WO2001084144A1 - Procede de dosage in vitro de medicaments a tester pour la prevention de la resorption osseuse - Google Patents

Procede de dosage in vitro de medicaments a tester pour la prevention de la resorption osseuse Download PDF

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Publication number
WO2001084144A1
WO2001084144A1 PCT/FI2001/000357 FI0100357W WO0184144A1 WO 2001084144 A1 WO2001084144 A1 WO 2001084144A1 FI 0100357 W FI0100357 W FI 0100357W WO 0184144 A1 WO0184144 A1 WO 0184144A1
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WO
WIPO (PCT)
Prior art keywords
antigen
drug
osteoclasts
bone
estrogen
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Application number
PCT/FI2001/000357
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English (en)
Inventor
Jussi Halleen
Vilhelmiina Parikka
Petri Lehenkari
Kalervo Väänänen
Pirkko Härkönen
Lauri Kangas
Original Assignee
Jussi Halleen
Vilhelmiina Parikka
Petri Lehenkari
Vaeaenaenen Kalervo
Haerkoenen Pirkko
Lauri Kangas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FI20001318A external-priority patent/FI20001318A0/fi
Application filed by Jussi Halleen, Vilhelmiina Parikka, Petri Lehenkari, Vaeaenaenen Kalervo, Haerkoenen Pirkko, Lauri Kangas filed Critical Jussi Halleen
Priority to AU2001254847A priority Critical patent/AU2001254847A1/en
Publication of WO2001084144A1 publication Critical patent/WO2001084144A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane

Definitions

  • the present invention provides a method for in vitro screening of drug candidates, particularly estrogens and SERMs, useful for the prevention of bone resorption.
  • 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.
  • osteoblasts which enhance the bone building
  • 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.
  • markers can be mentioned hydroxyproline, hydroxylysine and the collagen crosslinking compound 3-hydroxypyridinium.
  • 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).
  • 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 CrossLapsTM antigens.
  • a typical synthetic peptide is the CrossLapsTM 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.
  • Bisphosphonates represent a typical group of such drugs.
  • SERMs selective estrogen-receptor modulators
  • 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.
  • direct effects on the resorbing activity of mature osteoclasts have not been demonstrated.
  • estrogen replacement therapy prevents bone loss, it is also associated with side effects such as increased breast cancer risk.
  • pharmaceutical companies are developing therapeutic compounds that would act through estrogen receptors and have the good effects of estrogen without the unwanted side-effects.
  • SERMs selective estrogen-receptor modulators
  • 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- 1271 a, 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.
  • 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).
  • 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.
  • 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.
  • an antigen preferably a collagen degradation fragment
  • 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)
  • SERM selective estrogen-receptor modulator
  • 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
  • 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 " M, 10 "9 M and 10 "10 M, respectively.
  • Figure 2 shows the effect of the estrogen 17beta-estradiol on the release of a CrossLapsTM antigen, caused by the osteoclasts in the in vitro test.
  • the filled bar represents control, the dashed bar means the 17beta-estradiol concentration 10 "9 M, and the open bar the 17beta-estradiol concentration of
  • Figure 3 shows the effect of the SERMs FC-1271a and tamoxifen on relative amount of a released CrossLapsTM antigen, caused by the osteoclasts in the in vitro test.
  • the filled bar represents control, the dashed bar means the SERM concentration 10 "8 M, and the open bar the SERM concentration of 10 " M.
  • FIG 4 shows the effect of two antiresorptive agents, baf ⁇ lomycin 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
  • E64 inhibits cathepsin K.
  • the drug to be tested is an estrogen or a SERM
  • 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.
  • 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 ⁇ v ⁇ 3 , 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.
  • ROS reactive oxygen species
  • 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.
  • cathepsin K 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 CrossLapsTM 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 CrossLapsTM antigen from bone collagen.
  • the inventors' results show that one of the major targets of estrogen on bone are the mature resorbing osteoclasts.
  • 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 CrossLapsTM antigen and NTX released into the culture medium is measured as an index of the amount of estrogen-sensitive bone resorption.
  • the CrossLapsTM antigen is the amino acid sequence EKAHDGGR derived from the carboxy-terminal telopeptide region of the type I collagen alpha 1 chain, or the beta-aspartate form thereof.
  • 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.
  • label shall also be understood to cover a binding site able to bind to the marker mentioned.
  • 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: ⁇ -Minimal Essential Medium ( ⁇ -MEM) supplemented with 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 20 mM HEPES and 10% heat- inactivated fetal calf serum, pH 7.2.
  • ⁇ -MEM ⁇ -Minimal Essential Medium
  • 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.
  • 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.
  • FIG 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 "8 M caused a stronger effect than the lower concentration 10 "10 M.
  • Figure 2 shows the effect of the estrogen 17beta-estradiol on the release of the CrossLapsTM 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.
  • CTX CrossLapsTM antigen
  • Figure 3 shows the effect of the SERMs FC-1271a 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 "6 M), the compound FC-1271a showed a very clear inhibition at a concentration of 10 "6 M.
  • 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.
  • Kangas L Biochemical and pharmacological effects of toremifene metabolites. Cancer Chemother Pharmacol 27: 8-12, 1990.
  • Serum CTX A new marker of bone resorption that shows treatment effect more often than other markers because of low coefficient of variability and large changes with bisphosphonate therapy. Calif Tissue Int (2000) 66: 100-103.
  • Hentunen TA Lakkakorpi PT, Tuukkanen J, Lehenkari PP, Sampath TK, Vaananen HK (1995) Effects of recombinant human osteogenic protein-1 on the differentiation of osteoclast-like cells and bone resorption. Biochem Biophys Res Commun 209: 433-443. Laitala T, Vaananen HK (1994) Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H+-ATPase. J Clin Invest 93: 2311-2318.

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Abstract

L'invention concerne un procédé permettant de déterminer in vitro l'activité d'un médicament sur l'activité des ostéoclastes. Selon ce procédé, le médicament est un oestrogène ou un modulateur du récepteur de l'oestrogène sélectif et les ostéoclastes sont cultivés sur une surface osseuse en présence du médicament à tester. En outre, l'activité de ces ostéoclastes est déterminée en définissant la concentration d'un antigène dont la libération est réduite par l'activité d'inhibition de l'ostéoclaste dudit médicament, par un procédé immunométrique. En outre, le procédé selon l'invention consiste à comparer la concentration d'antigènes mesurée par rapport à une culture témoin dans laquelle aucun médicament n'a été ajouté.
PCT/FI2001/000357 2000-05-04 2001-04-11 Procede de dosage in vitro de medicaments a tester pour la prevention de la resorption osseuse WO2001084144A1 (fr)

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Application Number Priority Date Filing Date Title
AU2001254847A AU2001254847A1 (en) 2000-05-04 2001-04-11 Method for in vitro screening of drug candidates useful for the prevention of bone resorption

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US20173600P 2000-05-04 2000-05-04
US60/201,736 2000-05-04
FI20001318A FI20001318A0 (fi) 2000-06-02 2000-06-02 Menetelmä luuresorption estämiseen käyttökelpoisten lääke-ehdokkaiden seulomiseksi in vitro
FI20001318 2000-06-02

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005108551A1 (fr) * 2004-05-06 2005-11-17 Cambrex Bio Science Walkersville, Inc. Recipients de culture d'osteoclastes et leurs procedes d'utilisation
US8241628B2 (en) 2008-08-05 2012-08-14 Novartis Ag Compositions and methods for antibodies targeting complement protein C5
WO2014159813A1 (fr) 2013-03-13 2014-10-02 Moderna Therapeutics, Inc. Molécules polynucléotidiques à longue durée de vie
US9051365B2 (en) 2011-12-21 2015-06-09 Novartis Ag Antibodies that bind factor P

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A.C. RAMALHO ET AL.: "Effect of 17-beta estradiol and SERMs on osteoclastic differentiation in primary human bone marrow cell cultures", AMERICAN SOCIETY FOR BONE AND MINERAL RESEARCH MEETING 21, SU080 *
Q. QU, P.L. HARKONEN ET AL.: "Conditioned medium of estrogen-treated osteoblasts inhibits osteoclast maturation and function in vitro", BONE, vol. 25, no. 2, August 1999 (1999-08-01), pages 211 - 215 *
STEPHAN CHRISTGAU ET AL.: "Clinical evaluation of the serum crosslaps one stel ELISA, a new assay measuring the serum concentration of bone-derived degradation prods of type 1 collagen C-telopeptides", CLINICAL CHEMISTRY, vol. 44, no. 11, 1998, pages 2290 - 2300 *
T.J. HALL ET AL.: "The bone-specific estrogen centchroman inhibits osteoclastic bone resorption in vitro", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 216, no. 2, 1995, pages 662 - 668 *
Y.Z. BAGGER ET AL.: "Crosslaps-for-culture: An improved enzyme-linked immunosorbent assay (ELISA) for measuring bone resorption in vitro", AMERICAN SOCIETY FOR BONE AND MINERAL RESEARCH MEETING 21, SA248 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005108551A1 (fr) * 2004-05-06 2005-11-17 Cambrex Bio Science Walkersville, Inc. Recipients de culture d'osteoclastes et leurs procedes d'utilisation
US8241628B2 (en) 2008-08-05 2012-08-14 Novartis Ag Compositions and methods for antibodies targeting complement protein C5
US8883158B2 (en) 2008-08-05 2014-11-11 Novartis Ag Compositions and methods for antibodies targeting complement protein C5
US10407496B2 (en) 2008-08-05 2019-09-10 Novartis Ag Compositions and methods for antibodies targeting complement protein C5
US9051365B2 (en) 2011-12-21 2015-06-09 Novartis Ag Antibodies that bind factor P
US9988440B2 (en) 2011-12-21 2018-06-05 Novartis Ag Compositions comprising antibodies targeting factor P
US10865237B2 (en) 2011-12-21 2020-12-15 Novartis Ag Nucleic acids encoding anti-Factor P antibodies
WO2014159813A1 (fr) 2013-03-13 2014-10-02 Moderna Therapeutics, Inc. Molécules polynucléotidiques à longue durée de vie

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