WO1998004287A1 - Utilisation d'inhibiteurs de proteinase pour la prevention ou la reduction de la resorption osseuse - Google Patents

Utilisation d'inhibiteurs de proteinase pour la prevention ou la reduction de la resorption osseuse Download PDF

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WO1998004287A1
WO1998004287A1 PCT/EP1997/004110 EP9704110W WO9804287A1 WO 1998004287 A1 WO1998004287 A1 WO 1998004287A1 EP 9704110 W EP9704110 W EP 9704110W WO 9804287 A1 WO9804287 A1 WO 9804287A1
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mmp
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pro
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PCT/EP1997/004110
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Niels Taekker Foged
Jean-Marie Delaisse
Morten Meldal
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Center For Clinical & Basic Research
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Priority to AU42032/97A priority Critical patent/AU733104B2/en
Priority to IL12810397A priority patent/IL128103A0/xx
Priority to BR9710615-1A priority patent/BR9710615A/pt
Priority to EP97940041A priority patent/EP0915709A1/fr
Priority to JP10508510A priority patent/JP2001501594A/ja
Publication of WO1998004287A1 publication Critical patent/WO1998004287A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP

Definitions

  • the present invention relates to the reduction of the rate of bone resorption by targeting the action or production of proteases.
  • Osteoporosis is a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a subsequent increase in bone fragility and susceptibility to fracture.
  • Post- menopausal osteoporosis is a chronic disease which affects millions of women throughout the world and it has an enormous economical and social impact on society.
  • Reduction of bone resorption is believed to be an appropriate way to prevent and treat several metabolic bone diseases, including osteoporosis and osteolytic bone metastasis.
  • Agents such as steroid hormones (especially oestrogen) , calcitonin and bisphosphonates are able to suppress bone resorption and have been used for prevention and treatment of osteoporosis and/or osteolytic bone metastasis.
  • these therapeutic agents fail to achieve satisfactory effects in some cases, due to subject limitation or uncertain efficacy. There is therefore need of a new prophylactic/ therapeutic method for preventing or treating accentuated bone resorption.
  • Removal of the mineralised osseous substance i.e. organic matrix embedded in deposits of calcium phosphate salts, is a complicated process. Though still a controversial subject, it seems probable that osteoclasts are the only cells capable of bone resorption. The progressing bone loss in patients with osteoporosis is caused by an increase in the activity of osteoclasts.
  • the expected life cycle of osteoclasts involve the following major phases:
  • proteolytic enzymes have been known to play a role in degradation of the organic matrix of bone.
  • the knowledge about proteolytic enzymes involved m bone resorption mainly comes from in vitro and in vivo studies of the effects of natural and particularly synthetic enzyme inhibitors.
  • histochemical and lmmuno- cytochemical characterisation of enzymes in bone cells and tissues as well as more recently identification of enzyme- encoding mRNA in osteoclasts and other bone cells has increased the information about proteolytic enzymes involved in bone resorption.
  • the proteolytic enzymes of major relevance to osteoclastic bone resorption seem to be members of the families of cysteine proteinases and matrix metalloprotemases (MMPs).
  • proteinase inhibitors in disease control has been suggested in several scientific publications and in patents and patent applications.
  • MMP inhibitors the mam focus has been the potential of inhibitors in treatment 5 of cancer and tumour metastasis, but also diseases such as arthritis, ulcers, periodontal and bone diseases, HIV infection, corneal and other eye diseases, diabetes and myocardial infarction have been the target of these speculations and ensuing early experiments (reviewed by 0 Birkedal-Hansen et al, 1993 2 ) .
  • MT-MMPs Membrane-type matrix metalloprotemases
  • MTl-MMP also referred to in the literature as MT-MMP-1 and as MMP-14
  • anti-MTl- MMP antibodies have been suggested, though rather “ unspecifically, as useful for application not only in the diagnostic area but also in other medical fields (EP-A- 0685557 and W095/25171) .
  • the inhibition of cathepsms is considered another possible way of reducing bone resorption by using proteinase inhibitors.
  • Several cathepsins are produced by osteoclasts and though still somewhat controversial, they are apparently ⁇ involved in the degradation of organic matrix in the acidic environment of the sub-osteoclastic resorption zone.
  • cathepsm K cathepsm 0
  • 0C2 was cloned from osteoclasts and osteoclast-like cells by several independent groups. It was suggested that
  • hybrid molecules for conferring specificity to cell- and tissue-interacting agents has been proposed in several modifications including hybrids consisting of three parts including not only a cell-binding
  • 25 cells of peptides and proteinase inhibitors is by administering them as lipid conjugates (WO93/01828 ) .
  • interference by an inhibitor of a proteolytic enzyme involved in osteoclast migration and/or attachment might be more effective than inhibition of an enzyme involved directly in the resorptive process.
  • This type of interference will also be easier to accomplish since the secreted enzymes of the migrating cells are not protected from inhibition as they are when secreted into the tightly sealed resorption zone which is formed when the active polarised osteoclasts attach to bone.
  • osteoclasts an MT-MMP closely related to or identical to MTl-MMP, previously identified in cancer cells not related to bone, is expressed by osteoclasts. It may be expected that this osteoclast MTl-MMP plays an important role in the action of osteoclasts, probably being implicated in their migration to their site of action at which to degrade bone (see Examples 1, 2, 3-2 and 3-3 and Figures 1 to 3) . This finding indicates that also other membrane-associated metalloprotemases such as other MT-MMPs or members belonging to families of non-matrix type of membrane metalloprotemases (e.g. meltrins and "A disintegrm and metalloproteinase"' s, ADAMs) ) could be produced by osteoclasts.
  • membrane-associated metalloprotemases such as other MT-MMPs or members belonging to families of non-matrix type of membrane metalloprotemases (e.g. meltrins and "A disintegrm and metalloproteina
  • osteoclast etalloelastase MMP-12 a proteinase hitherto believed to be almost specifically expressed in macrophages, where it is obligatory for the invasion of these cells through basement membranes. Since macrophages and osteoclasts are closely related cell types both originating from the haematopoietic stem cell and differentiating late in its development, a similar role of MMP-12 in osteoclast invasion and migration s likely (see Example 3-4 and Figures 4 to 6) .
  • the present invention provides the use of an agent in the manufacture of a medicament for the treatment of bone metaoolic disease, characterised in that the agent acts by inhibition of the production or action of a membrane associated protease or the matrix metalloprotease MMP-12 involved in the resorptive activity of osteoclasts . More preferably, the invention provides the use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease by inhibition of the production or action of a metalloproteinase involved in the resorptive activity of osteoclasts. Particularly, inhibition of the production or action of an MT-MMP but also of other membrane-associated metalloprotemases such as a meltrin or an ADAM as well as a secreted MMP such as MMP-12.
  • the treatment may be for prevention or for cure of such diseases .
  • the metalloproteinase is involved m the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or m the migration, fusion, attachment, polarisation, activity in removal of mineralised osseous substance, or death of osteoclasts.
  • the invention also includes regulation of bone metabolism by inhibition of non- osteoclastic proteinases which influences the life cycle of osteoclasts.
  • Other bone cells such as osteoblasts and chondrocytes are able to produce both latent and active forms of MMPs, cathepsins and plasminogen activator as well as natural inhibitors of some of these enzymes.
  • the agent may be selectively inhibitory of MTl-MMP or MT-MMPs broadly, of MMP-12 or MMPs broadly, or of membrane- associated metalloprotemases or metalloprotemases broadly.
  • the agent may be an antibody selectively immunoreactive with an MT-MMP.
  • Such an agent may alternatively be an antisense oligo-nucleotide or oligo-nucleotide analogue directed against a gene involved m the production of an MT- MMP or an agent regulating MT-MMP activity.
  • It may be an MT-MMP substrate mimic inhibitor.
  • It may be a broad spectrum matrix metalloproteinase (MMP) inhibitor or a broad spectrum membrane-associated metalloproteinase inhibitor.
  • MMP matrix metalloproteinase
  • It may also be a peptide, peptide analogue or other peptide mimicking agent obtained by screening an appropriate library for compounds reactive with an MT-MMP, an MMP or a membrane- associated metalloproteinase.
  • a preferred inhibitor provided by the invention is the peptide S-K-Y-P-J-A-L-F-F-K (SEQ ID No .1 ) (J being the single letter code of hydroxyprolme) and inhibitory variants thereof such as the peptide analogue S-K-Y (N0 2 ) -P-J- A-L-F-F-K(Abz) (SEQ ID No.2).
  • the invention includes the use of an agent in the manufacture of a medicament for the treatment of bone metabolic disease by inhibition of the recruitment, proliferation, differentiation, or migration of osteoclast precursor cells or in the migration, fusion, attachment, polarisation, or death of osteoclasts.
  • said agent produces said inhibition by inhibiting the production or action of a proteinase.
  • the invention includes an anti-bone resporption agent comprising a proteinase inhibitor active against a proteinase involved in bone resorption operatively linked to a ligand having binding specificity targeting the inhibitor to said proteinase or to the environment of the proteinase.
  • the invention includes a new protease termed rabbit osteoclast MTl-MMP having the amino acid sequence given m
  • MTl-MMP are included also, as is human osteoclast MTl-MMP and isolated nucleic acid sequences encoding it.
  • the invention also includes a new protease termed rabbit osteoclast MMP-12 having the ammo acid sequence given Figure 4 and Figure 5, as well as an isolated nucleic acid coding for such a protein, e.g. one having the sequence set out in Figure 4.
  • Human osteoclast MMP-12 and isolated nucleic acid sequences encoding it as well as other proteins and nucleic acid sequences with a high homology (e.g. at least 50%, preferably at least 70, 80 or 90%) to rabbit osteoclast MMP-12 are also included in the invention.
  • Inhibition of proteolytic activity can be obtained several ways and by several classes of agents.
  • the inhibition could be direct, i.e. by an agent acting directly either on the proteinase in its active form(s) inhibiting its proteolytic activity or substrate recognition or on the latent form of the proteinase inhibiting its conversion into active proteinase.
  • the most relevant directly acting inhibitors of proteinases include:
  • antibodies or antibody fragments which e.g. neutralise the active site or block the substrate recognition site;
  • a-macroglobulins e.g. a-macroglobulins
  • the inhibition could also be indirect i.e. by an agent regulating either the expression and/or production of the proteinase (e.g. a natural transcription factor or its naturally regulating systemic or local factor, or a synthetic antisense probe specifically binding to and blocking tne mRNA encoding the proteinase) or by an agent influencing the level or activity of a natural regulator of the proteinase (e.g. an inhibitor of an enzyme responsible for catalytic activation of the target proteinase) .
  • an agent regulating either the expression and/or production of the proteinase e.g. a natural transcription factor or its naturally regulating systemic or local factor, or a synthetic antisense probe specifically binding to and blocking tne mRNA encoding the proteinase
  • an agent influencing the level or activity of a natural regulator of the proteinase e.g. an inhibitor of an enzyme responsible for catalytic activation of the target proteinase
  • the production of proteinases may be performed either directly cultures of isolated osteoclasts or indirectly by transfection of an expression plasmid containing proteinase encoding cDNA into a recipient cell line.
  • an expression plasmid containing proteinase encoding cDNA into a recipient cell line.
  • m osteoclasts the majority of e.g. MMP-9 is produced in its latent proform (pro-MMP-9) and therefore needs a s.Dsequent activation process if the active form is required.
  • the amount of proteinase obtained from production in osteociast is severely restricted by:
  • both latent and active proteinase can be produced airectly by recombinant techniques depending on whether me expression plasmid-transfected into the recipient cell is designed to contain the complete cDNA or a cDNA devoid of the region encoding the propeptide moiety of the latent enzyme. Since active proteinases are generally less stable than their corresponding latent pro-enzymes and particularly under cell culture conditions might be degraded, production of latent proteinases is often preferable.
  • the identification and cloning of cDNA encoding several osteoclastic MMPs or parts thereof, including MMP-9, MMP-12 and MTl-MMP is described
  • agents inhibiting metalloprotemases including MMPs and especially MT-MMPs and MMP-12) involved in one or more phases of the osteoclast life cycle can include:
  • substances stimulating the level or activity of a natural inhibitor of metalloproteinase or MMP and .
  • substances reducing the level or activity of a natural activator of metalloproteinase or MMP e.g. a substance analogous to the description in 1. and 2. but regulating a proteolytic enzyme responsible for activation of latent MMP.
  • Examples 5 and 6 below describe the development of inhibitory agents; the production and use of anti-protemase antibodies (Example 5) ; the production, identification and characterisation of synthetic, peptide-mimicking proteinase inhibitors (Example 6 a-e) ; and the design and use of antisense probes to proteinase mRNA (Example 6f) .
  • Anti-prote nase antibodies are central tools for the ⁇ evelopment of proteinase inhibitors and under appropriate conditions can be used as inhibitors themselves (see Example 5e and Figure 9) .
  • the applications for anti- proteinase antibodies and parts thereof are several and n particular anti-MMP antibodies and antibody fragments will be useful :
  • Synthetic peptide and peptide-mimicking inhibitors of proteinases are promising agents for use for treatment of bone metabolic disease by inhibition of the action of proteinases involved in the recruitment, proliferation, differentiation, or migration or osteoclast precursor cells or the migration, fusion, attachment, polarisation, removal of mineralised osseous substance, or death of osteoclasts.
  • Several methods for production of peptide and peptide mimicking inhibitory agents are available, two of which are described in Example 6 (a-e).
  • PEGA polyethylene glycol cross-linked polyamide
  • Example 6b we report the finding of a peptide-mimickmg molecule (CL-1) identified by incubation of MMP-9 with a PEGA bead substrate library, which has a low K m (3.4 ⁇ M) but also a low k cat / m ( ⁇ 500 M ⁇ s "1 ) suggesting its potential use as an inhibitor of osteoclastic MMP-9. Even better inhibitory characteristics of pseudo-substrates can be expected after modification of the originally identified substrates, e.g.
  • peptide-mimickmg substrates either by linking peptide-mimickmg substrates to chelatmg groups such as hydroxamates, thiols, phosphonamidates, phosphmates and phosphoramidates (reviewed by Birkedal-Hansen et al , 1993 2 ) or by designing pseu ⁇ o-substrates which easily forms acyl-proteinase complexes but which hydrolyse slowly due to interaction with the binding site on the enzyme for the leaving group (Baggio et al 1996 1 ) .
  • chelatmg groups such as hydroxamates, thiols, phosphonamidates, phosphmates and phosphoramidates (reviewed by Birkedal-Hansen et al , 1993 2 ) or by designing pseu ⁇ o-substrates which easily forms acyl-proteinase complexes but which hydrolyse slowly due to interaction with the binding site
  • synthetic peptide inhibitors can be identified among millions of randomly designed peptides in a PEGA bead synthetic peptide inhibitor library (Meldal and Svendsen, 1995 12 ; Meldal et al , 1997 21 ) .
  • the screening is based on the rare ability of some peptides to inhibit the hydrolysis of the established synthetic peptide-mimicking substrate.
  • this knowledge has been used in combination with the PEGA bead technology by extending the group of building blocks used for synthesis of putative inhibitory peptide analogues on the PEGA-beads from just natural amino acids (including hydroxyproline) and their corresponding D-forms to also including pseudo dipeptides such as NH 2 -P1 P / C P1 ' -COOH, NH 2 -P1 P / N P1 ' -COOH or NH : - P1 P ⁇ ?1 ' -COOH, where the two normal amino acids (Pl and Pl ' ) instead of being linked through the peptide bond are linked through the phosphinate, phosphonamidate or phosphonate bond ( p / c , P / N or p /°) .
  • pseudo dipeptides such as NH 2 -P1 P / C P1 ' -COOH, NH 2 -P1 P / N P1 ' -COOH
  • PEGA-bead substrate library technology By employment of the PEGA-bead substrate library technology, it has been possible to identify peptide sequences which are of use in the design of novel highly specific MMP-substrates (see Example 6 a and b) . These substrates facilitate the design and use of PEGA-bead hioitor libraries both through the use of one of these selective substrates in the library and through the use of the substrate sequence data for the design of the structure of the randomised inhibitors in the library (Meldal and Sven ⁇ sen, 1995 12 ; Meldal et al , 1997 21 ) . Particularly in the design of PEGA-bead inhibitor libraries based on inhibitors witr.
  • the substrate data were usec for determination of the two ammo acid R-groups around the phosphinate, phosphonamidate or phosphonate of the pseu ⁇ o dipeptide (see Example 6c). Furthermore, the design of selective inhibitors based on the characteristics of the novel MMP-substrate specificities will be facilitated (see data for CL-1, CL-21, CL-25 and CL-29 in Example 6b) .
  • the specific substrates could become important tools for selective detection and quantification of MMPs in tissue samples in diagnosis and research.
  • the other method for identification of peptide and peptide mimicking inhibitory agents is based on the use of positional combinatorial peptide inhibitor libraries.
  • a few members of these libraries of randomly synthesised peptides having in a single am o acid position an abnormal amino acid, such as a D-ammo acid instead of an L-amino acid, in some case will act m an inhibitory way to a particular enzyme, probably due to a pseudo-substrate effect.
  • an inhibitory signal is obtained by incubation of a positional combinatorial peptide inhibitor library with a proteinase or a biological model system including essential proteinase activity
  • the peptide (s) in the library responsible for this inhibition must be subsequently identified by systematic segmentation of the library as described in Example 6 (d-e) for incubation of positional combinatorial peptide inhibitor libraries with murine foetal metatarsal cultures.
  • Some preferred inhibitory libraries and peptide structures provided by the invention are the libraries X-X-w-X-X, X-X- 1-X-X and X-X-w-Y-X and the peptides C-L-w-Y-L, C-L-w-Y-M, C-Y-w-Y-L, V-Y-w-Y-M and -F-w-Y-L, where X are natural ammo acids including hydroxyprolme, and w and 1 are D- tryptophan and D-leucme, respectively (see Example 6e).
  • the major advantage and disadvantage of the PEGA bead library are the immediate identification of inhibitors and the need for incubation w th a preferably purified proteinase preparation in a test tube, respectively.
  • the major advantage and disadvantage of the positional combinatorial peptide inhibitor library is the possibility to screen directly for an inhibitory effect a biological test system and the need for several cumbersome segmentations of the initial library to identify the agent originally causing the inhibition, respectively.
  • one feature of the positional combinatorial peptide inhibitor library can be seen as both favourable and non- favourable, since the functional background for an inhibitory response induced in the biological system by this 5 type of library is uncertain i.e. the inhibitory peptides might not be proteinase inhibitors but have other regulatory functions .
  • the benefits of using antisense probes to proteinases can be divided into two major aspects, an early aspect and a l later aspect.
  • the antisense probes are important tools for evaluation of the role of the corresponding proteinase m a biological process, because they can be used at an early stage of a study when anything else than the oligonucleotide sequence of this proteinase is unknown, and this even with
  • Antisense probes were used successfully for inhibition of MMP synthesis by fibroblasts (Lin et al,
  • the inhibitor due to its intrinsic specificity selectively reacts with the proteinase present on these cells either because the proteinase at this target cell is particularly available to the inhibitor (due to e.g. the localisation of the cell, the localisation of the proteinase m the cell or simply by a local high concentration of the proteinase) or because the proteinase when produced by these cells is different from the corresponding proteinase as it is expressed in other cells and tissues (due to e.g. post-translational modifications).
  • hybrid molecules or conjugates combining one part of the agent having protemase-inhibitory characteristics with another part having antibody or ligand specificity for the particular cells or tissue.
  • hybrids can be made by recombinant expression of fusion-proteins after cloning of a hybrid cDNA.
  • a piece of cDNA encoding the osteoclast- specific ligand calcitonin (or a receptor-binding part thereof/ can be ligated to another piece of cDNA encoding a pept-.de inhibitor for an osteoclast proteinase.
  • Hybrids can also be conjugates of two compounds e.g.
  • Figure 1 shows the nucleotide (SEQ ID No.3) and deduced ammo acid sequence (SEQ ID No.4) of the MTl-MMP or MTl-MMP analogue identified in rabbit osteoclasts;
  • Figure 2 shows a comparison between the ammo acid sequence of the novel MT-MMP identified in rabbit osteoclasts (RaoDit) (SEQ ID No.4) and the previously reported amino aci ⁇ sequences of Human (SEQ ID No.5), Rat (SEQ ID No.6) and Mouse MTl-MMP (SEQ ID No. 7). Positions with an ammo acid identical in all 4 proteins are indicated (*);
  • Figure 3 shows schematically the structure of three MTl-MMP cDNA constructs and the corresponding control construct used in Example 3-2;
  • Figure 4 shows the nucleotide (SEQ ID No.8) and deduced 0 ammo acid sequence (SEQ ID No.9) of the MMP-12 or MMP-12 analogue identified rabbit osteoclasts;
  • Figure 6 shows schematically the structure of a MMP-12 cDNA construct and the corresponding control construct used in Example 3-4;
  • Figure 7 shows the effect of various proteinase inhibitors on the migration of purified osteoclasts through collagen coated membranes. The values are relative to the number of migrations observed in the absence of proteinase inhibitor.
  • Figure 8 shows the effect of an MMP-inhib tor on pit formation by purified osteoclast seeded on dentine slices which were either not coated or coated with collagen. The values are relative to pit formation in the absence of collagen coating and MMP-mhibitor;
  • Figure 9 shows the dose dependent inhibitory effect on MMP-9 proteolytic activity of sera from mice immunised with the conjugated femta-peptide RSGAPVDQMFPGVPL (SEQ ID No.13) 0 (peptide B, mimicking a region of the rabbit MMP-9 hemopexin domain) alone or together with purified intact rabbit osteoclast pro-MMP-9. No inhibitory effect was observed for sera from non-immunised mice and for mice immunised with another non-related femta-peptide (peptide A) .
  • the values -1 are relative to the average relative fluorescence generated during 30 minutes of incubation of the synthetic quenched fluorogenic substrate Mca-PLGL-Dpa-AR-NH (Bachem) (SEQ ID No. 14) with a pre-incubated mixture of purified activated MMP-9 and the appropriate dilutions of 9 different control 0 sera (non-immunised or immunised with non-relevant femta- peptides ) ;
  • Figure 11 shows inhibition of hydrolysis of CLl by the MMP-mhibitor RP59794, but not the cysteine proteinase inhibitor E-64.
  • MMP-9 80 pmol
  • subtilism 3.4 pmol
  • Figure 12 shows the synthesis of the phosphinate analogue to hydroxyprolme for use as a building block in the subsequent generation of a hydroxyprolme-methionine phosphinate pseudo dipeptide (see also Figure 13) .
  • the phosphmic acid analogue to trans-hydroxyproline is synthesised from potassium D- or L-erythronate . After bromination at the 2 and 4 position the acid is transformed into the methyl ester by methanol quenching. The 2-pos ⁇ t ⁇ on is reduced and the ester converted into the alcohol by sodium borohydride reduction. The primary alcohol is oxidized by sodium hypochlorite to the aldehyde and condensed with tritylamme. The lmine formed is reacted with bis-trimethylsilyloxyphosphine to yield the phosphinate. Upon acid hydrolysis and intramolecular substitution of the bromine the free hydroxyprolme is obtained;
  • Figure 13 shows the synthesis of the hydroxyprolme- methionme phosphinate pseudo dipeptide for use in preparation of the PEGA bead phosphinate inhibitor library Ila (see Example 6c).
  • the phosphmic acid analogue of hydroxyprolme (see Figure 12) is derivatised with oenzyloxycarbonyl chloride.
  • 2-methylene-4-methyl mercapto- outanoic acid ethyl ester was synthesised from diethylmalonate sodiation and reaction with methyl mercaptoethyl chloride followed by selective basic ester hydrolysis, acid decarboxylation and reaction with formaldehyde in the presence of piperidine. These reactions can be performed on a large scale.
  • Figure 14 shows the synthesis of the glycine-leucine phosphinate pseudo dipeptide for use in preparation of the PEGA bead phosphinate inhibitor library lib (see Example 6c) .
  • the phosphinic acid analogue of glycine is synthesised from tritylamine and formaldehyde to give the imine which is reacted with bis-trimethylsilyloxyphosphine obtained from ammoniumphosphinate and hexamethyl disilazane.
  • the product is deprotected by acid hydrolysis and is derivatised with benzyloxycarbonyl chloride.
  • Figure 15 shows the development and structure of the PEGA bead phosphinate inhibitor library (Ha) based on the hydroxyproline-methionine phosphinate pseudo dipeptide.
  • the invariable quenched fluorescent substrate here: Ac- Y(N0 2 ) PLJMKGK(Abz)G-"Linker"-) (SEQ ID No.17) and the randomly variable phosphinate inhibitor (here: X ⁇ X 2 J p / c MX 3 X4- "Linker"-) are independently associated to the PEGA bead.
  • an FmocLys (Aloe) residue can be used to obtain orthogonal protection and incorporation of the two compounds and the order of synthesis of the library and the substrate may be reversed. This gives the possioility to use the same library with several substrates.
  • the analogous 5 library (lib) was prepared similarly by using an invariable substrate corresponding to MR1 (see Table 3) and a randomly variable phosphinate inhibitor X ⁇ X 2 G p / c LX 3 X 4 -"L ⁇ nker"-;
  • Figure 16 shows inhibition of the 45 Ca 2+ -release from foetal 10 murine metatarsals cultured for 4 days in the presence of positional combinatorial pentapeptide inhibitor libraries.
  • the results for 5 selected libraries with the sequence X-X- D-X-X are shown.
  • D was either D-isoleucme, D-leucme, D-lysme, D-serine or D-tryptophan
  • X were lb randomly varying L-amino acids.
  • the pentapeptide libraries with a D-ile, D-leu or D-trp at the third position induced a significant reduction of bone resorption.
  • the MMP- hibitor RP59794 was included as a positive control.
  • the cells were washed with PBS and then treated with 0.001% pronase E and 0.02% EDTA for approximately 10 min. to release all non- osteoclastic cells.
  • the purified osteoclasts were cultured 10 for another 2 hrs before isolation of mRNA.
  • cDNA reverse-transcribed from mRNA from the purified osteoclasts was subjected to PCR with degenerate primers designed from conserved regions of MMP genes. Briefly, the poly(A) ⁇ RNA from purified osteoclasts was prepared using a mRNA purification kit (Pharmacia Biotech,
  • cDNA bands 330-340, 380-390 bp and 560-570 bp m length were identified by electrophoresis in a 1% agarose gel.
  • the cDNAs were purified and cloned into a pCRII vector
  • MMP-12 human metalloelastase
  • a rabbit cDNA library (Tezuka et al , 1994 15 ) was screened by colony hybridisation, using the random-primed 32P-labelled PCR product of A3 as a probe. By screening 1x10 s clones, one positive clone was identified and made into
  • Figure 1 shows the nucleotide sequence of the cloned insert.
  • the deduced amino-acid sequence of the insert showed 96% similarity with human MTl-MMP ( Figure 2).
  • Figure 2 There were no additions or deletions of specific sequences when compared to MTl-MMP of other species.
  • the isolated novel cDNA encoded the rabbit homologue of MTl-MMP or of a closely related but previously unreported human osteoclast MT-MMP.
  • the nucleotide sequence analysis of the A3 PCR fragment and of the rabbit MTl-MMP cDNA clone from the cDNA library was determined from both strands by the dideoxy cham- termmation method using the Qiagen-purifled plasmid DNA 5 (Qiagen, USA), the Sequenase kit (U.S.B., USA), and either pBluesc ⁇ pt SK primers (Stratagene, USA) or synthetic oligonucleotide primers.
  • the cDNA probes were radiolabelled with a multiprime DNA 0 labelling system (Amersham International pic, Buckinghamshire, England) using [alpha- 32 P] dCTP and the oligonucleotide probe was radiolabelled with a 5 ' -end labelling kit (Amersham) using [gamma- 32 P] ATP .
  • Hybridisation was performed as described previously (Tezuka et al , 1992 15 ) 5 and visualised by a Phosphorimager SF (Stratagene, La Jolla, CA) .
  • Digoxygenm-labelled antisense or sense RNA probes were prepared by use of a DIG RNA labelling kit 5 (Boehringer Mannheim) according to the instruction manual and compared to paraffin sections stained for tartrate- resistant acid phosphatase (Blavier and Delaisse, 1995 3 ) . Many tartrate-resistant acid phosphatase-positive multi- nucleated cells were positive for MTl-MMP, whether they were attached to calcified cartilage or to bone.
  • rabbit bone cells were seeded on glass coverslips. After 1.5 hr cultivation the non-adherent cells were discarded and the remaining cells were cultured for 1 to 18 hr, fixed and processed for immunocytochemistry. They were incubated for 90 mm in the presence of 1-3 ⁇ g/ml of the monoclonal MT1-
  • CDGNFDTVAMLRGEM SEQ ID No.21 which differs by 1 ammo acid from the corresponding rabbit sequence (V in rabbit
  • Rhodamme-labelled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) was used as secondary antibody at 200 times dilution.
  • Rhodamme-labelled donkey anti-mouse IgG Jackson ImmunoResearch Laboratories, Inc. West Grove, PA
  • fluorescence did not appear when the MTl-MMP antibody was replaced by non-immune IgG. All bright signals were in the focal plane where the cells were seen in contact with their suostrate. In moving cells, mainly the extremities of the lamellopodia were illuminated.
  • MTl-MMP antibody Therefore MTl-MMP appears to be localised on tne podosomes.
  • MTl-MMP stammg was however somewhat more diffuse as compared to the sharp actm sta mg, probably because the sharp actm dots are due to bundles of actm filaments m the core of the podosome and oriented perpendicularly to the attachment surface, while MTl-MMP might be on the surface of the podosome.
  • staining for actin illuminated also the extremities of the lamellopodia, as did the anti-MTl-MMP antibody. Similar localisations of MT-MMP were found when the osteoclast was cultured on bone slices.
  • the production of osteoclast proteinases can be performed in cultures of osteoclasts or in cell lines transfected with cDNA encoding the osteoclast proteinase or a part thereof. In all cases a purification of the product is needed and in those cases where the production leads to a latent pro-form of the proteinase a subsequent activation is also needed for some purposes. Exemplifying this process, the production, purification and activation of osteoclastic pro-MMP-9 was performed according to the following descriptions: a . Os teoclas t production of pro-MMP-9
  • osteoclastic pro-MMP-9 When cultured at 37°C and 5% C0 2 , under serum-free conditions to avoid contamination with serum-derived proteinases and natural inhibitors of proteinases, rabbit 5 osteoclasts secreted 92 kDa pro-MMP-9 into the culture medium. According to studies by gelatinase-zymography, addition of 40 nM of phorbol 12-myr ⁇ state 13-acetate (PMA) to the cell culture increased the yield of pro-MMP-9 at least 3-fold. 10 b . Purifica tion of osteoclastic pro-MMP-9
  • the osteoclast conditioned medium was concentrated by 10 kDa cut-off filtration (Amicon) and subsequently diluted in 2.5 mM sodium phosphate containing 0.04% Triton X-100
  • pro-gelatmases including pro-MMP-9 and pro-MMP-2 were observed to bind efficiently to the hydroxyapatite column.
  • pro-MMP-9 was eluted
  • the purified latent pro-MMP-9 was activated either by a traditional method based on incubation with 1 mM (4-am ⁇ no- phenyl) mercuric acetate (APMA) for 2-8 hrs at 37°C or by a method based on the activation of gelatmases as it is
  • the purified pro-MMP-9 was run into a slab gel by preparative SDS-PAGE.
  • the SDS was substituted by Triton X- 100 during subsequent incubation of the gel for 16 hrs in a Duffer containing 50 mM T ⁇ s-HCl, pH 7.5, 5 mM CaCl 2 , 1 ⁇ M t ⁇ - ZnCl., and 1% Triton X-100.
  • a part of the gel corresponding to an electrophoretic migration distance of compounds with an approximate molecular weight of 92 ⁇ 5 kDa (but including the by now activated approximate 68 kDa form of MMP-9) was excised.
  • the active MMP-9 was electroph ⁇ retically eluted from the excised gel.
  • GST glutathione S-transferase
  • tne cells were pelleted and resuspended in 25 ml of ice-cold IX PBS. All subsequent steps were carried out at 4°C or on ice. E.coli cells were lysed by sonication (5 bursts of 10 seconds/burst) . Cellular debris was pelleted by centrifugation at 3000 rpm after incubation with 1%
  • fusion proteins were eluted with 900 ⁇ l of Glutathione Elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0). The eluates were stored at -20°C until use .
  • the three fusion proteins migrated m SDS-PAGE as proteins of approx. 85, 60 and 55 kDa corresponding to their cDNA-deduced sizes of 87, 61 and 57 kDa, respectively.
  • the fusion proteins were confirmed to be GST-MT1-MMP fusion proteins by Western-blotting using an anti-GST antibody
  • Ec2, Ec3 and the GST tag alone were incubated with 2.7 pmol of human plasmin (Boehringer) at 25°C for 30 minutes in a final volume of 45 ⁇ l . The reactions were stopped by the addition of 10 ⁇ M aprot in.
  • Enzyma tic assay The proteolytic activities were evaluated by fluorescence measurements (excitation wavelength: 320 nm, emission wavelength: 387 nm) of the hydrolysis of the quenched fluorescent peptide substrate Mca-PLGL-Dpa-AR-NH 2 (Bachem) (SEQ ID No.14) after incubation at 37°C for 180 minutes in 150 mM NaCl, 10 mM CaCl 2 , 0.05 % (v/v) Bri]-35 in 50 mM Tris-HCl, pH 7.5 (see Table 1).
  • MMP-12 Due to the expression and use in cell invasion of MMP- 12 m macrophages as well as the common hematopoieitic stem cell origin of osteoclasts and macrophages, we investigated whether MMP-12 was also expressed in osteoclasts. As indicated in Example lb and shown in the present example, this was indeed the case, and we therefore expect that MMP- 12 plays a similar role in osteoclast invasion and migration as it does in macrophages.
  • MMP-12 cDNA isolated from the rabbit osteoclast cDNA library, and the subsequent steps of expression, characterisation and recombinant production of the MMP-12 fusion protein was done essentially as described for MTl-MMP cDNA (see Examples 1, 3-2 and 3-3) .
  • the osteoclast preparations were obtained from rabbit long bones and the reverse transcribed mRNA from these osteoclasts was amplified by PCR using degenerate primers based on regions conserved in the MMP family (see Example lb) .
  • one (B4) presented homology with a sequence of human MMP-12.
  • rabbit MMP-12 cDNA containing the open reading frame (bp 58-1437, see Figure 4) was amplified by PCR using 0 primers sense 5 -CGGGATCCCTGTGGGTCACTTCTTCT-3 (SEQ ID No.22) ana antisense 5 ' -CCGCTCGAGCTGGCACCATTACTAGC-3 ' (SEQ ID No.23).
  • the cDNA fragment was inserted into the BamHI and Xhol sites of the pGEX-6P-2 vector as described for MTl- MMP.
  • the cDNA was shown by direct sequence analysis to lie 5 ⁇ ust 5' to the GST-encodmg moiety of the vector and in proper reading frame with the plasmid translation initiation site ( Figure 6) .
  • E.coli strain BL-21, transformed with pGEX-6P-2 alone (control vector) and pGEX-6P-2/MMP-12 were plated on Luna 5 Broth (LB) agar plates with 50 ⁇ g/ml ampicillm at 37°C overnight. Single colonies were grown overnight in 50 ml of LB containing 50 ⁇ g/ml ampicillm in a shaking incubator at 30°C. Subsequently, the overnight cultures were diluted 1:100 in 400 ml of LB containing 50 ⁇ g/ml ampicillm and grown at
  • IPTG Sigma
  • the fusion protein according to SDS-PAGE was localised in the pellet (estimated molecular weight approx. 75 kDa corresponding well to the cDNA-deduced size of 83 kDa) .
  • the pellet was solubilized in 20 ml of buffer containing 8 M urea and then stirred ror 1 h at 4°C.
  • the ⁇ sample was clarified by centrifugation at 40,000 x g for 30 rutes at 4°C.
  • tne urea was removed completely by stepwise dialysis of the supernatant against the Tris-HCl buffer. The supernatant was subjected to SDS- PAGE and proteins stained by Coomass e Brilliant Blue R250.
  • osteoclast purification techniques did not allow the demonstration of whether these MMPs were from osteoclasts or other cells.
  • l z> Briefly, we seeded purified or non-purified osteoclasts on membranes (12 ⁇ m pore size) coated with type I collagen, and followed their migration to the lower surface of the membranes after an overnight culture the absence or presence of MMP inhibitors.
  • osteoclasts could extend cell processes into the pores of the membranes and spread over the lower surface of the membranes. This migration process was inhibited by MMP inhibitors of both the synthetic osteoclast preparations, but also when using purified preparations, osteoclasts could extend cell processes into the pores of the membranes and spread over the lower surface of the membranes. This migration process was inhibited by MMP inhibitors of both the synthetic osteoclast preparations, but also when using purified preparations, osteoclasts could extend cell processes into the pores of the membranes and spread over the lower surface of the membranes. This migration process was inhibited by MMP inhibitors of both the synthetic osteoclasts
  • MMPs is on the migration of the osteoclasts to their future
  • pro-MMP-9 purified from osteoclast cultures as described in Example 3-1 was used for immunisation either in its latent form or after activation by APMA or by m-gel treatment with SDS/Triton X-
  • femta-pepcides were synthesised by using Fmoc-ammo-acids-O- 5 pentafluorophenyl-esters in the presence of catalytic amounts of 3, 4-d ⁇ hydro-4-oxo, 1, 2, 3-benzotr ⁇ azm-3-yl m a fully automated custom made peptide synthesiser.
  • thyroglobulm prote aceous carrier molecular
  • thyroglobulin 0 and glutaric anhydride (1:2 w/w) were incubated for 2 hrs at 20°C in 0.1 M sodium borate, pH 9.0 and subsequently desalted on a Nap 10/Sephadex G-25 column (Pharmacia) and dried by vacuum centrifugation.
  • the carrier was resolubilized m 0.01 M sodium phosphate, pH 5.0 and incubated for 3 min at
  • CDI 1-ethyl- 3- (3-d ⁇ methylam ⁇ nopropyl) -carbodiimide
  • the CDI- activated thyroglobulm was incubated 4 hrs at 20°C m equal volumes and amounts (w/w) with the femta-peptide in 0.2 M sodium phosphate, pri 9.0.
  • the thyroglobulm/CDI/femta- peptide conjugates were dialysed and their protein content determined.
  • the thyroglobulin/CDI/femta-peptide conjugates were mixed with Freunds incomplete adjutant and injected intramuscularly once per month in female New Zealand White rabbits. Blood was collected and the immunoglobulin fraction purified from the corresponding serum by ammonium sulphate precipitation.
  • the thyroglobul /CDI/femta-peptide conjugates were mixed with Freunds incomplete adjuvant and injected mtraperitoneally every third week in female BALB/c-CFl murine hybrids. A final booster immunisation of the conjugate without adjuvant was given 3 days prior to splenectomy.
  • the spleen cells were fused with P3-X-63- Ag8.653 myeloma cells in the presence of 50% polyethylene glycol 4000 and the resulting hybridoma cells propagated and cloned according to standard proce ⁇ ures .
  • Monoclonal antibody was purified from the conditioned medium of hybridoma cultures by using protein A affinity cnromatography .
  • antisera and monoclonal antibodies were selected and initially characterised by enzyme-linked immunosorbent assay (ELISA) based on 96-well polystyrene plates coated with either purified intact or truncated MMPs or homologous or heterologous conjugated femta-peptides .
  • ELISA enzyme-linked immunosorbent assay
  • ELISA have several applications.
  • One example is their use in lmmunohistochemical identification of MMP-expression on the protein level by incubation of an anti-MMP antibody with
  • Example 2d the binding of a monoclonal antibody raised by immunisation with a MTl-MMP mimicking peptide to the actm- ⁇ ch membranous areas of an osteoclast shows that MMP-antibodies not only are tools of central importance to the identification of the 0 cells which produce a particular MMP, but also can demonstrate the cellular localisation of a MMP and thereby aid in the clarification of its biological role.
  • non-immunoglobulm inhibitors of osteoclast proteinase aimed at two main type of agents, one being peptide or peptide-mimick g proteinase inhibitors another being antisense probes specifically binding to 3b osteoclast proteinase mRNA.
  • the peptide and peptide mimicking agents were produced by two methods: a technology cased on PEGA bead peptide substrate and inhibitor libraries (see a-c, below) , the other being based on positional combinatorial peptide inhibitor libraries (see d-e, below).
  • the design and use of antisense probes is described in f (see below) :
  • K(Abz) is a quenching 3-n ⁇ trotyros ⁇ ne and a fluorogenic lysine (2- ammobenzoic acid), respectively.
  • the libraries were incubated at 37°C with purified and activated osteoclast proMMP-9 (approx. 0.1 ⁇ M) and fluorogenic beads subsequently isolated by a micropipette under fluorescence microscopy. The isolated beads were analysed by an ammo acid sequencer.
  • one of the fluorescent beads (A2 n Table 2) isolated from PEGA bead peptide substrate library ⁇ ) contained two similar peptides with the sequences S-K- 5 Y(N0 2 ) -P-J-A-L-F-F-K(Abz) -PEGA (SEQ ID No .2 ) and L-F-F- K(Abz)-PEGA (SEQ ID No.24) indicating hydrolysis by osteoclastic MMP-9 of the novel peptide-mimicking substrate S-K-Y(N0 2 )-P-J-A-L-F-F-K(Abz) (SEQ ID No.2) at the Pl-Pl' position: A-L. Based on this information several soluble
  • IC quenched fluorogenic peptides were synthesised (e.g., CL-1 and CL-6, see Table 3 and Figures 10 and 11) .
  • the first 30 soluble quenched fluorogenic peptide substrate was identified in the PEGA bead substrate libraries A and B.
  • ⁇ - can ⁇ idates for MMP-9 (named CL-1 to CL-30) were synthesised cy multiple column peptide synthesis. Their individual ⁇ net ⁇ c properties (k cat and K ra ) were determined by incubation at 37°C with MMP-9 and recombinant truncated MTl-MMP of osteoclast origin, and as controls recombinant truncated
  • 3_ -.mmobilised on the PEGA bead E.g., the putative substrate, L-1, was inhibitory to MMP-9 as would have been expected for a pseudo-substrate, i.e. with a low K m (3.4 ⁇ M) and a low ⁇ dt /K m (250 M ' V 1 ) (see Table 3) .
  • Table 3 Kinetic parameters for the hydrolysis of three established soluble MMP-9 substrates (B, MRl, MR2) and two soluble substrates (CLl, CL6) designed according to results from PEGA bead library (B).
  • Fable 4 Kinetic parameters for the hydrolysis of three soluble selective MMP-9 substrates (CL-21, CL-25 and CL-29) designed according to results from PEGA bead substrate library (B) .
  • the kinetic parameters are k 3 ./K in ⁇ M ' x min ' and relatively ( ) to the corresponding value for MMP-9.
  • PEGA bead peptide inhibitor library (I) was generated consisting of approx. 10° different beads, each containing many copies of a single well-defined substrate sequence as well as many copies of a randomly generated putative inhibitor sequence: N X1-X2-X3- D-X4-X5-X6-V C -PEGA, where XI to X6 are L-amino acids varying randomly from bead to bead, and D is a D-amino acid varying randomly from bead to bead.
  • the library was incubated at 37°C with active MMP-9 and beads remaining quenched (i.e. dark compared to the majority of brightly fluorescent beads) were isolated by a micropipette under fluorescence microscopy.
  • the isolated beads were analysed by an ammo acid sequencer and since the substrate sequence was not degraded by tne Edman degradation due to prior acylation at the N-termmus, the sequences obtained corresponded to potential peptide-mimickmg MMP-9 inhibitors.
  • PEGA bead inhibitor library A novel type of PEGA bead inhibitor library was developed in order to identify peptide substrate mimicking MMP-inhibitors with a phosphinate instead of a peptide bond at the susceptible cleavage site (i.e. between the expected Pl and Pl' sites of the corresponding substrate) .
  • Two PEGA bead phosphinate inhibitor libraries (Ila and lib) were generated. Each library consisted of approx.
  • each PEGA bead contained many copies of a single well-defined substrate sequence as well as many copies of a randomly generated putative inhibitor sequence: N X1-X2-J p / c M-X3-X4-"L ⁇ nker"-EGA (in Ila) or N Xl-X2-G p / c L-X3-X4-"L ⁇ nker"-PEGA (in lib), where XI to X4 are L-ammo acids varying randomly from bead to bead, and J P / C M and G P / C L is the phosphinate pseudo dipeptide used in library Ila and lib, respectively (see Figures 12-15) .
  • the design of the first two phosphinate pseudo dipeptides was based on the identity of suitable Pl and Pl' ammo acids m newly developed and existing MMP-9 substrates. Other combinations of pseudo ammo acids around the phosphinate bond will also be investigated according to the findings of MMP selective peptide substrates by use of e.g. PEGA bead substrate libraries.
  • the peptide libraries were purified by high performance liquid chromatography in order to remove salts and other substances which were toxic to bone tissue cultures before being tested for inhibitory effects on osteoclast migration and bone resorption in murine foetal metatarsal cultures.
  • Each of the 20 libraries contained 30 ⁇ mol pentapeptides composed of up to 21 4 (194,481) different structures.
  • the general MMP inhibitor, RP59794 which has been shown previously to inhibit the migration of osteoclasts and thereby reduce the release of Ca + in the metatarsal culture model (Blavier & Delaisse, 1995 ) was included as a positive control in all experiments.
  • Each of the 20 libraries was used in a concentration of 3 mM total peptide corresponding to a concentration of approx. 15
  • each of the 23 libraries was used in concentration of 3.2 mM total peptide, corresponding to approx. 340 nM for each of the 9,261 structures in a library. More than half of the 23 libraries did not significantly affect the bone resorption, whereas 11 of the 23 libraries showed significant and/or marginally significant reductions in the 45 Ca-release at Day 1, 2 and/or 4 (see Table 7) .
  • Table 7 Change (in %) of Ca-release due to the addition of a Z5-X- trp-X-X, X-Z2/5-trp-X-X, X-X-trp-Z2-X or X-X-trp-X-Z5 combinatorial library to 4-day metatarsal cultures
  • V-X-trp-X-X (3.2 mM) - 6% (0.33) -30% (0.17) -23% (0.11)
  • V-Y-w-Y-M -17% (0.21) -21% (0.04) -15% (0.02)
  • Antisense oligonucleotide probes against various MMPs were produced in order to study their influence on bone metabolism and osteoclast biology n bone cell and tissue cultures as well as in animal models.
  • the antisense oligonucleotide probes were designed by choosing sequences which were specific to a particular MMP and showing as little as possible similarity to any predictably relevant mammalian genes. In all cases a sense probe and/or a so-called scrambled probe was used as negative controls for comparison to the antisense probe.
  • the probes were used in the murine metatarsal system described in Example 6e and in a murine pre-osteoclast culture system.
  • the latter was based on unfractionated bone cells isolated from 12 day old mice and cultured for 7 days in the presence of 5% fettle calf serum in order to eradicate all multmucleated osteoclast leaving only stromal cells and osteoclast precursors.
  • Upon subsequent culture of approximately 10 days in the presence of 2 ⁇ g/ml PGE 2 new mature osteoclasts were formed.
  • the continuous differentiation of pre-osteoclasts to mature osteoclasts in this culture system correlated well to production of pro-MMP-9 according to gelat ase zymographical studies of the corresponding conditioned medium.
  • the probe was added to the culture medium in a concentration varying between 1 and 10 ⁇ g/ml and the medium was renewed every day.
  • Seven antisense probes (14- to 18-mers) to rabbit MMP-9 were constructed as shown in the Table below: Table 10: Selected probes for use m experiments with MMP-9 expression in rabbit cells and tissues:
  • MOLECULE TYPE DNA (genomic)
  • CTGCTCACAC TCGGCACCGC ACTCGCCTCC CTCGGCTCGG CCAAAAGCAA AACAGCTTCA 600
  • CTACGCCATC CAGGGCCTCA AATGGCAGAA CATAATGAGA TCACTTTCTG CATCCAGAAT 900
  • CAAGGTCTGA CCCCCACCGC TGGCCAACAC CCACTCCCAC CGCAAGGACT TTGCTCTTCC 2460
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO ⁇
  • ORIGINAL SOURCE
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Oryctolagus cuniculus
  • MOLECULE TYPE DNA (genomic) (111) HYPOTHETICAL: NO
  • ORGANISM Oryctolagus cuniculus
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL YES
  • ANTI-SENSE NO ( lx ) FEATURE :
  • MOLECULE TYPE peptide
  • HYPOTHETICAL YES
  • ANTI-SENSE NO

Abstract

L'invention concerne un traitement de maladies métaboliques des os, par inhibition de la production ou de l'action d'une métalloprotéinase matricielle de type membranaire (MT-MMP) ou de la métalloprotéinase matricielle 12 (MMP-12), impliquées dans l'activité de résorption des ostéoclastes. Les inhibiteurs de MT-MMP et de MMP-12 et de l'activité des métalloprotéinases associée aux membranes comprennent des peptides et des analogues de peptides produits à partir d'une banque de billes PEGA, des agents acides nucléiques antisens et des anticorps. On a découvert que les protéinases MT1-MMP et MMP-12 sont exprimées par des ostéoclastes et peuvent être inhibées de façon sélective.
PCT/EP1997/004110 1996-07-30 1997-07-29 Utilisation d'inhibiteurs de proteinase pour la prevention ou la reduction de la resorption osseuse WO1998004287A1 (fr)

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AU42032/97A AU733104B2 (en) 1996-07-30 1997-07-29 The use of proteinase inhibitors for prevention or reduction of bone resorption
IL12810397A IL128103A0 (en) 1996-07-30 1997-07-29 The use of proteinase inhibitors for prevention or reduction of bone resorption
BR9710615-1A BR9710615A (pt) 1996-07-30 1997-07-29 Uso de um agente na manufatura de um medicamento para o tratamento de doença metabólica óssea, e, agente de anti-absorção óssea.
EP97940041A EP0915709A1 (fr) 1996-07-30 1997-07-29 Utilisation d'inhibiteurs de proteinase pour la prevention ou la reduction de la resorption osseuse
JP10508510A JP2001501594A (ja) 1996-07-30 1997-07-29 骨吸収を防止もしくは減少させるためのプロテイナーゼ阻害剤の用途

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US6352976B1 (en) 1998-12-31 2002-03-05 Aventis Pharmaceuticals Inc. Selective inhibitors of MMP-12
WO2000061623A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. Soixante-deux protéines humaines sécrétées
FR2802945A1 (fr) * 1999-12-28 2001-06-29 Pf Medicament Nouvelle metalloprotease matricielle mmp-25 homologue de la matrilysine
WO2001074380A2 (fr) * 2000-04-05 2001-10-11 Ipf Pharmaceuticals Gmbh Medicament contenant un inhibiteur tissulaire des metalloproteinases-2 (timp-2) en tant que substance osteo- anabolisante
WO2001074380A3 (fr) * 2000-04-05 2002-02-07 Ipf Pharmaceuticals Gmbh Medicament contenant un inhibiteur tissulaire des metalloproteinases-2 (timp-2) en tant que substance osteo- anabolisante
US7232796B2 (en) 2000-04-05 2007-06-19 Ipf Pharmaceuticals Gmbh Medicament containing a tissue inhibitor of metalloproteinases-2 (TIMP-2) as an osteoanabolically active substance
EP1288297A1 (fr) * 2000-05-19 2003-03-05 Fujichemico Ltd. Regulation de l'activite de mt1-mmp
EP1288297A4 (fr) * 2000-05-19 2004-11-17 Daiichi Fine Chem Co Ltd Regulation de l'activite de mt1-mmp
US7094754B2 (en) 2001-08-16 2006-08-22 Kimberly-Clark Worldwide, Inc. Anti-aging and wound healing compounds
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US7186693B2 (en) 2001-08-16 2007-03-06 Kimberly - Clark Worldwide, Inc. Metalloproteinase inhibitors for wound healing
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WO2004009098A1 (fr) * 2002-07-18 2004-01-29 Index Pharmaceuticals Ab Composes antisens, procedes et compositions pour le traitement de troubles inflammatoires lies a la metalloproteinase matricielle-12 (mmp-12)
US7148194B2 (en) 2002-12-30 2006-12-12 Kimberly-Clark Worldwide, Inc. Method to increase fibronectin
US7189700B2 (en) 2003-06-20 2007-03-13 Kimberly-Clark Worldwide, Inc. Anti-chrondrosarcoma compounds
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US9051377B2 (en) 2005-12-30 2015-06-09 Dyax Corp. Method for treating breast cancer using antibody binding to MMP-14
EP2231186A2 (fr) * 2007-12-17 2010-09-29 Dyax Corp. Compositions et procédés pour traiter des troubles ostéolytiques comprenant des protéines de liaison à mmp-14
EP2231186A4 (fr) * 2007-12-17 2012-08-08 Dyax Corp Compositions et procédés pour traiter des troubles ostéolytiques comprenant des protéines de liaison à mmp-14
US8501181B2 (en) 2007-12-17 2013-08-06 Dyax Corp. Compositions and methods for treating osteolytic disorders comprising MMP-14 binding proteins
EP2262530A4 (fr) * 2008-03-03 2012-12-05 Dyax Corp Protéines de liaison à métalloprotéinase 12
EP2262530A1 (fr) * 2008-03-03 2010-12-22 Dyax Corp. Protéines de liaison à métalloprotéinase 12
US10314909B2 (en) 2011-10-21 2019-06-11 Dyax Corp. Combination therapy comprising an MMP-14 binding protein
EP2907512A1 (fr) 2014-02-14 2015-08-19 Commissariat A L'energie Atomique Et Aux Energies Alternatives Inhibiteurs de MMP-12 en tant qu'agents antiviraux

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GB9615976D0 (en) 1996-09-11
AU733104B2 (en) 2001-05-10
AU4203297A (en) 1998-02-20
CN1226174A (zh) 1999-08-18
IL128103A0 (en) 1999-11-30
JP2001501594A (ja) 2001-02-06

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