METHOD OF TREATING POST MENOPAUSAL DISEASES, INCLUDING OSTEOPOROSIS

METHODE DE TRAITEMENT DES MALADIES POST-MENOPAUSIQUES, DONT L'OSTEOPOROSE

English Abstract

A novel method for treating osteoporosis is described. Idoxifene is the preferred compound.

French Abstract

L'invention concerne une nouvelle méthode destinée à traiter l'ostéoporose. L'idoxifène est le composé préféré.

Claims

What is claimed is:

1. A method of preventing and treating postmenopausal diseases which
comprises administering to a subject in need thereof an effective amount of a
compound of formula I.

2. A method according to Claim 1 wherein the postmenopausal disease
is osteoporosis

3. A method according to Claim 1 wherein the compound of formula I is
(E)-1-[2-[4-[1-(4-Iodophenyl)-2-phenyl-1-butenyl]phenoxy]ethyl]pyrrolidine.

4. The use of a compound of formula I in the manufacture of a
medicament for use in the treatment of postmenopausal diseases.

5. A use according to Claim 4 wherein the postmenopausal disease is
osteoporosis.

6. A use according to Claim 1 wherein the compound of formula I is
(E)-1-[2-[4-[1-(4-Iodophenyl)-2-phenyl-1-butenyl]phenoxy]ethyl]pyrrolidine.

-12-

Description

WO 98/09519101520253035CA 02264775 1999-03-05METHOD OF TREATING POST MENOPAUSAL DISEASES. INCLUDING OSTEOPOROSISField of the InventionThe present invention relates to therapeutic agents that bind to the estrogenreceptor and have been found to be useful in the treatment of osteoporosis.B u the InventiThe decrease in estrogen that occurs at the menopause is an importantetiological factor in the increased incidence of osteoporotic fractures andcardiovascular disease in postmenopausal women. Although postmenopausal boneloss can be prevented by estrogen replacement therapy (ERT), unopposed ERTincreases the risk of endometrial cancer. An ideal therapy would retain the desirableskeletal and cardiovascular effects of estrogen without having the unwanted effectson reproductive tissues. Tamoxifen is an antiestrogen that has been shown to lowercholesterol levels and protect against bone loss in postmenopau-sal women.Tamoxifen is also effective in the ovariectornized rat model of osteoporosis.However, tamoxifen has been shown to have unwanted side effects, in particular bycausing endometrial hyperplasia and endometrial cancer. See 1. Love RR, WiebeDA, Newcomb PA, Cameron L, Leventhal H, Jordan VC, Feyzi J, DeMets DL.(1991). Effects of tamoxifen on cardiovascular risk factors in postmenopausalwomen. 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 breastcancer. New England Journal of Medicine, 326, 852,856. 3. Turner RT, WakelyGK, I-Iannon KS, Bell NI-I. ( 188). Tamoxifen inhibits osteoclast-mediated resorptionof trabecular bone in ovarian hormone—deficient rats. Endocrinology, 122, 1146-1150.Summa_ry of the InventionThis invention provides a method for the prevention and treatment ofpostmenopausal diseases without having an overt uterotrophic effect. The methodcomprises administering to a human in need thereof an effective amount of acompound of formula IPCT/U S97/ 15474 ‘CA 02264775 1999-03-05W0 98l095l9 PCT/US97/15474 -l/ROCHECHZQ a[1 O “‘/C=CCZHS x(I)wherein X represents 3- or 4- iodo or bromo and the R1 and R2 symbols, which maybe the same or different, represent C1_3 alkyl, especially methyl or ethyl, groups or5 R1 represents a hydrogen atom and R2 a C 1-3 alkyl group or R1 and R2 togetherwith the nitrogen atom to which they are attached represent a saturated heterocyclicgroup, typically having 5 or 6 ring atoms, especially a pyrrolidino, piperidino, 4-methylpiperidino or morpholino group, and their pharmaceutically acceptable acidaddition salts.10Detailed Description of the InventionThe present invention is a therapeutic method for treating postmenopausaldiseases with a group of compounds that have been previously prepared and15 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 isO\__0 O “C3 O20 I(E)-I-[2-[4-[1-(4-lodophenyl)-2-phenyl-l-butenyl]phenoxy]pyrrolidineSuch compounds are known to bind to the estrogen receptor and to causeeither estrogen agonist or antagonist effects depending on the tissue being studied.The term "postmenopausal diseases" refers to osteoporosis and25 atherosclerotic cardiovascular diseases such as myocardial infarction and stroke andan increase in plasma cholestrerol. The method of this invention is useful in-2-W0 98/095191015202530CA 02264775 1999-03-05PCT/US97/15474 ‘preventing bone loss and in producing a plasma lipid profile that is associated with areduced risk of atherosclerosis.The ability to prevent bone loss is assessed by studies in an ovariectomizedrat model of osteoporosis and by studies in postmenopausal women.Histomorphometric studies have shown that when the ovaries are removedfrom adult female rats, progressive bone loss occurs in the proximal tibialmetaphysis. Three months after ovariectomy (OVX). 60-70% of cancellous bone hasbeen removed through an increase in bone turnover in which excessive boneresorption predominates. Bone loss also occurs in the lumbar spine although at aslower rate. OVX—induced bone loss in rats (which can be completely prevented byreplacement doses of estrogen) forms the basis of the most widely used and bestcharacterized animal model of osteoporosis.Sprague-Dawley rats were used at the age of 7-8 months. Baseline bonemineral density (BMD) was measured by dual energy x-ray absorptiometry (DXA) inthe lumbar 3-6 region of the spine and the proximal tibial metaphysis. The rats werethen apportioned into groups of 8- 10 having approximately the same mean andstandard deviation values for lumbar BMD. Groups of rats were bilaterallyovariectomized (OVX) and in each experiment one group was sham operated.Idoxifene was prepared for oral dosing as a suspension in a 1% aqueoussolution of carboxymethyl cellulose. Rats were dosed by oral gavage once daily. Ineach experiment one OVX group and the sham group received oral dosing vehicle bygavage 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 weresacrificed, the uteri removed and the wet weight determined. Tibiae were collectedpost mortem and embedded and sectioned for histomorphometry (15). Cancellousbone area and perimeter were measured in the secondary spongiosa, 1.2 mm fromthe growth plate. Cancellous bone area (Cn.B.Ar) was expressed as a percent of themedullary area. The secondary structural parameters, trabecular width, trabecularnumber and trabecular separation, were calculated from the primary area andperimeter measurements using equations developed by Parfitt et al.WO 98/095191015202530CA 02264775 1999-03-05Initial dose-ranging study of the effect of idoxifene on bone loss, plasmacholesterol and uterine weight in the ovariectomized rat model of osteoporosis.The aim of this study was to determine the optimal dose of idoxifene for theprevention of bone loss in the OVX rat model of osteoporosis.BMD was measured after 1, 2 and 3 months of treatment. In addition toremoving the tibiae for histomorphometry, the femora and vertebrae (L1 and 2) wereremoved for the ex vivo measurement of BMD (femur only) and mechanical testing.idoxifene was closed at 2, 8, 40 and 200 micrograms/kg/d.The no-effect dose of idoxifene was 2 pg/kg according to all measuredparameters.Only the 200 pg/kg dose of idoxifene caused a significant prevention ofOVX—induced decrease in BMD in the lumbar spine. This dose was effective andcaused 100% inhibition of bone loss at 1 month. In the lumbar spine after 3 monthsof treatment, idoxifene at 200 pg/kg caused about a 50% inhibition of bone loss.In the proximal tibia at 1 month, doses of idoxifene from 8-200 ug/kg causedabout a 50% inhibition of bone loss. This degree of protection had fallen to about25% at three months which was not significant. This suggested that 200 pg/kg is notthe optimal dose of idoxifene at this skeletal site.Bone mineral density measured ex vivo showed that 200 ug/kg idoxifenemaintained proximal femoral BMD at the level of sham controls. This dose alsomaintained femoral mid-shaft medullary cross-sectional area at the level of shamcontrols, an indication that idoxifene prevents cortical as well as cancellous boneloss. Idoxifene did not adversely affect the mechanical strength of either the femoraldiaphysis in a 3-point bending test or the L2 vertebral body in an axial compressiontest.Histomorphometry revealed a small but non-significant effect of idoxifene onproximal tibial cancellous bone area after 3 months of treatment , which was inagreement with BMD measurements. There were no differences between any of thegroups with respect to trabecular width. Despite its lack of effect on tibial cancellousbone area in the long term, activity of idoxifene at doses as low as 40 pg/kg wereapparent with respect to trabecular number and separation.-4-PCT/US97/15474 ‘W0 98/095191015202530CA 02264775 1999-03-05PCT/U S97/ 15474 'Idoxifene (200 pg/kg) significantly reduced plasma cholesterol (figure 5).After 3 months of treatment, all doses of idoxifene caused a very slight butstatistically significant increase in uterine weight.Dose-refining study of the effect of idoxifene on bone loss, plasma cholesteroland uterine weight in the ovariectomized rat model of osteoporosis.The aim of this study was to determine the optimal dose of idoxifene for theprevention of bone loss in the OVX rat model of osteoporosis. BMD was measuredat l month and treatment continued for a further 2 weeks before collection of theuteri and tibiae.Idoxifene at 200 and 500 pg/kg completely prevented bone loss in the lumbarspine. There was no significant effect of idoxifene at 1000 pg/kg in the lumbarspine. Idoxifene at 200-1000 pg/kg completely prevented bone loss in the proximaltibial metaphysis.Histomorphometry revealed that idoxifene optimally prevented OVX-induced loss of cancellous bone at 500 pg/kg. Prevention of OVX-induced reductionin trabecular width occured significantly at 200 and 500 pg/kg idoxifene. Trabecularnumber was significantly preserved at 500 and 1000 pg/kg idoxifene. Idoxifene at200-1000 pg/kg significantly prevented the OVX-induced increase in trabecularseparation.All doses of idoxifene significantly reduced plasma cholesterol levels (figurel 1). There was no effect of idoxifene on uterine wet weight at any of the dosestested.The dose 500 pg/kg of idoxifene was consistently identified by all measured7 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 tobe 500 pg/kg. The minimally effective dose for prevention of bone loss in the spinewas 200 pg/kg and 100 pg/kg for its cholesterol-lowering effect. In summary, thebone-protective and cholesterol-lowering effects of idoxifene are useful in theprevention of postmenopausal diseases without having an overt uterotrophic effect.Three different doses of idoxifene (2.5, 5 and 10mg/day) were compared withplacebo in a study of three months duration in postmenopausal women. These-5-WO 98/09519101520CA 02264775 1999-03-05PCT/US97/15474 ‘women had evidence of low bone mineral density at the beginning of the study. Thepresence of an effect on bone that would be consistent with a reduction in the rate ofbone loss was detected by measuring changes in biochemical markers of boneresorption (urinary collagen crosslink excretion, measured as excretion of C-telopeptides, free crosslinks and total crosslinks) and bone formation (serumosteocalcin).The observed dose related reduction in the levels of these biochemical markers (seebelow) is representative of a reduction in bone turnover and is consistent with theeffects of estrogen on bone turnover in postmenopausal women. All changes aredescribed as percentage change from the baseline value. Statistically significantdifferences from placebo are designated by the following notation: *=p<0.0l,**=p<0.00l.Placebo 2.5 5.0 10.0C-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) 1 -6.4 (2.3)*Total Crosslinks 2.4 (2.9) -4.8 (2.7) -2.4 (4.8) -12.6 (2.6)**Osteocalcin -l (0.01) -6 (0.02) -9 (0.02)** -17 (0.02)**Changes in the levels of different lipid parameters, fibrinogen and othercoagulation/fibrinolysis parameters were also measured before and after treatment asan index of the likely effect of idoxifene on the risk of cardiovascular disease. Theresults of this study are described below.Placebo 2.5 5.0 10.0Total chol 0.5(1.l) -0.9(1.l) -4.2 (1.2)* -9.8 (l.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.l)**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.l)* -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)** -11.1 (1.6)**WO 98109519101520253035CA 02264775 1999-03-05PCT/US97/15474 ‘The compounds of the instant invention and their pharmaceutically acceptable saltswhich are active when given orally can be fonnulated as liquids, for example syrups,suspensions or emulsions, tablets, capsules and lozenges.A liquid formulation will generally consist of a suspension or solution of thecompound or pharmaceutically acceptable salt in a suitable liquid cairier(s) forexample, 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 suitablepharmacuetical carrier(s) routinely used for preparing solid formulations. Examplesof such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, pellets containing the active ingredient canbe prepared using standard carriers and then filled into a hard gelatin capsule;alternatively, a dispersion or suspension can be prepared using any suitablepharmaceutical caxrier(s), for example aqueous gums, celluloses, silicates or oils andthe dispersion or suspension then filled into a soft gelatin capsule.The compounds of the instant invention and their pharmaceuticallyacceptable salts which are active when administered parenterally (i.e. by injection ofinfusion) can be formulated as solutions or suspensions.A composition for parenteral administration will generally consist of asolution or suspension of the active ingredient in a sterile aqueous carrier orparentcrally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone,lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised andthen reconstituted with a suitable solvent just prior to administration.A typical suppository composition comprises a compound of the instantinvention or a pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent such as polymericglycols, gelatins or coca butter or other low melting vegetable or synthetic waxes orfats.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 amedicated plaster, patch or membrane.For topical administration, the pharmaceutical compositions adapted includesolutions, suspensions, ointments, and solid inserts. Typical pharmaceuticallyacceptable carriers are, for example, water, mixtures of water and water—misciblesolvents such as lower alkanols or vegetable oils, and water solubleophthalmologically acceptable non-toxic polymers, for example, cellulose-7-W0 98/09519101520253035CA 02264775 1999-03-05derivatives such as methyl cellulose. The pharmaceutical preparation may alsocontain non-toxic auxiliary substances such as emulsifying, preserving, wetting andbodying agents, as for example, polyethylene glycols; antibacterial components suchas quaternary ammonium compounds; buffering ingredients such as alkali metalchloride; antioxidants such as sodium metabisulfite; and other onventional .ingredients such as sorbitan monolaurate. 0Preferably the composition is in unit dose form. Doses of the compounds ofthe 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 .l - 100 mg/kg. The selected dose is administered to a human patient inneed of treatment or prevention of osteoporosis or in the lowering of plasmacholesterol or prevention of cardiovascular disease from 1-6 times daily, orally,rectally, topically, by injection, or continuously by infusion. Oral dosage units forhuman administration preferably contain from 10 to 500 mg of active compound.Lower dosages are used generally for parenteral administration. Oral administrationis used when safe, effective, and convenient for the patient.No unacceptable toxicological effects are expected when-compounds of theinvention are administered in accordance with the present invention.Example 1An oral dosage form for administering orally active Formula (I) compoundsis produced by screening, mixing and filling into hard gelatin capsules theingredients in proportions, for example, as shown below.Ingredients Arno n(E)- l -[2-[4-[ l —(4-lodophenyl)-2-phenyl- 1 -butenyl]phenoxy]pyrrolidine 100 mgmagnesium stearate 10 mglactose 100 mgExamThe sucrose calcium sulfate dihydrate and orally active Fonnula (1)compounds are mixed and granulated with a 10% gelating solution. The wetgranules are screened, dried, mixed with the starch, talc and stearic acid, screenedand compressed into a tablet.Ingredients Amounts(E)— 1-[2-[4-{ l -(4-lodophenyl)-2-phenyl— l—butenyl]-3-PCT/US97/15474 -9CA02264775 1999-03-05Example 3PCT/US97/15474 975 mg100 mg15 mg8 mg4 mg2 mg(E)-1-[2-[4-[1-(4-lodophenyl)-2-phenyl-1-butenyl]phenoxy]pyrrolidine, 50mg, is dispersedin 25 ml of normal saline to prepare an injectable preparation.Exam 4This experiment was conducted to compare the mechanism of action ofidoxifene and raloxifene in osteoblasts. Using a construct containing an estrogenresponse element (ERE) upstream of a luciferase reporter gene (described below) ithas now been shown that idoxifene, like estrogen, is a pure agonist through the EREin osteoblasts. The potency of agonist action was similar between the natural steroidhormone estrogen and idoxifene. Raloxifene, at the same concentrations as idoxifene(0.01 to 10},tM), gave an extremely weak signal, which was of similar magnitude toCompetition experiments were performed to confirm the mechanism ofaction through this response element. Raloxifene inhibited the agonist activity ofboth estrogen and idoxifene through the ERE. At doses of ligand (either estrogen oridoxifene ) at 100nM, there was a maximum agonist response. Co-treatment withWO 98/09519phenoxy]pyrrolidinecalcium sulfate dihydratesucrosestarch5 talcstearic acid101520 the to vehicle control, through the ERE.25500nM raloxifene reduced the reporter gene activity to vehicle control levels. Incontrast, idoxifene at 500nM did not diminish the maximal agonist action ofestrogen at 100nM in osteoblasts. If submaximal concentrations of idoxifene andestrogen were used (< 100nM) there was additive actions of the two agonists throughthe ERE in osteoblasts.WO 98/09519101520253035CA 02264775 1999-03-05Experimental ProceduresCells were seeded in either 6-well plates at 1.5 x l05cells/well or in 24-wellplates at 1.5 x 104 cells/well in phenol red-free medium. A DNA constructcomprising a mouse mammary tumor virus promoter in which the glucocorticoidresponse elements have been replaced with five copies of a 33-base pair vitellogeninestrogen response element was employed. This is upstream of the Luciferase reportergene (MMTV-ERE-Luc) "(Wen, D.X., Y-F. Xu, M.K. Goldman and P. McDonnell.1994. The A and B isoforrns of the human progesterone receptor operate throughdistinct signalling pathways within target cells. Molec. Cell. Biol. 14: 8356-8364)".The renilla-Luciferase vector was used to correct for transfection efficiency using thedual-luciferase detection method (Promega, Madison, WI). DNA was introducedinto rat osteosarcoma (Ros 17/2.8) cells by the lipofectin method (Life Technologies,Gaithersburg, MD). Cells were co-transfected with 2 pg per well in 6-well platesand l40ng per well in 24-well plates of MMTV ERE—Luc and 25ng of the controlrenilla-Luciferase vector (pRL-CMV). Transfection efficiency was corrected for byco-transfection with a renilla-Luciferase vector, which utilizes a different substrate,coelenterazine, for its bioluminescent readout (Promega, Madison WI). Cells wereincubated overnight. Transfection medium was then removed and cells wereincubated for 48 with or without hormones. Cells were washed in phosphatebuffered saline and then lysed with 500uUwell 1x passive lysis buffer (PLB) for 15minutes while rocking sample on a rocking platform. Lysates were centrifuged for30secs. at l2,000g and the clear lysate was transferred to a tube prior to reporterenzyme analysis. Sarnples(20y.1l) were transfered to a 96 well luminescence detectionplate and reacted with l0Op.l of each assay reagent (Promega, Madison, WI). Eachassay reagent was injected by a rnicrolumat LB96P luminometer (Wallac,Gaithersburg, MD), which measured luciferase activity. Luciferase activity providesa surrogate of transcriptional activation of estrogen responsive gene that contains theestrogen response element (ERE). Therefore upregulation of luciferase activity isindicative of an agonist effect, whereas down-regulation indicates antagonismthrough the ERE.Discussion:Idoxifene is an agonist through the estrogen response element (ERE) inosteoblasts. In contrast to idoxifene, raloxifene is an antagonist through the ERE inosteoblasts at the doses tested, which suggests a distinct mechanism for the bonesparing effects seen with raloxifene. Thus, raloxifene is able to exert its biological-10-PCT/US97/15474 9WO 98/09519l015CA 02264775 1999-03-05effect through a non-ERE containing sequence present on the 5’-untranslated regionof the human TGFB3 promoter. In the same cellular system, raloxifene inhibited theERE—containing vitellogenin promoter expression and exhibited therefore pureestrogen antagonism. The raloxifene response element‘ (Yang, N, N., Venugopalan,M., Hardikar, S., and Glasebrook, A. (1996) Identification of an estrogen responseelement acti)/ated by metabolites of 17beta-estradiol and raloxifene Science 273:1222-1225) is not present on the same genes as the ERE suggesting modulationthrough this response element will result in effects on different genes. Thisdistinguishes the mechanism of action of idoxifene from that of the selective-estrogen receptor modulator (SERM) raloxifene and aligns idoxifene to the moreclassic estrogenic type mechanism exerting its biological agonist effect in osteoblaststhrough the ERE. The effects of idoxifene and estrogen are specific to reporter geneconstructs that carry the classical ERE. This system was shown to be sensitive to cellspecific factors and is thus a valid model for effects on endogenous genetranscription.-11-PCT/US97/15474 J