BACKGROUND OF THE INVENTION The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

Osteoporosis accompanied by increased suspectibility of fractures results from a progressive reduction on skeletal bone mass. The costs for treating osteoporosis-related injuries increase every year as result of the ageing of the population in the industrialized society.

Collagen type I accounts for more than 90 % of the organic matrix of bone.

The collagen molecules consist mainly of a triple-helical domain having terminal telopeptide sequences which have an important function as sites of intermolecular cross-linking.

There are two kinds of cells regulating the bone bilding and the bone resorption process, i. e. osteoblasts which enhance the bone building, and osteoclasts, which cause the degradation of the bone mass. The osteoclasts are dominant for elderly people and cause, for persons over 50 years, a bone mass decrease of 5 to 10 % per decennium.

Attempts have been made to estimate the degree of bone resorption and thus to predict the suspectibility of bone fractures, by determining certain bone resorption related markers in the body fluid samples. As examples of such markers can be mentioned hydroxyproline, hydroxylysine and the collagen crosslinking compound 3-hydroxypyridinium. The International Patent Application WO 9508115 discloses an immunassay for the determination of a linear crosslinkable collagen fragment in a body fluid sample. The method disclosed is a competitive assay wherein the sample is brought into contact with a binding antibody and a synthetic peptide (a competiting antigen). Such synthetic peptides have potential sites for crosslinking, and are typically collagen type I chains comprising 6 to 9 amino acids. These synthetic peptids are also called CrossLaps antigens. A typical synthetic peptide is the CrossLaps antigen aI (I) Cl amino acid sequence EKAHDGGR. The patent publication WO 9612193 disclose the use of two different immunoassays for the determination of collagen fragments in body fluid samples in order to obtain improved diagnostic results.

The patent publication WO 9808098 discloses a competitive assay for the determination of collagen degradation products in which D-amino acids occur in stead of the normal L-form. The synthetic peptide used contains also the D-amino acid. The D-form is used as a marker of the rate of the bone resorption. The patent publication WO 9826286 discloses a non-competitive immunoassay for the determination of a collagen fragment. The first antibody (the capturing antibody) is reactive with an epitope located in the amino acid sequence EKAHDGGR. The second antibody (the detecting antibody) may be raised against an epitope in the same amino acid sequence or a different epitope in the fragment to be determined.

Attempts have been made to develop drugs effective in the prevention of bone resorption. Bisphosphonates represent a typical group of such drugs. As

other groups can be mentioned estrogens and related compounds called SERMs (selective estrogen-receptor modulators).

Estrogen deficiency in postmenopausal women causes increased penetrative resorption leading to trabecular instability and fractures. Although the efficiency of estrogen replacement therapy in preventing bone loss has been known for a long time, the exact cellular and molecular mechanisms of estrogen in bone are not clear. Both osteoblasts and osteoclasts express estrogen receptors, and estrogen has been shown to affect to the formation of both cell types, by decreasing osteoclast formation and increasing osteoblast formation. However, direct effects on the resorbing activity of mature osteoclasts have not been demonstrated. Although estrogen replacement therapy prevents bone loss, it is also associated with side effects such as increased breast cancer risk. Thus, pharmaceutical companies are developing therapeutic compounds that would act through estrogen receptors and have the good effects of estrogen without the unwanted side-effects. These compounds are known as selective estrogen-receptor modulators (SERMs) which are antiestrogens and have both estrogen-like and antiestrogenic properties (Kauffman & Bryant, 1995). The effects may be tissue-specific as in the case of tamoxifen and toremifene which have estrogen-like effects in the bone, partial estrogen-like effect in the uterus and liver, and pure antiestrogenic effect in breast cancer. Raloxifene and droloxifen are similar to tamoxifen and toremifene, except that their antiestrogenic properties dominate. The compound (deaminohydroxy) toremifene, which also is known under the code FC-1271a, has relatively weak estrogenic and antiestrogenic effects in the classical hormonal tests (Kangas, 1990). It has antiosteoporosis actions and it decreases total and LDL cholesterol levels in both experimental models and in human volunteers (International patent publications WO 96/07402 and WO 97/32574).

The effect of drugs suitable for the prevention of bone resorption have normally been studied by in vivo tests. Body fluid samples have been taken at certain intervals after the start of the drug administration, and a marker specific for bone resorption has been determined. Comparisons to controls without drug administration show the effect of the drug. As an example of such studies can be mentioned H N Rosen el al., 2000, where serum CTX (carboxy terminal collagen crosslinks) has been determined for patients obtaining the drug pamidronate and for controls. However, such in vivo studies of drug effects are very tedious and time wasting. There is thus a need to provide fast and reliable in vitro tests for routine drug screening.

The resorbing activity of mature osteoclasts can be determined using in vitro models where isolated osteoclasts are cultured on bone slices. The formed resorption pits are visualised using peroxidase-conjugated WGA-lectin (Wheat Germ Agglutin lectin). The amount of bone resorption can be quantitated by microscopic counting of the amount of resorption pits formed and by determining total resorbed area using image-analysis techniques. The effect of bisphosphonates can be studied by this model because the presence of the bisphosphonate will prevent the osteoclasts from attaching to the bone surface. Thus, in the presence of bisphosphonates the osteoclasts will not give rise to any kind of collagen degradation products.

Results using the above in vitro techniques have, however, suggested that estrogen would not have any effects on the resorbing activity of mature osteoclasts. Estrogens and SERMs are generally believed to increase the generation of osteoblasts and to suppress the formation of osteoclasts. It has, however, not been shown that estrogens and SERMs also could inhibit the activity of osteoclasts already existing. Attempts to measure such activities have failed, mainly due to the method used. In the methods used, osteoclasts have been grown on bone and after a certain period of time the number of pits

in the bone surface, caused by the osteoclasts, has been counted. The presence or absence of estrogen has not caused any difference in the number of pits generated. Therefore it has been concluded that these compounds do not affect the activity of the osteoclasts.

Bagger et al., 1999, have recently developed an in vitro test for the determination of bone resorption. This test is a non-competitive immunoassay for the determination of a bone degradation marker, the CrossLaps antigen EKAHDGGR. In this study the new in vitro test has been compared to normal bone resorption assay. No drug screening tests were reported in this study.

The present invention is an in vitro drug screening method based on the immunodetermination of an antigen (preferably a collagen degradation fragment) which is caused by osteoclasts and the formation of which is more or less prevented by the presence of the drug to be screened.

SUMMARY OF THE INVENTION This invention relates to a method for in vitro determination of the activity of a drug on the activity of osteoclasts, wherein -said drug is an estrogen or a selective estrogen-receptor modulator (SERM), and -osteoclasts are cultured on bone surface in the presence of the drug to be tested, and -the activity of said osteoclasts is determined by determining the level of an antigen, the release of which is decreased by the osteoclast inhibiting activity of said drug, by an immunometric method, and -comparing the measured antigen level to a control culture into which no drug has been added.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of the estrogen 17beta-estradiol on the resorption area and of the depth of the resorption pits caused by the osteoclasts in the in vitro test. E-8, E-9 and E-10 means the 17beta-estradiol concentration 10-8M, 10-9M and 10-1°M, respectively.

Figure 2 shows the effect of the estrogen 17beta-estradiol on the release of a CrossLaps antigen, caused by the osteoclasts in the in vitro test. The filled bar represents control, the dashed bar means the 17beta-estradiol concentration 10-9M, and the open bar the 17beta-estradiol concentration of 10-7M.

Figure 3 shows the effect of the SERMs FC-1271a and tamoxifen on relative amount of a released CrossLaps antigen, caused by the osteoclasts in the ill vitro test. The filled bar represents control, the dashed bar means the SERM concentration 10-8M, and the open bar the SERM concentration of 10-6M.

Figure 4 shows the effect of two antiresorptive agents, bafilomycin Al (BafAl, 10-7 M) and E64 (10-6 M) on the release of CrossLaps into the culture medium in in vitro bone resorption assay. BafAl inhibits the vacuolar ATPase proton pump, and E64 inhibits cathepsin K.

DETAILED DESCRIPTION OF THE INVENTION According to a preferable embodiment, the drug to be tested is an estrogen or a SERM, and the collagen fragment is a fragment the level of which is influenced by said drug to be tested, and said fragment is determined as an

antigen in an immunoassay. Typically the antigen is a collagen type I carboxy-terminal peptide (CTP) or a collagen type I N-terminal peptide (NTP).

Osteoclasts are multinucleated cells derived from hemopoietic stem cells.

Their function is to resorb bone by dissolving both the organic and inorganic components of the bone matrix. Based on current knowledge, osteoclastic bone resorption requires the following steps : 1) Tight attachment of osteoclasts to bone surface (mediated by integrins, especially the vitronectin receptor OCVß3, and some other and as yet unidentified mechanisms) ; 2) Generation of protons in the cytoplasm by carbonic anhydrase II ; 3) Transportation of the protons from the cytoplasm to intracellular vesicles by a vacuolar ATPase proton pump ; 4) Transportation of the acidic vesicles to the cell membrane facing the bone matrix ; 5) Fusion of the acidic vesicles to the cell membrane, and release of the protons to the extracellular space ; 6) Solubilization of inorganic bone matrix by the secreted acid ; 7) Secretion of proteases, including MMP-9 and cathepsin K, into the extracellular space ; 8) Degradation of organic bone matrix by the secreted proteases ; 9) Endocytosis and transcytosis of the degradation products ; 10) Completion of the degradation of matrix components by reactive oxygen species (ROS) generated by TRAP during transcytosis. All of the steps described above are potential targets of antiresorptive drugs.

Osteoclasts resorb bone by secreting acid and proteolytic enzymes into an extracellular space called the resorption lacuna. The enzymes involved in the degradation of bone matrix are not known. The most promising candidate as a bone degrading protease in the resorption lacuna is a serine protease known as cathepsin K. Recent evidence suggests that degradation of bone collagen by cathepsin K produces circulating C-and N-terminal fragments known as CrossLaps and NTX.

The inventors of the present invention have shown that the estrogen 17beta- estradiol almost completely inhibits cathepsin K-mediated bone resorption, resulting in significant decrease of the depth of resorption pits formed, and significantly decreased release of CrossLaps antigen from bone collagen.

The inventors'results show that one of the major targets of estrogen on bone are the mature resorbing osteoclasts. In the following is presented a novel method for the screening of the effects of estrogens and SERMs on bone in vitro using osteoclast cultures, where the amount of cathepsin K-generated collagen degradation products such as CrossLaps antigen and NTX released into the culture medium is measured as an index of the amount of estrogen-sensitive bone resorption.

Preferably, the CrossLaps antigen is the amino acid sequence EKAHDGGR derived from the carboxy-terminal telopeptide region of the type I collagen alphal chain, or the beta-aspartate form thereof.

Competitive as well as non-competitive assay methods can be employed.

According to a preferred embodiment, the antigen is measured by non- competitive methods employing at least two different antibodies, preferable monoclonal antibodies, where the first antibody, i. e. the capture antibody is bound to a solid phase. The second antibody, i. e. the detecting antibody, is labelled with a detectable label.

Suitable lables are for example enzymes, radio-isotopes, fluorescent, fosforescent or luminescent markers, and coloured particles that are visibly detectable. The word"label"shall also be understood to cover a binding site able to bind to the marker mentioned.

The invention will be illuminated by the following non-restrictive Experimental Section.

EXPERIMENTAL SECTION The method of osteoclast culture on bone slices was originally described by Boyde and co-workers (1984) and by Chambers and co-workers (1984). For cell culture, we have used a method slightly modified from the original methods (Lakkakorpi et al. 1989, Lakkakorpi and Vaananen, 1991). The rate of bone resorption in the cultures was originally determined by counting the number of resorption pits on each bone or dentine slice using a microscope with phase contrast objectives (Sundquist et al. 1990). Later, the pits were visualized using Wheat Germ Agglutinin lectin that specifically binds to the resorbed area in bone (Selander et al. 1994), making it possible to quantitate the total resorbed area using a microscope and computer-assisted image analysis system (Laitala and Vaananen 1994, Hentunen et al. 1995). We use a commercially available method (CrossLaps for cultures, Osteometer Biotech, Herlev, Denmark) to detect the amount of collagen cross-links released into the culture medium as an index of the bone resorption rate (Bagger et al., 1999).

To visualise the depth of the resorption pits cells were cultured on tetracycline labelled (Salo et al., 1997) bone slices which, after the removal of the cells, were stained with TRITC-conjugated WGA-Lectin and analysed using a laser scanning confocal microscope (Leica Aristoplan CLSM, Leica Lasertechnik, Heidelberg, Germany).

Transverse 0. 1 mm thick slices of cortical bone are cut from the diaphysis of fresh bovine femurs using a low-speed diamond saw, cleaned by ultrasonication in multiple changes of sterile distilled water, and stored at 4°C before use. Long bones are removed from 1 to 2-day-old rat pups killed by decapitation. The bones are dissected free of adherent soft tissues, and the

endosteal surfaces are curetted with a scalpel blade into osteoclast culture medium : a-Minimal Essential Medium (a-MEM) supplemented with 100 IU/ml penicillin, 100 pg/ml streptomycin, 20 mM HEPES and 10% heat- inactivated fetal calf serum, pH 7. 2. The resulting suspension of dispersed cells and bone fragments is agitated using a plastic pipette. Larger fragments are allowed to sediment for a few seconds and the supernatant is seeded onto the bone slices pre-wetted in the medium. After a settling period of 30 minutes at 37°C, the bone slices are washed by dipping in fresh medium, and then transferred to wells in 24-well culture dishes containing osteoclast culture medium, and the test substance or vehicle. The bone slices are incubated in a humidified atmosphere of 95 % air and 5 % carbon dioxide at 37°C for 48-72 hours.

After the culture period, the amount of bone resorption is determined by measuring the amount of collagen cross-links released into the culture medium using a commercial kit (CrossLaps for cultures, Osteometer Biotech) according to the manufacturer's instructions.

Results : The procedure described above was performed in the absence (control) and in the presence of various drugs.

Figure 1 shows that the estrogen tested (17beta-estradiol) did not affect the resorption area caused by the osteoclasts. On the other hand, this estrogen significantly decreased the depth of the individual resorption pits. The estradiol concentration 10-8M caused a stronger effect than the lower concentration 10-l°M.

Figure 2 shows the effect of the estrogen 17beta-estradiol on the release of the CrossLaps antigen (CTX), caused by the osteoclasts in the in vitro test.

The release of this antigen was almost completely inhibited by the presence of the estrogen.

Figure 3 shows the effect of the SERMs FC-1271 a and tamoxifen on relative amount of released of a CrossLaps antigen, caused by the osteoclasts in the in vitro test. While tamoxifen did not show any effect at the tested concentrations (10-8 and 10-6M), the compound FC-1271a showed a very clear inhibition at a concentration of 10-6M.

The above experiments clearly show that it is possible to test the inactivating effect of estrogens and SERMs on the osteoclasts by simple in vitro testing.

Although the effect in principle can be measured by measuring the depth of the pits, a far more rapid method is the immunometric quantification of a released antigen.

The information on the inactivating effect of estrogens and SERMs on the osteoclasts can further be combined by data on the inhibiting effect of such compounds on the formation of osteoclasts. The effect on the formation of osteoclasts can be studied by well known methods. Estrogens or SERMs with both strong inhibiting effect a) on the formation of new osteoclasts and b) on the activity of existing osteoclasts would therefore be excellent drug canditates in the prevention of bone resorption.

Although the above method is especially useful for testing estrogens and SERMs, its use is not restricted to these types of drugs.

It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which

are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention.

Thus, the described embodiments are illustrative and should not be construed as restrictive.

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