EP1465619A1

EP1465619A1 - Suppression of cartilage degradation via the estrogen receptor

Info

Publication number: EP1465619A1
Application number: EP03702427A
Authority: EP
Inventors: Claus Christiansen; Stephan Christgau
Original assignee: Nordic Bioscience AS
Current assignee: Nordic Bioscience AS
Priority date: 2002-01-14
Filing date: 2003-01-13
Publication date: 2004-10-13
Also published as: WO2003063859A1; JP2005524630A

Abstract

The present invention relates to the pharmaceutical use of selective estrogen receptor modulators (SERMs) alone or in combination with progestins for the treatment or prevention of diseases associated with elevated cartilage degradation. In particular this invention relates to the pharmaceutical use of chroman derivatives in combination with moretindrone for the treatment or prevention of osteoarthritis or rheumatoid arthritis.

Description

Suppression of cartilage degradation via the estrogen receptor

Field of the invention; The present invention relates to the pharmaceutical use of selective estrogen receptor modulators (SERMs) alone or in combination with other pharmaceutical agents, especially progestins, for the treatment or prevention of a wide range of diseases but especially those associated with elevated cartilage degradation. In particular this invention relates in part to the pharmaceutical use of chroman derivatives for the treatment or prevention of osteoarthritis or rheumatoid arthritis.

Background;

Osteoarthritis (OA), which is also called "degenerative joint disease", is the most common rheumatic disease. The prevalence of this chronic disease increases with age and it is a prominent cause of disability and poor quality of life among the elderly. The prevalence of the disease is higher in women than in men, and in women the incidence of osteoarthritis increases after the menopause. These observations have suggested that estrogens may have an effect on cartilage metabolism, and that loss of endogenous estrogen production after the menopause increases the risk for subsequent development of OA (D Felson & M Nevitt 1998). Furthermore, most observational studies suggest that postmenopausal estrogen use is associated with decreased occurrence of OA (Nevitt et al 1996), although some studies challenge this conclusion (Oliveria et al 1996, Erb et al. 2000, Nevitt et al 2001). Thus, until now a potential role of estrogen for preserving cartilage integrity and preventing OA development has not been conclusively demonstrated. A central element of the disease process in OA is the non-reversible degradation of cartilage. This process is likely to be initiated many years prior to clinical diagnosis of the disease, and it persists until the end stage of the disease where almost all articular cartilage of the affected joints is lost. At this stage the mobility of the joint is severely compromised, and joint replacement surgery remains the only treatment option. Due to this prolonged development of the pathological joint changes associated with OA it is very difficult to elucidate the factors involved in the early phases of the disease process. Rheumatid arthritis (RA) is an inflammatory condition where articular cartilage of affected joints are being degraded. The etiology of RA is complex and a number of environmental 5 and genetic factors have been suggested a role in the development of the disease. Also RA is more prevalent in woman. It has been suggested that estrogen may also have a potential beneficial effect in RA, as estrogen has a positive role as an inhibitor of inflammation. Studies in humans and animal models of OA an RA have demonstrated a progressive depletion of articular cartilage matrix macromolecules as the disease develops. In OA 0 cartilage degeneracy and osteophytes (small abnormal bone outgrowths in the affected joints) occur and develop on the stripped part of the articular bones. Symptoms of OA are pain, swelling and stiffness of the articulation. In RA the cartilage degradation tends to occur more rapidly. The progression of joint destruction varies widely between individual patients with a marked cyclical pattern characterized by periods of elevated disease activity 5 (flare ups) intermittent with more 'silent' periods. This cyclical pattern of disease activity is especially prominent for RA.

The most commonly used drugs for the treatment of RA are the non-steroidal anti- inflammatory agents (NSAJD) and disease modifying anti-rheumatic drugs (DMARD's) as o well as more specific anti-inflammatory agents such as steroids or TNF-alfa antagonists. In OA treatment NSAID and DMARD's also plays an important role. The aim of current therapies of these diseases is mainly to relieve pain and disease symptoms. NSAJD and DMARD's have proven effectiveness in relieving the symptoms of OA and RA but their effect on decreasing cartilage catabolism has not been well documented. Some of them, 5 like sodium salicylate, have shown inhibiting properties in relation to proteoglycan synthesis which may jeopardize the cartilage repair process. Other drugs, such as tiaprofenic acid, which do not inhibit the proteoglycan synthesis have shown in vitro that they are able to decrease OA cartilage catabolism, (Jean-Pierre Pelletier et al. The Journal of Rheumatology 1989;16:5, 646-655). However they have been unable to provide any o significant protective effect in development of OA when administrated to patients suffering from the latter, (Edward C. Huskisson et al. The Journal of Rheumatology 1995; 22:10- 1941-1946). Doxycycline, a member of the tetracycline family, was also shown to reduce, in vivo, the severity of O A lesions in the dog ACL model while reducing metalloprotease activity, (Yu LP Jr et al. Arthritis Rheum 35:1150-1159, 1992). Recent data suggests that the action of corticosteroids is associated with a reduction in the synthesis of stromelysin-1 by chondrocytes. (see: Pelletier et al., J Arthritis Rheum 37:414-423, 1994; and Pelletier et al., J Lab Invest 72:578-586, 1995).

In both OA and RA, estrogen and compounds acting through the estrogen receptor may have a potential beneficial effect.

Selective Estrogen Receptor Modulators (SERMs) have been used for several years in hormone replacement therapy in the treatment of e.g. osteoporosis as an alternative to the natural estrogen to avoid the side effects such as breast cancer and endometrium growth while maintaining the beneficial effects of the female sex hormone. A SERM is a synthetic compound that possesses some, but not all, of the actions of estrogen. For example, raloxifene is classified as a SERM because it prevents bone loss and lowers serum cholesterol as an estrogen agonist but act as an estrogen antagonist by not stimulating the endometrial lining of the uterus. However, SERM's with potent estrogen agonistic activity on e.g. bone tissue tend to be less selective in their antagonistic action on tissues such as the endometrium. Thus, many potent SERM's are not used as drugs in prevention, treatment of e.g. osteoporosis because of unwanted side effects. An example of this is the chroman compound levormeloxifene as disclosed in patent No. US 3822287 and US 5977158.

Progestins includes progesterone related steroid hormones, their derivatives and compounds having progestogenic effect. They are widely used in hormone replacement therapy in combination with other steroid hormones to reduce the (side) effect on the endometrium lining growth. Only recently progestins have been proposed for the treatment of bone related diseases as disclosed in WO 00/74684 and EPO 0474374. It has been found that the combination of the SERM raloxifene and the progestin norethinedrone acetate has a synergistic effect on osteoporosis by increasing the bone mass index significantly as disclosed in EPO 0665015.

While the beneficial actions of estrogen replacement therapy on the skeleton are clearly 5 significant, there are indications but no conclusive evidence for a positive effect of estrogen on OA or rheumatoid arthritis (Felson et al. 1998). However, the SERM raloxifene has been demonstrated as an effective inhibitor of cartilage degradation in rodents (US Pat No. 5418252). Recently, coumarin derivatives have been proposed as inhibitors of rheumatoid arthritis (US Pat. No. 6291456 and 6331562). None of these l o compounds however has been approved as clinically useful drugs for the treatment of arthritis. Thus, there is a continuing need for investigating the nexus between estrogens and OA and for the development of potent estrogen agonists, which can selectively target joint tissue suffering from OA and which display a high bioavailability and at the same time minimize the side effects.

15

This invention provides in a first aspect a method of preventing, treating or alleviating conditions involving elevated cartilage degradation comprising administering to a subject an effective amount of a selective estrogen receptor modulator (SERM) selected from droloxifene, levormeloxifene, chroman derivatives, nafoxidine, miproxifene, arzoxifene, 2 o lasofoxifene, basedoxifene, MDL-103323, EM-800, fulvestrant, ICI 183,780, ICI 164,384, 19-nor-testosterone derivatives and pharmaceutically acceptable esters, ethers and salts thereof.

Preferably, the SERM comprises one or more of levormeloxifene, a chroman derivative, or 5 a 19-nor-testosterone derivative or a pharmaceutically acceptable salt thereof.

As used herein "progestin or progestin agent" means a compound having progestational activity (i.e., induce the formulation of a secretory endometrium), such as, for example, norethindrone (also referred to as norethisterone), ethynodiol, desogestrel, levonorgestrel, 0 norgestrel, norgestimate, medroxyprogesterone, danazol, lynoestrenol, dydrogesterone, chlormadinone, promegesterone, gestrinone, algestone acetophenide, allyloestrenol, cyproterone acetate, demegestone, gestodene, osaterone, hydroxyprogesterone hexanoate, medrogestone, megestrol, nomegestrol, ethynylnortestosterone, no regneninolone, NSC- 9564, norethynodrel, dexnorgestrel, gestodene, progesterone, chlormadinone acetate, drospirenone (dihydrospirorenone), or 3-ketodesogestrel or salts thereof, and other compounds having progestational activity. Progestin agents are well described in the art (See, for example, Martindale: The Extra Pharmacopoeia, 30th edition, 1993 incorporated herein by reference). Preferred salts are acetate salts. A more preferred progestin agent is norethindrone, with norethindrone acetate being most preferred. A progestin agent is also referred to as a progestogen, a gestagen or a progestational hormone.

"Osteoarthritis" or "OA" is a type of arthritis that is caused by breakdown of cartilage with eventual loss of the cartilage of the joints. Cartilage is a protein substance that serves as a "cushion" between the bones of the joints. Osteoarthritis is also known as degenerative arthritis.

"Rheumatoid arthritis" or "RA" is an inflammatory condition where articular cartilage of affected joints are being degraded. The etiology of RA is complex and a number of environmental and genetic factors have been suggested a role in the development of the disease.

A "selective estrogen receptor modulator (SERM)" is a synthetic compound that possesses some, but not all, of the actions of estrogen and exhibits activity as an agonist or antagonist of an estrogen receptor (e.g., ER.α. or ER.β) in a tissue-dependent manner. Thus, as will be apparent to those of skill in the biochemistry and endocrinology arts, compounds of the invention that function as SERMs can act as estrogen receptor agonists in some tissues (e.g., bone, brain, and/or heart) and as antagonists in other tissue types, such as the breast and or uterine lining.

As used herein "chromans" means 3,4-chroman derivatives. As used herein "coumarins" means 2-chromen-2-one derivatives, [is 'one' correct?]

As used herein " cartilage specific biochemical marker" means a metabolite of cartilage catabolism which can be quantified in a body fluid, where said metabolite is indicative of 5 systemic cartilage degradation.

According to the preferred practice of the first aspect of the invention, the selective estrogen receptor modulator is selected from chroman derivatives. Any chroman derivatives presently known in the art, or subsequently developed may be used in l o practising the claimed methods. The synthesis of exemplary receptor antagonists is described, by way of example only, in U.S. Pat. Nos. 5,919,817; 6,043,269; and EP 937057; EP 937060; and EP 937062, incorporated herein by reference in their entireties.

In a preferred embodiment of the present invention the selective estrogen receptor modulator 15 is selected from a group of chroman derivatives of the formula I

wherein

0 R¹ is H, SO₂ NR₂ ⁴, SO₂ NHR⁴ ,C1, CH₃ or benzyl;

R² is phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of OH, halogen, nitro, cyano, SH, SR⁴, trihalo-Cι-C6 alkyl, Cι-C₆ -alkyl, d-Cβ alkoxy and phenyl; 5

R is phenyl substituted with — X~(CH2)_n — Y, wherein: X is a valency bond, O or S,

n is an integer in the range of 1 to 12,

Y is H, halogen, OH, OR⁴, NHR⁴, NR₂ ⁴, NHCOR⁴, NHS0₂ R⁴,C0NHR⁴,

CONR⁴, COOH, COOR⁴, SO₂ R⁴, SOR⁴, SONHR⁴, SONR₂ ⁴, a pyrrolidinyl ring, optionally being substituted with 1 to 3 substituents independently selected from the group consisting of H, OH, halogen, nitro, cyano, SH, SR⁴, trihalo-d -C₆ -alkyl, Ci -C₆ -alkyl and Ci -C₆ -alkoxy; and

R⁴ is d -Ce -alkyl;

and optical and geometrical isomers, pharmaceutically acceptable esters, ethers and salts thereof as disclosed in WO 98/18773.

In another embodiment of the present invention is provided use of compounds of the formula I in which substituents R² and R³ are arranged in trans-configuration such as centchroman (levormeloxifene) or (-)-3,4-trans-7-methoxy-2,2-dimethyl-3-phenyl-4 {4[2- (pyrrolidin-l-yl)ethoxy]phenyl}chromane.

In another preferred embodiment, the present invention is concerned with use of the compound of the formula I wherein:

R¹ is H, SO₂NR₂ ⁴ ,C1, CH₃ or SO₂NHR⁴;

R² and R³ are arranged in cis-configuration;

R² is phenyl optionally substituted with 1 to 3 substituents independently selected from the group consisting of OH, halogen, nitro, cyano, SH, SR₄, trihalo-d -C₆ -alkyl, d -C₆ -alkyl and CI -C6 -alkoxy;

R³ is phenyl substituted with ~X~(CH₂)_n ~Y, wherein:

X is a valency bond, O or S,

n is an integer in the range of 1 to 12,

Y is H, OH, OR l⁴⁴,, NNHHRR44,, NNRR₂₂*⁴,, NHCOR⁴, NHSO₂ R⁴, CONH⁴, CONR₂ ⁴, COOH,

COOR\ SO₂ R.⁴, SOR⁴' SONHR , SONR ⁴, a pyrrolidinyl ring, optionally being substituted with 1 to 3 substituents independently selected from the group consisting of H, OH, halogen, nitro, cyano, SH, SR⁴, trihalo-d -C₆ -alkyl, d-C₆ -alkyl and d -C₆ -alkoxy; and

R⁴ is Ci -Q; -alkyl;

and optical and geometrical isomers, pharmaceutically acceptable esters, ethers and salts thereof.

hi another preferred embodiment, the present invention is concerned with use of compound of the formula

wherein R¹, R² and R³ are as defined above.

In another preferred embodiment, the present invention is concerned with use of the compound of the formula

wherein R¹ is as defined above and m is an integer from 0 to 10.

wherein R¹ is as defined above and R⁶ represents one or more of the following substituents: methoxy, hydroxy, trifluormethyl, fluoro and chloro.

In a more preferred embodiment the invention is concerned with the following compounds:

(+)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(3-piperidinopropoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-phenyl-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-phenyl-4-(4-(3-piperidinopropoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-(4-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane (+)-cis-7-Methoxy-3-(4-phenyl-phenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-(4-methylphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-(4-methylphenyl)-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+)-cis-7-Memoxy-4-(4-(2-piperidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) chromane

(+)-cis-7-Methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) chromane

(+)-cis-7-Methoxy-3-(3-methylphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-3-(3-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-(3-memoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(3-piperidinopropoxy)phenyl)chromane

(+)-cis-7-Methoxy-4-(4-(2-pyrrolidinoe oxy)phenyl)-3-(3-(trifluoromethyl)phenyl) chromane

(+)-cis-7-Methoxy-4-(4-(2-piperidinoethoxy)phenyl)-3-(3-(trifluoromethyl)phenyl) chromane

(+)-cis-3-(2-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane (+)-cis-7-Methoxy-3-(2,3,4,5,6-pentafluorophenyl)-4-(4-(2-pyrrolidinoethoxy) phenyl)chromane

(+)-cis-7-Methoxy-3-(2, 3,4, 5,6-pentafluorophenyl)-4-(4-(2-piperidinoethoxy) ρhenyl)chromane

(+)-5-(cis-7-Methoxy-3-(4-methylphenyl)-chroman-4-yl)-2-methyl-benzoxazole

(+)-cis-6-Methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+^)-cis-6-Methoxy-3-(3-hydroxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+)-cis-4-(4-Hexylphenyl)-7-methoxy-3-phenylchromane

(+)-cis-7-Methoxy-3-phenyl-4-{4-{2-(pyrrolidin-l -yl)ethoxy}phenyl} chromane

(-)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(2-piperidinoethoxy)phenyl)chromane

(-)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(3-piperidinopropoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-phenyl-4-(4-(2-piperidinoethoxy)phenyl)chromane

(-)-cis-.sup.7 -Methoxy-3-phenyl-4-(4-(3-piperidinopropoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(4-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(4-phenyl-phenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane (-)-cis-7-Methoxy-3-(4-methylphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(4-methylphenyl)-4-(4-(2-piperidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-4-(4-(2-piperidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) chromane

(-)-cis-7-Methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) chromane

(-)-cis-7-Methoxy-3-(3-methylphenyl)4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-3-(3-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(2-piperidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(3-piperidinopropoxy)phenyl)chromane

(-)-Cis-7-Me oxy-4-(4-(2-pyrrolidinoethoxy)phenyl)-3-(3-(trifluormethyl)phenyl) chromane

(-)-cis-7-Methoxy-4-(4-(2-piperidinoethoxy)phenyl)-3-(3-(trifluoromethyl)phenyl) chromane

(-)-cis-3-(2-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-7-Methoxy-3-(2, 3,4, 5,6-pentafluorophenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl) chromane (-)-cis-7-Methoxy-3-(2,3,4, 5,6-pentafluorophenyl)-4-(4-(2-piperidinoethoxy)phenyl) chromane

(-)-5-(cis-7-Methoxy-3-(4-methylphenyl)-chroman-4-yl)-2-methyl-benzoxazole

(-)-cis-6-Methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-6-Methoxy-3-(3-hydroxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(-)-cis-4-(4-Hexylphenyl)-7-methoxy-3-phenylchromane

(-)-cis-7-Methoxy-3 -phenyW- {4- {2-(pyrrolidin- 1 -yl)ethoxy}phenyl} chromane .

(+,-)-cis-3 -(4-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+,-)-cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(3-piperidinopropoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-phenyl-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-phenyl-4-(4-(3-piperidinopropoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3 -(4-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-(4-phenyl-phenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-(4-methylρhenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane (+,-)-cis-7-Methoxy-3-(4-methylphenyl)-4-(4-(2-piperidinoethoxy)phenyl)chromnane

(+,-)-cis-7-Methoxy-4-(4-(2-piperidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) chromane

(+,-)-cis-7-Methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) chromane

(+,-)-cis-7-Methoxy-3-(3-methylphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-3-(3-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3 -(3 -methoxyphenyl)-4-(4-(2-piperidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-(3-methoxyphenyl)-4-(4-(3-piperidinopropoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)-3-(3-(trifluoromethyl)phenyl) chromane

(+,-)-cis-7-Methoxy-4-(4-(2-piperidinoethoxy)phenyl)-3-(3-(trifluoromethyl)phenyl) chromane

(+,-)-cis-3 -(2-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-7-Methoxy-3-(2,3,4,5,6-pentafluorophenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl) chromane

(+,-)-cis-7-Methoxy-3-(2,3,4, 5,6-pentafluorophenyl)-4-(4-(2-piρeridinoethoxy)phenyl) chromane

(+,-)-5-(cis-7-Methoxy-3-(4-methylphenyl)-chroman-4-yl)-2-methyl-benzoxazole

(+,-)-cis-6-Methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-6-Methoxy-3-(3-hydroxyphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane

(+,-)-cis-4-(4-Hexylphenyl)-7-methoxy-3-phenylchromane,

(+,-)-(cis-4-(4-(2-Pyrrolidinoethoxy)phenyl)-3-(4-(trifluoromethyl)phenyl) -chroman-7-yl) 2,2-dimethylpropanoate,

(+,-)-(cis-4-(4-(2-Pyrrolidinoethoxy)phenyl)-3 -(3 -(trifluoromethyl)phenyl) -chroman-7-yl) 2,2-dimethylpropanoate hydrochloride,

(+,-)-(cis-3 -(4-Methylphenyl)-4-(4-(2-pyrrolidinoethoxy)phenyl)-chroman-7- yl) N,N- diethyl carbamate,

(+,-)-(cis-3 -(4-Methylphenyl)-4-(4-(2-pyrrolidinoethoxy)ρhenyl)-chroman-7- yl) N,N- dimethylsulfamic acid ester,

(+,-) cis 7-Benzyloxy-4-(4-hydroxy-phenyl)-3-phenyl-chroman,

7-Benzyloxy-4-[4-(4-chlorobutyloxy)-phenyl]-3-phenyl-chroman,

7-Benzyloxy-4-[4-(2-chloroethyloxy)-phenyl]-3-phenyl-chroman,

7-Benzyloxy-4-[4-(6-bromohexyloxy)-phenyl]-3-phenyl-chroman, 7-Benzyloxy-4-[4-( 10-bromodecyloxy)-phenyl]-3-phenyl-chroman,

(+,-: -cis-3-(4-Fluorophenyl)-7-methoxy-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane,

(+- •5-(cis-7-Methoxy-3-(4-methylphenyl)-chroman-4-yl)-2-methyl-benzoxazole

(+.^■ •cis-6-Methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane,

(+,-: ■cis-4-(4-Hexylphenyl)-7-methoxy-3-phenylchromane,

(+.- cis [4-(7-Benzyloxy-3-phenyl-chroman-4-yl)-phenoxy] -acetic acid methyl-ester,

(+.^■ cis 7-Benzyloxy-4-[4-(6-morpholinohexyloxy)-phenyl]-3-phenyl-chroman,

(+,^• cis 7-Benzyloxy 4-[4-(6-dibutylamino-hexyloxy)-ρhenyl]-3-phenyl-chroman,

⁽+. cis 7-Benzyloxy 4-[4-(dimethylaminodecyloxy)-phenyl]-3-phenyl-chroman,

(+,-: cis 7-Benzyloxy 4-[4-(morpholinodecyloxy)-phenyl]-3-phenyl-chroman,

(+,^■ cis 7-Benzyloxy 4-[4-(2-dibutylaminoethyloxy)-phenyl]-3-phenyl-chroman,

⁽+, cis 7-Benzyloxy 4-[4-(butylaminoethyloxy)-phenyl]-3-phenyl-chroman,

(+»^■ cis 7-Benzyloxy 4-[4-(moφholinoethyloxy)-phenyl]-3-phenyl-chroman,

(+.^■ cis 7-Benzyloxy 4-[4-(N-methylpiperazinoethyloxy)-phenyl]-3-phenyl-chroman,

(+,^■ cis 7-Benzyloxy 4-[4-(morpholinobutyloxy)-phenyl]-3-phenyl-chroman, including the pure enantiomers thereof.

In a most preferred embodiment the present invention is concerned with the following compounds:

(-)-cis-{3-phenyl-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-chroman-7-ol

(-)-cis- {3-(4-trifluormethyl-phenyl)-4-[4-(2-pyrrolidin-l -yl-ethoxy)-phenyl] } -7- methoxychroman

(-/+)-cis-{3-(3-methoxy-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-7- methoxychroman

(-/+)-cis-{3-(3-hydroxy-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-chroman-7-ol

(-/+)-cis- {3 -(4-trifluormethyl-phenyl)-4-[4-(2-pyrrolidin-l -yl-ethoxy)-phenyl] } -chroman- 7-ol

(-)-cw-7-methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane

(+/-)-cz5-7-memoxy-3-(3-memoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane

(+y-)- s-7-methoxy-3-(4 rifluoromethylphen^

(-)-czs-7- e&oxy-3-(4-trifluoromethylρhenyl)-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane

This invention includes, the use of a SERM as defined above for the preparation of a 5 medicament for use in the treatment, prevention, or alleviation of a condition involving elevated cartilage degradation.

In an alternative aspect, the present invention relates to a method for preventing, treating or alleviating conditions involving elevated cartilage degradation comprising administering to a l o subject an effective amount of the combination of

1) a selective estrogen receptor modulator (SERM) or a pharmaceutically acceptable salt thereof and;

2) a progestin or a pharmaceutically acceptable salt thereof such that the combination decreases cartilage degradation.

15

The invention also provides pharmaceutical formulations for treatment of arthritis comprising (1) a chroman or a coumarin derivative or a pharmaceutically acceptable salt or solvate thereof; and (2) a progestin or a salt thereof and other compounds having progestational activity; in amounts such that the combination inhibits OA or RA, together 2 o with one or more pharmaceutically acceptable carriers.

In particular, the present invention relates to a method for treating or alleviating arthritis comprising administering to a subject an effective amount of the combination of a selective estrogen receptor modulator (SERM) selected from the group consisting of raloxifene, droloxifene, tamoxifen, 4-hydroxy-tamoxifen, 4'-iodotamoxifen, toremifene, (deaminohydroxy)toremifene, chlomiphene, levormeloxifene, chroman derivatives, coumarin derivatives, idoxifene, nafoxidine, miproxifene (TAT-59), arzoxifene (LY-353381), lasofoxifene (CP-336156), MDL-103323, EM-800, fulvestrant (0-182,780) ICI 183,780, ICI 164,384, diethylstilbesterol, genistein, nafoxidine, GW 5638, panomifene, mtromifene citrate, moxesterol, diphenol hydrochrysene, erythro-MEA, allenolic acid, cyclophenyl, chlorotrianisene, ethamoxytriphetol, triparanol 19-nor-progesterone derivatives, 19-nor- testosteone derivatives and pharmaceutically acceptable esters, ethers and salts and a progestin selected from the group consisting of norethindrone, ethynodiol, desogestrel, levonorgestrel, norgestrel, norgestimate, medroxyprogesterone, danazol, lynoestrenol, dydrogesterone, chlormadinone, promegesterone, gestrinone, algestone acetophenide, allyloestrenol, cyproterone acetate, demegestone, gestodene, osaterone, hydroxyprogesterone hexanoate, medrogestone, megestrol, nomegestrol, ethynylnortestosterone, noφregneninolone, NSC-9564, norethynodrel, dexnorgestrel, gestodene, progesterone, chlormadinone acetate, drospirenone (dihydrospirorenone), or 3- ketodesogestrel and optical and geometrical isomers, pharmaceutically acceptable esters and salts thereof.

Preferred SERM's include droloxifene, tamoxifen, levormeloxifene, chroman derivatives, nafoxidene, toremifene, TAT-59, LY-353381, CP-336156, MDL-103323, EM-800, ICI-182, ICI 183,780 and 19-nor-testosteone derivatives or a pharmaceutically acceptable salt thereof.

This aspect of the present invention is in particular related to the observation that the side effects of hormone replacement therapy administering specific selective estrogen receptor modulators (SERMs) alone such as increased incidence of breast cancer and growth of the endometrial lining can be reduced by combined administration of progestins. In the treatment, prevention or alleviation of diseases related to elevated cartilage degradation such as osteoarthritis and rheumatoid arthritis a long lasting and continued administration of an effective inhibitor of cartilage degradation is mandatory. Thus even minor side effects of said inhibitor may have dramatic impact on the patients health over time and must be avoided or minimized to improve the life value of patients and save the 5 health system for expensive complications.

According to a preferred practice of the second aspect of the invention, the selective estrogen receptor modulator is selected from chroman derivatives.

o Preferred chroman derivatives are as described above.

In another preferred embodiment of the invention said progestin is progesterone, norethindrone acetate, ethynodiol, desogestrel, levonorgestrel, norgestrel, norgestimate, medroxyprogesterone and its esters or a pharmaceutically acceptable salt thereof. 5

In a more preferred embodiment of the invention said progestin is norethindrone or norethindrone acetate and its esters or a pharmaceutically acceptable salt thereof.

In another aspect the present invention is concerned with a pharmaceutical composition for o use in the therapeutic or prophylactic treatment of diseases wherein decreased estrogen production is a factor, said composition comprising an effective amount of the combination of a 3,4 chroman or coumarin derivative or a pharmaceutical acceptable salt thereof and a progestin or a pharmaceutical acceptable salt thereof and a pharmaceutical carrier or diluent. 5

In one embodiment the invention is related to the use of said pharmaceutical composition for the therapeutic or prophylactic treatment of conditions such as is breast cancer, testicular cancer, osteopenias or osteoporosis induced bone loss, endometriosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, prostatic carcinomas, 0 obesity, hot flashes, skin effects, mood swings, memory loss, urinary incontinence, hairloss, cataracts, natural hormonal imbalances, and adverse reproductive effects associated with exposure to environmental chemicals.

In a preferred embodiment the invention is concerned with a pharmaceutical composition for use in the therapeutic or prophylactic treatment of diseases wherein elevated cartilage degradation is a factor, said composition comprising an effective amount of the combination of a 3,4 chroman or a coumarin derivative or a pharmaceutical acceptable salt thereof and a progestin or a pharmaceutical acceptable salt thereof and a pharmaceutical carrier or diluent.

In a even more preferred embodiment the present invention is concerned with a pharmaceutical composition for use in the therapeutic or prophylactic treatment of diseases wherein elevated cartilage degradation is a factor, said composition comprising an effective amount of the combination of a 3,4 chroman or a coumarin derivative or a pharmaceutical acceptable salt thereof and norethindrone or norethindrone acetate or a pharmaceutical acceptable salt thereof and a pharmaceutical carrier or diluent.

In a most preferred embodiment the present invention is concerned with a pharmaceutical composition for use in the therapeutic or prophylactic treatment of diseases wherein elevated cartilage degradation is a factor, said composition comprising an effective amount of a cis-3,4 diarylchromane or a pharmaceutical acceptable salt thereof in combination with an effective amount of norethindrone or norethindrone acetate or a pharmaceutical acceptable salt thereof and a pharmaceutical carrier or diluent.

In a preferred embodiment of the invention the effective amount of said selective estrogen receptor modulator or a pharmaceutical acceptable salt thereof from 0.01 to about 100 mg per individual per day, preferably from 0.1 to about 10 mg per individual per day and most preferably from 0.2 to about 1.0 mg per individual per day.

In another preferred embodiment of the invention the effective amount of said progestin or a pharmaceutical acceptable salt thereof and a pharmaceutical carrier or diluent thereof is from 0.01 to about 100 mg per individual per day, preferably from 0.1 to about 10 mg per individual per day and most preferably from 0.2 to about 1.0 mg per individual per day.

5 In a most preferred embodiment of the invention the effective amount of said progestin or a pharmaceutical acceptable salt thereof and a pharmaceutical carrier or diluent thereof is in an amount sufficient to significantly reduce SERM induced estrogenic side effects.

In still another preferred embodiment the invention is concerned with the pharmaceutical l o composition as described above in the form of an oral dosage unit or parenteral dosage unit.

In a further preferred embodiment the invention is concerned with administering to a mammal in need thereof a therapeutically effective amount compound I in combination 15 with a progestin.

In a most preferred embodiment the invention is concerned with administering to a human in need thereof a therapeutically effective amount compound I in combination with norethindrone or norethindrone acetate.

20

In another embodiment the invention relates to a method of treating osteoarthritis or rheumatoid arthritis, said method comprising administering to a human in need thereof a therapeutically effective amount of a compound (I) in combination with a progestin.

25 In another aspect the invention is concerned with use of a cartilage specific biochemical marker for titration of the doses described above. Titration of a therapeutically effective dose is based on quantification of one or more fragments of a cartilage matrix protein as described above. Different doses of the pharmaceutically composition is given to a mammal for at least 2-4 weeks, and the cartilage degradation response is assayed for each dose for selection of o the minimally therapeutic effective dose. Therapeutically effective dose is defined as the dose giving a significant decrease in the cartilage specific biomarker.

hi a preferred embodiment said therapeutically effective dose leads to a reduction of the cartilage specific biomarker of 20-90 %, more preferably 30-70% and most preferably 40- 50% of the control level.

In a preferred embodiment said marker is CartiLaps ELISA as disclosed by Christgau etal. 2001.

In yet another aspect the invention is concerned with the use of a composition for preventing, treating or alleviating conditions involving elevated cartilage degradation comprising a selective estrogen receptor modulator (SERM) selected from droloxifene, tamoxifen, levormeloxifene, chroman derivatives, coumarin derivatives, idoxifene, nafoxidine, toremifene, TAT-59, LY-353381, CP-336156, MDL-103323, EM-800, ICI-182, ICI 183,780 and 19-nor-testosteone derivatives or a pharmaceutically acceptable salt thereof.

h another embodiment the present invention relates to a method of preventing, treating or alleviating conditions involving elevated cartilage degradation comprising administering to a subject a selective estrogen receptor modulator (SERM) selected from droloxifene, tamoxifen, levormeloxifene, chroman derivatives, coumarin derivatives, idoxifene, nafoxidine, toremifene, TAT-59, LY-353381, lasofoxifene, MDL-103323, EM-800, ICI- 182 jα 183,780, ICI 164,384, ICI 183,780, GW 5638, 19-nor-testosteone derivatives and pharmaceutically acceptable esters, ethers and salts thereof in amounts such that the combination decreases cartilage degradation.

In a preferred embodiment the present invention relates to a method of preventing, treating or alleviating conditions involving elevated cartilage degradation comprising administering to a subject an effective amount of a selective estrogen receptor modulator (SERM) selected from droloxifene, levormeloxifene, chroman derivatives, nafoxidine, TAT-59, LY-353381, lasofoxifene, MDL-103323, EM-800, ICI-182,780, ICI 183,780, ICI 164,384, GW 5638, ICI 183,780, 19-nor-testosteone derivatives and pharmaceutically acceptable esters, ethers and salts thereof.

It will be understood that in accordance with each aspect of the invention, preferred SERM's are SERM'S which contain the core structure shown below as Formula 2:

Formula 2

The dotted bond indicates a single or double bond.

It will be appreciated that this is present in chromans and also in numerous other (but not all) known SERM's. The core structure may in particular be extended at the positions marked R in Formula 3 below:

Formula 3

Each extension at a position R may be any structure attached to the core structure of Formula 2 to produce a SERM.

Preferably, a SERM for use in the invention in any of its aspects should have a binding affinity Ki for the estrogen receptor alpha of not less than 10 μM, preferably not less than 1 μM, andor a binding affinity for the estrogen receptor beta of not less than 10 μM, preferably not less than 1 μM.

In a most preferred embodiment of the present invention said SERM comprises levormeloxifene, chroman derivatives and 19-nor-testosteone derivatives or pharmaceutically acceptable salts thereof.

Compounds referred to above by trivial chemical names are more full defined as follows:-

trans-l-(2-(4-(-methoxy-2,2-dimethyl-3-phenyl)-5H-benzopyran-4-yl)-

Ormeloxifene phenoxy)ethylyl)-pyrrolidene

Raloxifene/Evi 6-hydroxy-2-(4-hydroxyphenyl)-benzo(p)-thien-3-yl-4-(2-(l-piperidinyl)- sta LY 117018 ethoxy)-phenylmethane

Toremifene/Fa FC- reston 1157/NK622 Z-2-(4-(l,2-diρhenyl-l-4-chloro-butenyl)-phenoxy)-N,N-dimethyl ethanamine

Tamoxifene ICI 46,474 Z-2-(4-( 1 ,2-diphenyl- 1 -butenyl)-ρhenoxy)-N,N-dimethyl ethanamine

6-hydroxy-3-(4-(2-(l-piperinydyl)-ethoxy)-phenoxy)-2-(4-hydroxyphenyl)-

Arzoxifene LY-353381 benzo(p)-thiophene

Cis-lR-[4'-pyrrolidino-ethoxyphenyl]-2S-phenyl-6-hydroxy-l,2,3,4,-

Lasofoxifene CP 336,156 tetrahydronaphtalene D-(-)-tartrate

ERA-923/TSE- lH-indol-5-ol l-[[4-[2-(hexahydro-lH-azepin-l-yl)ethoxy]phenyl]rnethyl]-2-

Bazedoxifene 424 (4-hydroxyphenyl)-3-methyl- monoacetate

Tesmilifene DPPE N,N-diethyl-2-[4-(phenylmethyl)-phenoxy] etaneamine

N-n-butyl-N-methyl-1 l-(3,17b-dihydroxyoesra-l,3,5 (10)-trien-7a-

ICI 164,384 yl)undecanamide

Ospemifene FC-1271a Z-2-(4-(4-chloro- 1 ,2-diphenyl-but- 1 -enyl)phenoxy)ethanol

3-phenyl-4-[[4-[2-(l-piperidinyl)ethoxy]phenyl]methyl]-2H-l-benzopyran-7-

CHF-4056 ol

(E)-4-[ 1 -[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4-isopropyl)phenyl- 1 -

Miproxifene TAT-59 butenyl]-ρhenyl-monophosphate

Idoxifene SB 223 030 Pyrrolidino-4-iodo-tamoxifene

7a-(9-(4,4,5,5,5-pentaflouro-pentyl-sulphinyl)-nonyl)-estra-l,3,5(10)-triene-

Faslodex ICI 182,780 3,17b-diol. l-(2-(4-(3,4-dihydro-6-methoxy-2-phenyl-naphtyl)-phenoxy)-ethyl)-

Nafoxidene 11100A pyrrolidine

(E)-l-butanamine, 4-[4-(2-chloro-l,2-diphenylethenyl)phenoxy]-N,N-diethyl-

MDL-103323 dihydrogen citrate

(s)-(+)-4-[7-(2,2-dimethyl-l-oxoρro-poxy)-4-methyl-2-[4-[2-(l-

EM- piperinidyl)ethoxy]phenyl] 2H- 1 -benzopyran-3 -yl]-phenyl 2,2-dimethyl

800/SCH57050 propanoate

Droloxifene Z-2-(4-( 1 -3 -phenol-2-ρhenyl- 1 -butenyl)-phenoxy)-N,N-dimethyl ethanamine

EM- 7-hydroxy-2-(4-(2-piperidinoethoxy)ρhenyl)-3-(4-hydroxyphenyl)-4-methyl-

652/SCH57058 2H-l-benzopyran

(E)-l,2,-diphenyl-l-[4-[2(2-hydroxyethylamino)-ethoxy]-ρhenyl]-3,3,3-

Panomifene GYKI 13504 triflouropropene

Chlomiphene 2-(4-(2-chloro- 1 ,2-diphenyl ethenyl)phenoxy-N,N-diethyl ethanolamine

Zindoxifene lH-indol-5-ol-2-(4-acetoxy) phenyl)-l-ethyl-3-methyl acetate

GW 5638 3-[4-(l,2-diphenylbut-l-enyl)ρhenyl]acrylic acid The compounds of the invention may be prepared by resorting to the chroman chemistry which is well-known in the art, for example in P. K. Arora, P. L. Kole and S. Ray, Indian J. Chem. 20 B, 41-5, 1981; S. Ray, P. K. Grover and N. Anand, Indian J. Chem. 9, 727- 5 8,1971; S. Ray, P. K. Grover, V. P. Kamboj, S. B. Betty, A. B. Kar and N. Anand, J. Med. Chem. 19, 276-9, 1976; Md. Salman, S. Ray, A. K. Agarwal, S. Durani, B. S. Betty, V. P. Kamboj and N. Anand, J. Med. Chem. 26, 592-5, 1983; Teo, C, Sim, K., Bull. Singapore Natl. Inst. Chem. 22, 69-74, 1994.

o However, the invention is furthermore concerned with a general method for the preparation of compounds of formula (I) as described in US 6043269.

Pharmaceutical formulations

The present invention also relates to pharmaceutical compositions comprising an effective 5 amount of a compound according to the invention and a pharmaceutical carrier or diluent. Such compositions are preferably in the form of an oral dosage unit or parenteral dosage unit.

The compounds with which the invention is concerned may also be prepared for administration by any route consistent with their pharmacokinetic properties. The orally o administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, 5 sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, o emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p- hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

The dosage unit involved in oral administration may contain from about 0.01 to 100 mg, preferably from about 0.1 to 10 mg, and most preferably from about 0.2 to 1 mg of a compound of the invention. A suitable daily dose for a mammal may vary widely depending on the condition of the patient. However, a dose of a compound of general formula I of about 0.1 to 10 mg/kg body weight, particularly from about 0.2 to 1 mg/kg body weight may be appropriate.

For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

For vaginal administration, the drug may include, in addition to the conventional vaginal suppositories, soft capsules containing a liquid oily base or ointment and tubes containing same for injection when in use. As the base, exemplified are oily base, water-soluble base, emulsion base, ointment-like base and so on. For example, the oily base includes fats and oils such as peanut oil, olive oil, corn oil, castor oil, cacao butter, laurin fat, glycerol fatty acid ester, specifically Pharmasol (product of NOF Corp.), Witepsol (product of Dinamite Nobel Inc.), SB-base (product of Kaneka Corp.) and lanolin fat. Examples of the water- soluble base include polyethylene glycol, polypropylene glycol, glycerin and glycerogelatin. The base emulsion is an emulsified base of water-soluble base and oily base and may be of an O/W type or W/O type. In the present invention, an oily base is preferably used, by which PG is highly stabilized and superior pharmacological effects of PG can be achieved. These bases may be used solely or in an appropriate combination. The compounds of the invention may also be included in an implant, a vaginal ring, a patch, a gel, and any other preparation for sustained release.

Nasal administration may be applied from solution as a nasal spray and may be dispensed as a spray by a variety of methods known to those skilled in the art. Preferred systems for dispensing liquids as a spray are disclosed in U.S. Pat. No.4,511,069. Such systems were used in carrying out the work described in the examples set forth hereinafter. Such nasal spray solutions comprise the drug or drug to be delivered, a nonionic surfactant which enhances absorption of the drug, polysorbate-80, and one or more buffers. In some embodiments of the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is preferably between pH 6.8 and 7.2.

Legends of the figures

Figure Legends and Tables

Figure 1. Cartilage erosion in the four different condyles in 5-month-old (top) and 7-month- old (bottom) female rats maintained for 9 weeks after OVX or sham treatment. The erosion is expressed as percentage of total cartilage surface. The erosion is presented as mean erosion + SEM values for the two groups (ovx - dark green, sham - light green). Each of the four condyles were scored: Medial Tibia (Medial T), Medial Femur (Medial F), Lateral Tibia (Lateral T), and Lateral Femur (Lateral F). Finally the average of all four areas are presented (Total).

Figure 2. Cartilage and bone turnover in the OVX and sham treated rats. Cartilage turnover was assessed using the CTX-II marker (panels A, B) and bone resorption was determined by measurement of RatLaps (CTX-I) (panels C, D). The left panels (A, C) are measurement from rats that were 5-month old at start and followed for 9 weeks. The right panels (B, D) are measurements from rats that were 7-month old at start and followed for 9 weeks. The X-axis indicates weeks after OVX.

Figure 3. Correlation between the biomarker CartiLaps expressed as % of baseline (4 weeks after operation), and histological scoring of erosion (n = 16) for the experiment with 7-months old animals. Linear regression gave a correlation coefficient of R² = 0.27, and a significant p-value of 0.039.

Figure 4. Percentage of erosion in animals with low or high CartiLaps values (at week 4) in the 7-month old cohort.

Figure 5. Effect of levormeloxifene on cartilage turnover (left) and bone resorption (right) in OVX rats. Results are expressed as average % of baseline within the groups, and error bars represent standard error of mean (SEM).

Figure 6. Effect of levormeloxifene, levormeloxifene + NETA, and NETA on bone resorption (left) and Cartilage degradation (right) in ovariectomised rats. Results are expressed as average percentage of baseline within the groups, and error bars present standard error of mean (SEM).

Figure 7. Chemical structure of SERM #0781 , (-)-cis-7-hydroxy-3-phenyl-4-[4-[2- (pyrolidin-1-yl) ethoxy] phenyl] chroman.

Figure 8. CartiLaps (left panel) and RatLaps (right panel) levels in 6 month old OVX rats treated with three doses of (-)-cis-3,4-diaryl-hydroxychromane (SERM #0781), as well as vehicle and estradiol.

Figure 9. Area under curve (AUC) measurements of the CartiLaps data in figure 8. Student's T-test was performed to test significance of the difference to the vehicle treated group, *) p>0.05; **) p<0.01; NS) not significant.

Figure 10.. Spleen weights at the termination of the CIA-2 experiment. A) low dose SERM #0781, B) medium dose SERM #0781, C) high dose SERM #0781, E) estrogen, S) Sham, V) vehicle. Values are presented as mean with SEMs.

Figure 11. Cumulative incidence of arthritis in CIA animals treated with or without SERM #0781 A) low dose SERM #0781, B) medium dose SERM #0781, C) high dose SERM #0781, E) estrogen, S) Sham, V) vehicle. Values are presented as mean within the group. Arthritis index is the mean clinical arthritis score for all rats in the group. The X- axis indicates days after immunization with collagen type II (performed 1 week after OVX).

Figure 12. Average survival time in CIA animals treated with or without SERM #0781 A) low dose SERM #0781 , B) medium dose SERM #0781 , C) high dose SERM #0781 , E) estrogen, S) Sham , V) vehicle. Values are presented as percent surviving rats within each group. The X-axis indicates days after immunization with collagen type II (performed 1 week after OVX). Figure 13. A) Levels of CartiLaps in CIA animals treated with or without SERM #0781 A) low dose SERM #0781, B) medium dose SERM #0781, C) high dose SERM #0781, E) estrogen, S) Sham, V) vehicle. Values are presented as mean with SEM within each group for the % of baseline calculated for each animal with its own baseline value as reference. In the right panel (B), the total animal cohort is divided according to arthritis score in animals with mild or very mild disease (score < 2) during the study period, animals with a late onset of arthritis (after day 25) and animals with early onset of disease or very severe arthritis. Again the mean response calculated as % of individual baselines is plotted with error bars representing SEM. On the X-axis week 0 indicates time of OVX. Figure 14. Difference in CartiLaps levels separated according to absence or presence of clinical arthritis in CIA animals treated with or without SERM #0781. The groups are labelled as follows: A) low dose SERM #0781, B) medium dose SERM #0781, C) high dose SERM #0781, E) estrogen, S) Sham, V) vehicle. Values are presented as mean creatinine corrected values with SD for each group.

Figure 15: Changes in the cartilage degradation marker urinary CartiLaps measuring degradation products of collagen type II C-telopeptides during treatment with Raloxifene (upper panel) and Levormeloxifene (lower panel). Designs and treatment groups are described in methods. Error bars represent SEM.

The invention having been described, the following examples are offered by way of illustration and not limitation.

Examples:

Example 1: Cartilage erosion is induced by ovariectomv.

Several animal models of osteoarthritis have been described in the literature (Bendele et al

2001). Spontaneous OA occurs in a number of animal strains such as mice, Syrian Hamsters, Guinea pigs and non-human primates. Furthermore, various transgenic OA prone mouse strains has been developed, and surgically induced joint damage has also been extensively studied. Models that demonstrate a slowly progressing disease are probably most relevant for the slowly developing forms of the human disease. These models have been extremely useful in identifying factors important in OA development, such as the role of matrix metallo-proteinases (MMPs) in cartilage degradation, the importance of TGF-β in osteophyte formation and the role of Nitric oxide for induction of

MMP production and chondrocyte apoptosis. However, the models have so far not been applied to the study of the potential role of estrogen in preserving cartilage integrity. A recent study in cynomologous monkeys demonstrated OA like pathological changes within articular joints of ovarectomized animals, which were prevented by exogenous estrogen production (Ham et al 2002). This points to a potential chondro-protective role of estrogen, and compounds acting on the estrogen receptor such as SERMs.

We established a model for in vivo assessment of postmenopausal OA, by performing ovariectomy (OVX) on female rats. The validity of this model was assessed in 5 and 7 months old rats by quantifying articular cartilage damage by histological evaluation using a scoring system developed to quantify erosive changes in knee joints. Furthermore, the levels of cartilage and bone metabolism was assessed by specific Biochemical markers; CartiLaps (CTX-II) specific for collagen type II degradation products as an indicator of cartilage turnover (Christgau et al 2001), and RatLaps (CTX-I) specific for bone resorption derived degradation products of collagen type I. The animals in this study consisted of two cohorts of 20 female Sprague-Dawley rats (obtained from Charles River laboratory, Germany). One cohort was 5-month old animals, and in the second cohort the animals were 7-month old. Each cohort was divided in two groups, 10 in each group, and kept for 9 weeks. The animals were received in the animal facilities 2 months prior to study start to enable a thorough acclimatization. At baseline the weight was determined and the animals were randomised to the two groups. Ovarectomy is performed on all rats by first anaesthetising with Hypnorm-Dormicum (1 part Hypnorm® + 1 part Dormicum® + 2 part sterile de-ionized water). The rats are given 0.15-0.2 ml/100 g body weight) and a standard sham operation or ovariectomy are performed. The weight of the animals was determined weekly throughout the experiment. Throughout the study the animals were weighed, and blood as well as urine samples taken at regular intervals. Urine samples were taken as spot urine samples, by either inducing urination by gently rubbing the belly of the animal or by placing it in a metabolic cage for 30 - 60 min and collecting the resulting urine sample. At termination the knees were isolated and the cartilage analysed for erosion by histology.

The knees were decalcified in 10% formic acid, 2% formaldehyde. The decalcified knee joints were cleaved along the medial collateral ligament into two sections and embedded in paraffin. The embedded knees were then cut in three different depths (0, 250, and 500 μm) from the medial collateral ligament each section was then stained in Toluidine Blue. The cartilage sections were scored blinded for the following parameters: Erosion, loss of proteoglycan, black dead chondrocytes, loss of chondrocytes, cysts, fibrillation, and bone formation in the cartilage. All changes were measured as percentage of total cartilage surface and the incidence of erosion in the groups (OVX/sham) were also calculated. RatLaps (CTX-I, bone resorption) and CartiLaps (CTX-II, cartilage degradation) levels were measured in serum and urine samples respectively.

CartiLaps ELISA is measured in urine using a competitive ELIS A employing a monoclonal antibody (MabF46) specific for collagen type π C-telopeptide fragments (Christgau et al 2001). The assay was performed by first incubating biotinylated collagen type II C-telopeptide derived peptide (EKGPDP) on a streptavidine microtitre plate and then sample as well as primary antibody was added. Monoclonal antibody MabF46 specific for collagen type U C-telopeptide fragments was used in a competitive ELISA format developed for measurement of urine samples. Briefly described the assay was performed as follows: Biotinylated collagen type II C-telopeptide derived peptide (EKGPDP) was diluted in coating buffer (1.5 mg/L) and 100 μL was pipetted into each well of 5 streptavidine coated micro-liter plates. (Micro-Coat, Munich, Germany). The plates were incubated for 30 ± 5 min. at 18 - 22°C. The plates were washed 5 times with washing buffer and 40 μL of calibrators, controls or unknown samples were pipetted into the wells. All samples were measured in duplicate. One hundred μL of primary antibody (MabF46) diluted in assay buffer to a concentration of 19 mg/L was pipetted into each well. After o incubation over night at 4°C, the plates were washed and a peroxidase labelled anti mouse antibody was added, followed by visualization of bound antibody with a chromogenic peroxidase substrate. The absorbency at 450 nm. (650 nm. reference) was measured in an ELISA plate reader and the concentration of the unknown samples was determined by constructing a standard curve from measurement of the calibrators with known 5 concentrations of type II collagen peptide. The concentration of the CartiLaps ELISA [ng/L] was standardized to the total urine creatinine [mmol/1]: Concentration/creatinine [ng/mmol]. Measurement precision of the assay was 7.1 and 8.4 % for intra and inter-assay variation, respectively. RatLaps ELISA measures collagen type I C-telopeptide degradation products using a 0 specific monoclonal antibody in a competitive ELISA form. The assay is applicable for measurement of both urine and serum samples, but only serum samples were assessed in this study. The assay is commercially available (Nordic Bioscience Diagnostics, Herlev, Denmark). All serum samples measured in the assay were from animals, which had been fasting for at least 6 hours prior to sampling. Briefly described, the assay is performed by 5 incubating a biotinylated form of a synthetic peptide representing the C-telopeptide epitope

EKSQDGGR. This is followed by addition of sample and primary antibody and after overnight incubation; the amount of bound antibody is visualized using a peroxidase labeled secondary antibody and a chromogenic peroxidase substrate. The concentrations in the samples were determined from construction of a calibration curve based on o measurement of synthetic peptide standards . In both cohorts, The OVX group had significant increased erosion in the medial femur compared to the sham group (p=0.037 and p = 0.08 respectively for 5- and 7-month old animals). Cartilage in other areas of the knee joints showed more erosion in the OVX, but only in the medial tibia of the 5-month old cohort and the lateral femur of the 7-month old cohort did the differences reach statistical significance (p<0.01 and p=0.009 respectively). The total score over the four areas showed significant increased erosion in the OVX group compared to the sham group in both 5- and 7-month old animals (p=0.09 and p = 0.008 respectively) (fig. 1). In both 5- and 7-month old animals, OVX induced a highly significant increase in CTX-I and CTX-II levels (Fig. 2). The results are in accordance with the histology data. Whereas the increase in bone resorption measured with the CTX-I marker is sustained throughout the 9-week study period in OVX animals, the increase in CartiLaps is most pronounced at week 4 after OVX and then shows a decreasing tendency. The correlation between change in CartiLaps and erosion was assessed (fig. 3). The correlation coefficient R² = 0.27 indicates a significant (p = 0.039) correlation between the two parameters, which would be expected. The correlation of CartiLaps to erosion in the Lateral Femur (where the highest erosion were seen) was also significant (R² = 0.25, p = 0.048; data not shown). Another way to assess the association between CartiLaps and erosion quantified by histology is to evaluate the association between levels as well as changes in CartiLaps levels and subsequent cartilage erosion. In figure 4 the data for the 7-month-old cohort has been processed in this way. The animals were divided into two groups according to CartiLaps at week 4 after the OVX: Low CartiLaps (28-75 ng mmol) and high values (97 - 520 ng/mmol). The animals with low CartiLaps data have only small erosion whereas the animals with high CartiLaps values have a higher percentage of erosion (fig. 4) as well as higher incidence of erosion. Similar results were obtained when data from the 5-month-old animals were analysed.

In conclusion, the results demonstrate that significant increases in both bone and cartilage turnover is induced in OVX treated rats, and that cartilage erosion appears in OVX treated rats after 9 weeks. This points to the important role of estrogen and compounds acting through the estrogen receptor for maintaining normal cartilage turnover and it points to a cartilage preserving potential of estrogen and SERM's as demonstrated in the following examples. Furthermore, the results validate the use of the OVX model as a relevant in vivo model of postmenopausal OA. Finally the results demonstrate that the CartiLaps assay may be used as a relevant biochemical marker of subsequent articular cartilage degradation in rat models of arthritis.

Example 2: Levormeloxifene is a potent inhibitor of cartilage degradation in the ovariectomized rat.

Levormeloxifene was assessed in the OVX model of postmenopausal OA (see example 1) to assess potential cartilage protective effects of this SERM. Seventy 6-month old female Sprague-Dawley rats were obtained from Charles River, Germany and used in the study. 10 animals were subjected to sham operation and the remainder (60 animals) were ovarectomized. The ovarectomy and handling of animals were performed as described in example 1. The OVX animals were divided in 5 groups of 12 animals treated with vehicle, 17α-ethynyl-estradiol (Sigma, E-8875, Lot # 21K1267) 0.1 mg/kg day or three doses of levormeloxifene (0.2, 1 or 5 mg/kg day). All treatments were administered orally in suspension (50% propylene glycol (Unikem, Copenhagen, Denmark, Cat. # 291443), 0.075 M NaCl) daily for 5 days a week, with no administration in the weekend. Throughout the study the animals were weighed, and blood/urine samples taken at regular intervals. Urine samples were taken as spot urine samples, by either inducing urination by gently rubbing the belly of the animal or by placing it in a metabolic cage for 30 - 60 min and collecting the resulting urine sample. At termination the knees were isolated and the cartilage analysed for erosion by histology. The lowest dose (0.2 mg/kg/day given 5 days a week) showed an identical development in body weight as sham or OVX treated animals, i.e. no significant change in body weight over the 9 week study duration, whereas the medium dose (1 mg/kg/day given 5 days a week) and the high dose (5 mg/kg/day given 5 days a week) displayed somewhat lower growth than these reference groups (data not shown). Levormeloxifene induced a significant reduction in CartiLaps levels in the OVX animals to the same levels as those seen in estrogen treated or Sham animals (fig. 5). No difference was seen between the three levormeloxifene treatment groups, indicating that a reduction in dose is possible while maintaining full response on cartilage, hi contrast the compound showed lower potency for reducing bone resorption. Only the highest dose of levormeloxifene was able to significantly reduce levels of RatLaps (CTX-I) to the same levels as observed in the sham or estradiol treated groups.

In conclusion this indicated that Levormeloxifene can reduce cartilage turnover, and thus cartilage erosion, in OVX treated rats to the levels seen in normal animals. Furthermore this SERM show a preferential effect on cartilage compared to bone and thus it may be highly useful as a potential chondro-protective treatment in postmenopausal OA.

Example 3: Levormeloxifene, and levormeloxifene + Norethisterone acetate (NETA) are potent inhibitors of cartilage degradation, but have no effect on uterus weight in 314-month old ovariectomised rats.

Levormeloxifene alone or combined with the gestagene analogue Norethisterone acetate (NETA) was assessed in the OVX model of postmenopausal OA in young animals (314 month) to assess potential uterus and cartilage protective effects. Fifty 314-month old female Sprague-Dawley rats were obtained and used in this study. 10 animals were subjected to sham operation and the remainders (40 animals) were ovariectomised. The ovarectomy and handling of animals were performed as described in example 1. The OVX animals were divided in 4 groups of 10 animals treated with vehicle, levormeloxifene (1.0 mg/kg/day given 5 days a week), NETA (1.0 mg/kg/day given 5 days a week) or levormeloxifen + NETA (both compounds given as 1.0 mg/kg/day given 5 days a week) and treated for 5 weeks. All treatments were administered orally in suspension (50% propylene glycol, 0.075 M NaCl) daily for 5 days a week, with no administration in the weekend. Throughout the study the animals were weighed, and blood/urine samples taken at regular intervals. Urine samples were taken as spot urine samples, by either inducing urination by gently rubbing the belly of the animal or by placing it in a metabolic cage for 30 - 60 min and collecting the resulting urine sample. Treatment with levormeloxifene showed an identical development in body weight as sham animals, whereas the levormeloxifene + NETA treatment lowered the growth in the animals. Treatment with vehicle or NETA increased the bodyweight with the vehicle group having the highest growth rate (table 1). Treatment with levormeloxifene, levormeloxifene + NETA, or NETA did not have any effect on uterus weight in OVX rats compared to the vehicle treated rats (table 1). The sham rats had an increased uterus weight compared to the OVX rats, which 5 is due to endogenous estrogen production. Treatment with levormeloxifene, NETA or levormeloxifen + NETA induced a significant reduction in CartiLaps levels in the OVX animals to the same level as those seen in the sham animals (Fig. 6). When comparing the CartiLaps and RatLaps data to examples 1, 2 and 4, it must be taken into account that the cohort in this experiment was much younger (314 months at study start), and thus have a 0 substantial skeletal growth which will contribute to the measurement both in the RatLaps assay and in the CartiLaps assay due to the presence of collagen type U in the growth plate. Hence, in the sham group there was decrease in CartiLaps of approximately 40 % over the 4- week observation period due to the gradual decrease in skeletal growth at this age range (fig. 6). Thus the increase in bone and cartilage turnover induced by OVX as seen in the vehicle 5 treated group has to be considered in relation to the sham group. Treatment with levormeloxifene or levormeloxifen + NETA induced a significant reduction in CartiLaps and RatLaps levels in the OVX animals to the same level as those seen in the sham animals (fig. 6). Treatment with NETA alone did not reduce the CartiLaps levels significantly from the vehicle treated animals. However, there was a trend towards lower CartiLaps levels in the o group of animals treated with NETA alone.

In conclusion, the addition of the gestagene analogue NETA to levormeloxifene does not impair the potential chondro-protective effects of this SERM. This ^"potential combination therapy may hold clinical advantages in human subjects where estrogenic effects on uterus and breast tissues of SERMs is a significant concern, and thus addition of a gestagene 5 analogue could be desirable as this provides a well recognized method of preventing estrogenic stimulation of these tissues.

Table 1: Effect of levormeloxifene (Levo), levormeloxifene + NETA, and NETA on bodvweight and uterus weight in ovariectomsied (OVX) rats. Results are expressed as mean weight + SEM values

Example 4: (-)-cis-3,4-diaryl-hvdroxychromane is a potent inhibitor of cartilage degradation in the ovariectomized rat.

The chroman SERM compound (-)-cis-3,4-diaryl-hydroxychromane (compound #0781, fig 7) was assessed in the OVX model of postmenopausal OA (see example 1) to assess potential cartilage protective effects of this SERM. Seventy 6-month old female Sprague- Dawley rats were obtained and used in the study. 10 animals were subjected to sham operation and the remainder (60 animals) were ovariectomized. The ovarectomy and handling of animals were performed as described in example 1. The OVX animals were divided in 5 groups of 12 animals treated with either vehicle, 17 -ethynyl-estradiol (Sigma, E-8875, Lot # 21K1267) 0.1 mg/kg/day given 5 days a week or three doses of (-)-cis-3,4- diaryl-hydroxychromane (0.2, 1 or 5 mg kg/day given 5 days a week). All treatments were administered orally in suspension (50% propylene glycol, 0.075 M NaCl) daily for 5 days a week, with no administration in the weekend. Throughout the study the animals were weighed, and blood/urine samples taken at regular intervals. Urine samples were taken as spot urine samples, by either inducing urination by gently rubbing the belly of the animal or by placing it in a metabolic cage for 30 - 60 min and collecting the resulting urine sample. RatLaps and CartiLaps measurements (fig. 8, 9) demonstrate a significant effect of the compound on both bone resorption and cartilage turnover. Whereas the highest dose of the SERM suppressed CartiLaps levels to the level of estrogen or sham animals (fig. 8 left panel), it was not able to suppress bone resorption to the same extent as estrogen. This indicates that this compound has a stronger action on cartilage than on bone, and thus may exert a potential chondro-protective role in the treatment of postmenopausal OA.

Example 5: Delay of onset and reduction in severity and incidence of inflammatory arthritis induced by (-)-cis-3,4-diarvI-hvdroxychromane in collagen type II immunized Lewis rats.

One of the most commonly employed animal models of RA is the Lewis-rat Collagen Induced Arthritis (CIA) animal model. In this model, joint inflammation is induced by immunization with collagen type II which provoke a severe inflammatory response to joint cartilage apparent as paw joint swelling and subsequent destruction of joints when histological analysis is performed. In this study we determined the effect of estrogen as well as SERM #0781 ((-)-cis-3,4-diaryl-hydroxychromane, fig. 7) on the disease development and severity in CIA. Arthritis was monitored by macroscopic scoring of swelling and redness of the paws. Cartilage and bone erosion was monitored by quantifying urinary levels of CartiLaps and serum levels of RatLaps. Furthermore, the uterine weights of the animals were determined to assess estrogenicity of the estrogen.

The study cohort comprised 66 female Lewis HanHsd rats (Harlan, The Netherlands). After 1-week acclimatization ovarectomy is performed on 55 of the rats by first anaesthetising with Hypnorm-Dormicum (1 part Hypnorm® + 1 part Dormicum® + 2 part sterile de-ionised water). The rats are given 0.15-0.2 ml/100 g body weight) and a standard ovariectomy are performed. Eleven rats are subjected to sham operation using the same anaesthesia procedure. The rats are immunized 1 week after OVX. Each rat is immunized with 150 μg bovine collagen type II (Cat. Nr. # 2002-1; Chondrex, Redmond, WA, USA) emulsified in Incomplete Freunds Adjuvant (IF A). 150 μl emulsion is injected intra- dermally at the base of the tail. The paws of the rats are scored daily from day 11 by visual observation of the paws. Each paw is scored on a scale 0-4 and the score for the four paws are added. If the combined arthritis score reached a score of 10 for a given animal or developed very severe arthritis in one paw or had CIA for more that 16 days, the animal was killed for ethical considerations, as more severe arthritis is associated with significant pain and discomfort. The remainder of the animals were maintained for 42 days after OVX before being terminated.

After OVX and sham operations 59 animals remained which were administered randomly to 6 groups (see table 3). 10 animals were in the sham group (S), 10 animals were in the group given vehicle alone (50 % propyleneglycol, 0.15 M NaCl) (V), 11 animals were in the group give 0.5 mgkg/day estrogen (17 -ethynyl-estradiol, Sigma, E-8875, Lot # 21K1267)(E). And 10, 8 and 10 animals respectively were in the groups treated with 0.2, 1 and 5 mg kg/day of SERM #0781 (A, B and C). The compounds are suspended in propylenglycol (Unikem, Copenhagen, Denmark, Cat. # 291443) by vortex and ultrasound. When a homogenous suspension is obtained the compound is diluted in the same amount (1:1) of 0.15M NaCl. The compounds were given orally by gavages for five days a week, with no administration during weekends. Treatment with vehicle, estrogen and SERM #0781 in three different doses was started the day after OVX, i.e. prior to collagen type II immunization. Uterus weights were measured at the end of the experiments to determine the uterotrophic effect of SERM #0781. Highest weights were observed in the sham animals (S) representing normal uterus weights. OVX resulted in a more the 50% reduction of uterus weight as seen in the vehicle treated groups (V). Estrogen treatment (E) restores the uterus weight to some extent, however not completely. SERM #0781 treated rats show a slight increase in uterus weight regardless of dose compared to vehicle treated rats. Spleen weights were determined at the end of the CIA experiment. Spleen weights were reduced in all SERM #0781 treated groups and the estrogen group compared to the sham and vehicle groups. Statistical analysis of the medium dose SERM #0781 (B) compared to the vehicle group (V) results in p=0,014 (n=5) (Fig 10). This may indicate an immunosuppressive effect of the SERM #0781 that would be of potentially great importance for CIA development.

The first signs of arthritis were observed day 12 after immunisation in the sham (S), vehicle (V) and medium dose SERM #0781 group (B). OVX rats were more susceptible to CIA in accordance with previously published studies. Day 15 after immunisation the vehicle group had the highest incidence and the highest severity of CIA (table 2).

Table 2. Arthritis incidence and average severity score in CIA animals treated with or without SERM #0781 for day 15, 23 and 40. Groups are as follows: A) low dose SERM #0781, B) medium dose SERM #0781, C) high dose SERM #0781, E) estrogen, S) Sham, V) vehicle.

The high dose SERM #0781 (C) was completely protected until day 18 (fig. 11 ). All three SERM #0781 treated groups remained protected and showed a lower arthritis index until day 25 after immunisation. However the estrogen treated rats showed the most severe disease from day 25. As the rats reached a score of 10 (out of 16) or developed very severe arthritis in one paw or had CIA for more that 1 days, they had to be terminated. Figure 12 shows that all three SERM #0781 groups had an increased survival during the first five weeks after immunisation compared to the vehicle group. Also the estrogen treated group showed an increased survival during the first four weeks. At the end of the experiment 50% of the low dose SERM #0781 treated rats remained whereas all other groups were reduced to 20%. Taken together, figure 11 and 12 shows that SERM #0781 has a protective effect on CIA development. Interestingly SERM #0781 treated rats were less susceptible to CIA than the sham group, indicating that the SERM #0781 had additional protective effects than just compensating for the lack of estrogen.

The CartiLaps analysis of articular cartilage turnover in this young animal cohort (9 week old at study start) was hampered by the significant skeletal growth occurring during the study period. Skeletal growth is associated with rapid turnover of the growth plate, which is made up of a cartilage like collagen type π containing matrix, and metabolites from this process will contribute to the measurement in the CartiLaps assay. Hence there was an overall decrease of approximately 50 % in the sham treated animals over the 7-week observation period (fig. 13 A). OVX rats treated with vehicle only showed increased levels of CartiLaps after three weeks compared to the sham group where as estrogen treated rats showed reduced levels (fig. 13 A). SERM #0781 treatment reduced the CartiLaps levels in a dose dependent fashion. The low dose group behaved similar to the vehicle group and high dose treatment resulted in a significant reduction of CartiLaps levels compared to vehicle (p=0,012) in similarity to estrogen treatment (p=0,0021). The great impact of arthritis on the CartiLaps levels shown in figure 13 B explains the large variability in the CartiLaps levels in figure 13 A. At three weeks after OVX (= two weeks after immunisation with collagen type H) there was a significant difference between the treatment groups, but already at this time point the subsequent disease course influenced the CartiLaps levels (fig. 13 A). The time point of immunisation, week 1 in figure 13, is one week after OVX. Rats that remain healthy during the six weeks of observation have an endpoint value of 26,5 % of the baseline level of CartiLaps. This group (n=10) also includes rats with mild symptoms (score < 2) of CIA that appeared during the last week of observation. Significantly higher endpoint values were found in arthritic rats (p<0,005). 41 rats had to be terminated earlier that planned because of very severe inflammation or because they had shown signs of CIA for more that 15 days (CIA severe/early). Eight rats had a milder disease or developed CIA later and could be kept until the final termination day week 6 (CIA moderate late). Both these CIA groups showed significantly increased CartiLaps levels week 4 after immunisation (p=0,0034 and p=0,014 respectively). Interestingly the CIA moderate late group had significantly increased levels already week 2 (p=0,012), which corresponds to two weeks before CIA onset. This may indicate that increased CartiLaps levels do not reflect the erosion of cartilage caused by joint inflammation, but rather an altered cartilage metabolism that precedes arthritis onset. This altered cartilage metabolism may be an early pathologic event without clinical symptoms that with time will progress and lead to arthritis development. Treatment with a SERM as used in this experiment may decrease this 'pre-clinical' cartilage turnover thus decreasing susceptibility for subsequent development of inflammatory arthritis. Thus the SERM compound may have a prophylactic potential for treatment of individuals at risk for development of inflammatory arthritis (RA).

Figure 14 shows the absolute values of CartiLaps at the end of the CIA experiment. The different treatment groups are shown separately. This demonstrates that also the absolute values of CartiLaps reflect the arthritis status of the rat. In the OVX OA model an increase of CartiLaps levels were induced by OVX (see examples 1 - 4). This homeostatic imbalance was restored within six weeks and could be prevented by estrogen levormeloxifene or SERM #0781 treatment. This period of imbalance in cartilage metabolism following OVX may be the reason for increased susceptibility to develop cartilage erosions and CIA during this period. Additional beneficial effect of the SERM treatment as exemplified by the effect of SERM #0781 in this experiment may be that they result in a general immunosuppressive effect (see fig. 10), which may also have a significant therapeutic potential for treatment of inflammatory arthritis.

In conclusion the experiment indicate a significant prophylactic and/or therapeutic effect in inflammatory arthritis of SERMs such as the hydroxy-chromane compound SERM #0781 (fig. 7).

Example 6: Suppression of cartilage turnover in humans induced by selective estrogen receptor modulators.

The effects of selective estrogen receptor modulators on cartilage turnover were assessed in a population of 235 healthy postmenopausal women aged 49 - 65 years. Cartilage turnover was quantified by measurement of degradation products of collagen type II C- telopeptides released during cartilage catabolism. For comparison serum levels of C- telopeptides of collagen type I ('CrossLaps') as measured as a specific marker of bone resorption.

The study participants had engaged in two different dose-finding studies in which, the effect of two different SERMs on the prevention of osteoporosis had been assessed. Fasting urine samples was collected regularly throughout the trial period and frozen for later study purposes. All participants had been postmenopausal for more than one year and had concentrations of serum estiadiol below 25 pg/ml. All participants were without clinically significant acute or chronic diseases and none had received medications known to affect bone or cartilage metabolism for 6 months prior enrolment. Table 3 demonstrates the physical demographics and relevant biomarker levels of the participants at baseline. Both studies were performed according to the Helsinki Declaration II and the European standard for Good Clinical Practice. 5 Designs: The raloxifene study included five treatment groups and lasted five years. One group received placebo and three groups were tieated with raloxifene, in the doses of 30, 60 or 150 mg/day p.o. throughout the 5-year study period. One group was given placebo treatment for the first three years and then changed to raloxifene 60 mg/day. The levormeloxifene study included five treatment groups and lasted two years. One group o received placebo and four treatment groups received levormeloxifene in the doses of 1.25, 5,

10 and 20 mg/day p.o., respectively. The treated groups received levormeloxifene for 12 months and were followed for an additional 12 months without treatment. All participants in both studies received a calcium supplement of 400 - 600 mg/day during the total trial period. Random number tables were used in both studies as method for allocating participants to 5 double blinded treatment.

Demographics: Age and years since menopause (YSM) was registered through questioning at baseline. Body height and weight was measured to the closest 0.1 cm and 0.1 kg, respectively on participants wearing light indoor clothes and no shoes. Body mass index (BMI) was calculated as BMI = m/h², where m is the weight of the patient in kg and h is the height in 0 meters.

Urinary levels of collagen type II C-telopeptide fragments was measured by the CartiLaps ELISA as described in example 1.

Serum CrossLaps ELISA: Serum CrossLaps was included for comparison. The Serum CrossLaps One Step ELISA is a sensitive indicator of bone resorption and the assay was 5 performed as described by the manufacturer (Nrodic Bioscience Diagnostics, Herlev,

Denmark). Serum CrossLaps measurements were performed at baseline and after 3, 6 and 12 months of treatment in both trials.

Statistical analyses: Comparative analyses between study-groups at baseline were performed using independent t-tests and ANOVA. Correlation between CartiLaps and CrossLaps values o was performed by Spearman's correlation. Results in text are mean ± SD. Statistical calculations were performed using SPSS for Windows 10.1 (SPSS Inc. Chicargo, IL, USA). Differences were considered significant if p<0.05.

Table 3 provides the baseline values for the two study populations divided into the treated 5 groups and their controls. Independent T-tests performed between the raloxifene study population and the levormeloxifene study population demonstrated a slight difference between the two groups with regard to age. The levormeloxifene group was thus 1.1 ± 0.5 years (p<0.05) older compared to the raloxifene group and had on average been postmenopausal for a corresponding 1.9 ± 0.5 years (p<0.05) longer than the raloxifene l o group. Serum CrossLaps values reflecting osteoclast mediated bone resorption were in accordance with this slight difference in age and YSM statistically higher in the levormeloxifene group. There were no statistically significant differences between the two study populations with regard to BMI and urinary CartiLaps levels reflecting cartilage collagen type II degeneration. The variance between the listed parameters in table 1 was

15 evaluated by ANOVA for both study populations individually and no statistical significant differences were found between the treated groups and their controls. The upper panel of figure 15 display the results of the CartiLaps measurements from baseline to the 5-year visit in the raloxifene group. All three doses of raloxifene (30, 60 and 150 mg/day) induced a significant and sustained decrease in CartiLaps levels to

2 o approximately 65% of baseline, whereas CartiLaps levels in placebo treated women showed a slight increase during the five-year study period. The treated groups reached a plateau of the CartiLaps marker after 12 months with no apparent dose response. The group that received placebo for the first 3 years and thereafter was given raloxifene 60 mg/day demonstrated a similar response to the institution of therapy. 5 Results of CartiLaps measurements from the levormeloxifene study are shown in the lower panel of figure 15. In this study a 50 % decrease in CartiLaps levels was observed after 3 to 6 months of levormeloxifene treatment. There were only relatively small and nonsignificant differences between the responses in the four treatment-groups during the 12 months of therapeutic intervention. After cessation of the levormeloxifene treatment all

3 o four treated groups reverted to baseline levels of CartiLaps. When CrossLaps measurements, quantifying bone resorption, were correlated to their corresponding CartiLaps values, only a weak correlation of r = 0.42 was found. The effect of estrogen and related compounds, such as SERMs, on estrogen responsive tissues like the endometrium, bone and breast has been extensively studied. Cartilage is not generally viewed as an estrogen responsive tissue and as described previously conflicting reports exists in the literature on whether estrogen exerts beneficial protective effects on cartilage. Here we show that two different SERMs induce a significant decrease in urinary levels of collagen type II C-telopeptide fragments demonstrating, that these compounds have a marked chondro-protective effect. Specific proteolytic degradation of collagen type II is a key event in cartilage degradation both in vitro and in vivo. Metabolites derived from this protein are thus well suited as biochemical markers of cartilage degradation. The CartiLaps assay has been demonstrated to correlate with other known biochemical markers assessing cartilage turnover and in a longituidinal study of knee OA patients, CartiLaps measurements were correlated with radiologically evaluated joint space narrowing and standardized scores on pain and function. Thus, the effect of the SERM treatment on CartiLaps levels, as we observe in this study, represent a direct effect on systemic cartilage turnover.

The study populations included in the two studies comprise postmenopausal women that were not selected according to a specific degree of OA. However, it has previously been shown that postmenopausal women experience an increased incidence of OA. If cartilage degradation is reduced in postmenopausal women this would probability involve a reduced risk of developing OA. Hence, postmenopausal women, that have not been selected to have symptomatic OA, represent a relevant group for investigating preventive chondroprotective therapies. Postmenopausal women also represent the key target group for SERM therapy, as the documented and approved indications for such a therapy includes prevention of postmenopausal osteoporosis. The raloxifene treated study population was followed for five years. In the three continously treated groups the reduction in CartiLaps levels was fully expressed after 12 months of raloxifene treatment and this level was maintained throughout the study period (Fig. 15). This demonstrates, that the potential chondroprotective effect of this SERM is maintained over long term therapy, but also that the chondroprotective effect of raloxifene is relatively slowly expressed. Therapies over extended periods of time are probably required in order to provide significant therapeutic benefits. There were no significant differences between the three investigated doses (30, 60 and 150 mg) suggesting that even at the lowest dose used in this study, the maximal chondroprotective effect was obtained. When placebo treated women were changed to raloxifene they responded similarly to the SERM with a 35% decrease in CartiLaps levels.

In the levormeloxifene study the SERM was given for 12 months and the patients were then followed for an additional 12 months. There were signs of a slight dose-response effect with 20 mg daily giving the most pronounced decrease in CartiLaps levels. The decrease was fully expressed after 3-6 months therapy demonstrating, that levormeloxifene given in tested doses has a faster chondroprotective effect compared to raloxifene. Also the suppression of the cartilage degradation marker was more pronounced compared to the effects seen with raloxifene. When treatment with levormeloxifene was stopped after 12 months of therapy the CartiLaps levels returned to baseline values after 12 months demonstrating that the potential suppressive effect of levormeloxifene on cartilage degradation is reversible.

Bone resorption is reduced by both raloxifene and levormeloxifene, measured by biomarkers and in the case of raloxifene also by a long-term reduction in fracture occurrence rates. The SERMs effect on bone resorption as assessed with the CrossLaps ELISA was of a similar magnitude as the effect on cartilage degradation we here demonstrate with the CartiLaps assay.

Although the responses to SERM measured by the CartiLaps and CrossLaps assays appear similar, the two assays clearly represent different processes. First, the correlation between CartiLaps and CrossLaps measurements was weak (r=0.42). More important, several differences can be pointed out between the effect of SERMs on cartilage and bone turnover. For raloxifene all three tested doses gave a maximal effect on cartilage degradation, whereas the effect on bone resorption gave an indication of dose response. Furthermore, where 12 months of treatment was required for raloxifene to depress cartilage degradation maximally, the bone resoφtion marker was fully depressed after only 3-6 months. For levormeloxifene, there was a tendency for a dose-response effect in CartiLaps, whereas levormeloxifene in all doses induced an identical decrease in CrossLaps levels. Thus, the responsiveness of bone and cartilage to levormeloxifene is quantatively different indicating that different doses are required to obtain maximal effects on bone and cartilage turnover. These observations demonstrate different dynamics of the effects exerted by these SERMs on bone and cartilage.

In conclusion the results demonstrate that treatment with a compound such as estrogen or an estrogen like substance such as a SERM, which acts like an estrogen agonist induces a significant reduction in cartilage catabolism. As degradation of cartilage is one of the most prominent features associated with OA, an inhibition or reduction of this process will decrease the prevalence or severity of the pathological processes leading to OA. Thus SERMs constitute a novel therapeutic option for treatment or prevention of OA.

Table 3. Demographics and biomarker levels at baseline.

Raloxifene Levormeloxifene

Treatment

Placebo 30, 60 and Placebo 1.25, 5, 10 150 mg/day and 20 mg/day

Randomized patients 35 107 21 71

Age [years] 55.6 (3.5) 56.1 (3.1) 57.4 (4.1) 56.9 (3.8)

BMI^* [kg/m²] 25.9 (3.8) 26.0 (4.0) 25.4 (3.3) 26.1 (3.7)

YSM° [years] 4.1 (2.1) 5.0 (2.5) 7.7 (4.0) 6.3 (4.4)

U-CartiLaps/Creatinine 211 (141) 179 (132) 232 (93) 214 (104)

[ng/mmol]

S-CrossLaps [pmol/1] 3335 3386 (1479) 4412 (1350) 3963 (1700) (1265)

Results are mean with (SD). Body Mass Index, ^D Years Since Menopause References:

(D Felson & M Nevitt 1998): Curr Opin Rheumatol. 1998, 10: 269-272

(M Nevitt et al 1996): Arch.Intern.Med. 1996;156(18):2073-80

(Oliveria et al 1996): Epidemiology. 1996 Jul;7(4):415-9.

(Erb et al. 2000): Ann.Rheum.Dis. 2000;59(2): 105-9

Nevitt et al 2001): Arthritis & Rheumatism. 2001, 44: 811-818

(D Felson & M Nevitt 1998): Curr Opin Rheumatol. 1998, 10: 269-272

Bendele A. (2001) Animal models of Osteoarthritis. J Musculoskel Neron Interact, 1(4):

363-376

Ham KD, Loeser RF, Lindgren BR, Carlson CS. (2002) Effects of long-term estrogen replacement therapy on osteoarthritis severity in cynomolgus monkeys. Arthritis Rheum,

46(7): 1956-64

Christgau S, Garnero P, Fledelius C,Moniz C, Rosenquist C, Ensig M, Gineyts E,

Christiansen C, Qvist P. (2001) Collagen type II degradation products in urine as an index of cartilage degradation. Bone, 29: 209-215

Claims

1. A method of preventing, treating or alleviating conditions involving elevated cartilage degradation comprising administering to a subject an effective amount of a selective estrogen receptor modulator (SERM) selected from droloxifene, levormeloxifene, chroman

5 derivatives, nafoxidine, basedoxifene, miproxifene, arzoxifene, lasofoxifene, MDL-103323, EM-800, fulvestrant, ICI 183,780, ICI 164,384, GW 5638, 19-nor-testosteone derivatives and pharmaceutically acceptable esters, ethers and salts thereof.

2. A method according to claim 1, wherein said SERM comprises levormeloxifene, a l o chroman derivative or a 19-nor-testosteone derivative or a pharmaceutically acceptable salt thereof.

3. A method according to claim 1, wherein the SERM contains the core structure shown in Formula 2:

15

20

A method according to claim 1 or claim 3, wherein said SERM is a chroman derivative.

5. A method according to the claim 4, wherein said selective estrogen receptor modulator comprises a chroman derivative of the formula I

wherein:

R¹ is H, SO₂ NR₂ ⁴, S0₂ NHR⁴ ,C1, CH₃ or benzyl;

R is phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of OH, halogen, nitro, cyano, SH, SR⁴, trihalo-Cι-C₆ alkyl, -Cg -alkyl, d-Cβ alkoxy and phenyl;

R³ is phenyl substituted with ~X~(CH₂)_n — Y, wherein:

X is a valency bond, O or S,

n is an integer in the range of 1 to 12,

Y is H, halogen, OH, OR⁴, NHR⁴, NR₂ ⁴, NHCOR⁴, NHSO₂ R⁴,CONHR⁴,

CONR⁴, COOH, COOR⁴, SO₂ R⁴, SOR⁴, SONHR⁴, SONR₂ ⁴, a pyrrolidinyl ring, optionally being substituted with 1 to 3 substituents independently selected from the group consisting of H, OH, halogen, nitro, cyano, SH, SR⁴, trihalo-d -C₆ -alkyl, C\ -C₆ -alkyl and Ci -C₆ -alkoxy; and

R⁴ is Cι -C₆ -alkyl;

6. A method according to claim 5, wherein R² and R³ are arranged in trans- configuration.

7. A method according to claim 6, comprising the use of a chroman compound which is

(-)-3 ,4-trans-7-methoxy-2,2-dimethyl-3-phenyl-4 {4-[2-(pyrrolidin- 1 -yl)ethoxy] phenyl} chromane.

8. A method according to claim 5, wherein:

R¹ is H, SO₂ NR₂ ⁴ , CH₃ or SO₂ NHR⁴;

R _> ²2 a. nd R³ are arranged in cis-configuration;

R² is phenyl optionally substituted with 1 to 3 substituents independently selected from the group consisting of OH, halogen, nitro, cyano, SH, SR^ trihalo-Ci -C₆ -alkyl, Ci -C₆ -alkyl and CI -C6 -alkoxy;

R³ is phenyl substituted with — X~(CH₂)_n ~Y, wherein:

X is a valency bond, O or S, n is an integer in the range of 1 to 12,

Y is H, OH, OR⁴, NHR4, NR₂ ⁴, NHCOR⁴, NHSO₂ R⁴, CONH⁴, CONR₂ ⁴, COOH, COOR⁴, SO₂ R.⁴, SOR⁴' SONHR⁴, SONR₂ ⁴, a pyrrolidinyl ring, optionally being substituted with 1 to 3 substituents independently selected from the group consisting of H, OH, halogen, nitro, cyano, SH, SR⁴, trihalo-d -C₆ -alkyl, d-Cβ -alkyl and d -C₆ -alkoxy; and

R⁴ is d -C₆ -alkyl;

9. A method according to claim 8, comprising use of the compound of the formula la or lb having the formula:

wherein R¹, R² and R³ are as defined above.

10. The method according to claim 8, comprising use of the compound of the formula

wherein R¹ is as defined above and m is an integer from 0 to 10.

11. A method according to claim 8, comprising use of the compound of the formula

12. A method according to claim 11, comprising the use of a chroman compound 5 which is

(-)-cis- {3-phenyl-4-[4-(2-ρyrrolidin- 1 -yl-ethoxy)~phenyι] } -chroman-7-ol;

0 (-)-cis-{3-(4-1rifluormethyl-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-7- methoxychroman;

(-/+)-cis-{3-(3-methoxy-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-7- 5 methoxychroman;

(-/+)-cis- { 3 -(3 -hydroxy-phenyl)-4-[4-(2-pyrrolidin- 1 -yl-ethoxy)-phenyl] } -chroman-7-ol; 0

(-/+)-cis- {3-(4-trifluormethyl-phenyl)-4-[4-(2-pyrrolidin- 1 -yl-ethoxy)-phenyl] } -chroman- 7-ol; or

(+,-)-(cis-3 -(4-Methylphenyl)4-(4-(2-pyrrolidinoethoxy)phenyl)-chroman-7-y 1) N,N- 5 dimethylsulfamic acid ester;

(-)-cw-7-methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane .

o (+/-)-cz-f-7-methoxy-3-(3-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane .

(47-)-cώ-7-me1hoxy-3-(4-trrfluoromethy . _and

5

(-)-czs-7-methoxy-3-(4-trifluoromethylphenyl)-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane _

13. The use of SERM as defined in any one of claims 1 to 12, for the preparation of a medicament for use in the treatment, prevention, or alleviation of a condition involving elevated cartilage degradation.

14. A method for preventing, treating or alleviating conditions involving elevated cartilage degradation comprising administering to a subject an effective amount of the combination of

1) a selective estrogen receptor modulator (SERM) or a pharmaceutically acceptable salt thereof together with; 2) a progestin or a pharmaceutically acceptable salt thereof.

15. A method according to claim 14, wherein said selective estrogen receptor modulator (SERM) is selected from the group consisting of raloxifene, droloxifene, tamoxifen, 4- hydroxy-tamoxifen, 4'-iodotamoxifen, toremifene, (deaminohydroxy)toremifene, chlomiphene, levormeloxifene, chroman derivatives, coumarin derivatives, idoxifene, nafoxidine, basedoxifene, miproxifene, arzoxifene, lasofoxifene, MDL-103323, EM-800, fulrestrant, ICI 183,780, ICI 164,384, GW 5638,diethylstilbesterol, genistein, nafoxidine, nitromifene citrate, moxesterol, diphenol hydrochrysene, erythro-MEA, allenolic acid, cyclophenyl, chlorotrianisene, ethamoxytriphetol, dehydroepiandrosterone, triparanol 19-nor- progesterone derivatives, 19-nor-testosteone derivatives and pharmaceutically acceptable esters, ethers and salts and said progestins are selected from the group consisting of norethindrone, ethynodiol, desogestrel, levonorgestrel, norgestrel, norgestimate, medroxyprogesterone, danazol, lynoestrenol, dydrogesterone, chlormadinone, promegesterone, gestrinone, algestone acetophenide, allyloestrenol, cyproterone acetate, demegestone, gestodene, osaterone, hydroxyprogesterone hexanoate, medrogestone, megestrol, nomegestrol, ethynylnortestosterone, noφregneninolone, NSC-9564, norethynodrel, dexnorgestiel, gestodene, progesterone, chlormadinone acetate, drospirenone (dihydrospirorenone), or 3-ketodesogestrel and optical and geometrical isomers, pharmaceutically acceptable esters and salts thereof.

16. A method according to claim 15, wherein said SERM comprises raloxifene, droloxifene, tamoxifen, levormeloxifene, chroman derivatives, coumarin derivatives, idoxifene, nafoxidine, basedoxifene, toremifene, (deaminohydroxy)toremifene, miproxifene, arzoxifene, lasofoxifene, MDL-103323, EM-800, fulvestrant ICI 183,780, GW 5638 and 19- nor-testosteone derivatives or a pharmaceutically acceptable salt thereof.

17. A method according to claim 15, wherein said SERM incoφorates the core structure shown in Figure 2:

I

Formula 2

18. A method according to claim 15 or claim 16, wherein said SERM is a chroman derivative.

19. A method according to the claim 18, wherein said selective estrogen receptor modulator comprises a chroman derivative of the formula I

wherein:

R¹ is H, SO₂NR₂ ⁴, S0₂ NHR⁴ , CI, CH₃ or benzyl;

R is phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of OH, halogen, nitro, cyano, SH, SR⁴, trihalo-d-C₆ alkyl, d-C₆ -alkyl, Ci- C₆ alkoxy and phenyl;

R is phenyl substituted with ~X~(CH₂)_n — Y, wherein:

X is a valency bond, O or S,

n is an integer in the range of 1 to 12,

Y is H, halogen, OH, OR⁴, NHR⁴, NR₂ ⁴, NHCOR⁴, NHSO₂ R⁴,CONHR⁴,

CONR⁴, COOH, COOR⁴, SO₂ R⁴, SOR⁴, SONHR⁴, SONR₂ ⁴, apyrrolidinyl ring, optionally being substituted with 1 to 3 substituents independently selected from the group consisting of H, OH, halogen, nitro, cyano, SH, SR⁴, trihalo-d -C₆ -alkyl, Ci -C₆ -alkyl and Ci -C₆ - alkoxy; and

R⁴ is ^■Qs -alkyl;

20. The method according to claim 19, wherein R² and R³ are arranged in trans- configuration.

5

21. A method according to claim 20, comprising the use of a chroman compound which is

(-)-3,4-trans-7-methoxy-2,2-dimethyl-3-phenyl-4{4-[2-(pyrrolidin-l-yl)ethoxy] l o phenyl} chromane.

22. A method according to claim 19, wherein:

R¹ is H, SO₂NR₂ ⁴ , CH₃ or SO₂NHR⁴; 15

R² and R³ are arranged in cis-configuration;

R² is phenyl optionally substituted with 1 to 3 substituents independently selected from the group consisting of OH, halogen, nitto, cyano, SH, SR₄, trihalo-Ci -C₆ -alkyl, Ci -C₆ -alkyl 20 and CI -C6 -alkoxy;

R³ is phenyl substituted with ~X~(CH₂)_n — Y, wherein:

X is a valency bond, O or S,

25 n is an integer in the range of 1 to 12,

Y is H, OH, OR⁴, NHR4, NR₂ ⁴, NHCOR⁴, NHSO₂ R⁴, CONH⁴, CONR.Λ COOH, COOR⁴, SO₂ R.⁴, SOR⁴' SONHR⁴, SONR₂ ⁴, a pyrrolidinyl ring, optionally being 30 substituted with 1 to 3 substituents independently selected from the group consisting of H, OH, halogen, nitio, cyano, SH, SR , trihalo-Ci -C₆ -alkyl, d-Cβ -alkyl and Ci -C₆ -alkoxy; and

R⁴ is d -Ce -alkyl;

23. A method according to claim 22, comprising use of the compound of the formula la or lb having the formula:

wherein R .1 , τ R>2 and ^• R are as defined above.

24. The method according to claim 22, comprising use of the compound of the formula

wherein R¹ is as defined above and m is an integer from 0 to 10.

25. A method according to claim 22, comprising use of the compound of the formula

26. A method according to claim 25, comprising the use of a chroman compound which is

(-)-cis-{3-phenyl-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-chroman-7-ol;

(-)-cis- {3-(4-trifluormethyl-phenyl)-4-[4-(2-pyrrolidin- 1 -yl-ethoxy)-phenyl] } -7- methoxychroman;

(-/+)-cis-{3-(3-methoxy-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-7- methoxychroman;

(-/+)-cis-{3-(3-hydroxy-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-chroman-7-ol;

(-/+^)-cis-{3-(4-trifluormethyl-phenyl)-4-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]}-chroman- 7-ol; or

(+,-)-(cis-3 -(4-Methylphenyl)4-(4-(2-pyrrolidinoethoxy)phenyl)-chroman-7-y 1) N,N- dimethylsulfamic acid ester;

(-)-c s^,-7-methoxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane .

(+/-)-cfs'-7-methoxy-3-(3-methoxyphenyl)-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane .

(+/-)-cw-7-methoxy-3-(4-frffluoromethy . _and

(-)-cz 7-me1hoxy-3-(4-trifluoromemylphenyl)-4-(4-(2-pyrrolidinoethoxy)-phenyl)chromane

27. A method according to any one of claims 14 to 26, wherein said progestin is progesterone, norethindrone acetate, ethynodiol, desogesttel, levonorgestiel, norgestrel, norgestimate, medroxyprogesterone or an ester or a pharmaceutically acceptable salt thereof.

28. A method according to claim 27, wherein said progestin is norethindrone or norethindrone acetate or an ester or a pharmaceutically acceptable salt thereof.

5

29. A pharmaceutical composition for use in the therapeutic or prophylactic treatment of diseases wherein decreased estrogen production is a factor, said composition comprising an effective amount of the combination of a SERM which is a 3,4 chroman or coumarin derivative or a pharmaceutical acceptable salt thereof and a progestin or a pharmaceutical o acceptable salt thereof and a pharmaceutical carrier or diluent.

30. A method of therapeutic or prophylactic treatment of conditions such as is breast cancer, testicular cancer, osteopenias or osteoporosis induced bone loss, endometriosis, cardiovascular disease, hypercholesterolemia, prostatic hypertrophy, prostatic carcinomas, 5 obesity, hot flushes, skin effects, mood swings, memory loss, urinary incontinence, hairloss, cataracts, natural hormonal imbalances, and adverse reproductive effects associated with exposure to environmental chemicals comprising administering to a mammal in need thereof an effective amount of a pharmaceutical composition according to claim 27. 0

31. A pharmaceutical composition for use in the therapeutic or prophylactic treatment of diseases wherein elevated cartilage degradation is a factor, said composition comprising an effective amount of the combination of a SERM which is a 3,4 chroman or a coumarin derivative or a pharmaceutical acceptable salt thereof and a progestin or a pharmaceutical 5 acceptable salt thereof and a pharmaceutical carrier or diluent.

32. A pharmaceutical composition according to claim 27 or claim 29 wherein said progestin is norethindrone or norethindrone acetate or a pharmaceutically acceptable salt thereof. 0

33. A pharmaceutical composition according to claim 29 or claim 31 in a dosage form suitable to administer from 0.01 to about 100 mg as an effective amount of said chroman or coumarin derivative or a pharmaceutical acceptable salt thereof per individual per day.

34. A pharmaceutical composition according to claim 32, in a dosage form suitable to administer from 0.01 to about 100 mg as an effective amount of norethindrone or norethindrone acetate or a pharmaceutical acceptable salt thereof per individual per day.

35. The pharmaceutical composition according to any one of claims 29 to 34, in the form of an oral dosage unit or parenteral dosage unit.

36. A method of treating osteoarthritis, said method comprising administering a therapeutically effective amount of the combination of a compound of Formula (I) and a progestin to a mammal in need thereof.

37. A method of treating rheumatoid arthritis, said method comprising administering a therapeutically effective amount of the combination of a compound of Formula(I) and a progestin to a mammal in need thereof.

38. A method of treating osteoarthritis or rheumatoid arthritis according to the claim 34 or claim 35, wherein said mammal is human.

39. Use of a cartilage specific biochemical marker for titration of the doses according to claim 34 or claim 35.