguishes the mechanism of action of idoxifene from that of the selective- estrogen receptor modulator (SERM) raloxifene and aligns idoxifene to the more classic estrogenic type mechanism exerting its biological agonist effect in osteoblasts through the ERE. The effects of idoxifene and estrogen are specific to reporter gene constructs that carry the classical ERE. This system was shown to be sensitive to cell specific factors and is thus a valid model for effects on endogenous gene transcription.