WO2014180851A1

WO2014180851A1 - Inhibitor of calcifying disorders

Info

Publication number: WO2014180851A1
Application number: PCT/EP2014/059245
Authority: WO
Inventors: Frederic CAILOTTO; Rik Lories; Frank Luyten
Original assignee: Katholieke Universiteit Leuven, KU LEUVEN R&D
Priority date: 2013-05-06
Filing date: 2014-05-06
Publication date: 2014-11-13
Also published as: GB201308117D0

Abstract

Compounds for treatment or prevention of calcifying disorders and a screening method for identification of agents useful in the treatment or prevention of calcifying disorders or calcification in vascular, skeletal or skin disorders.

Description

INHIBITOR OF CALCIFYING DISORDERS FIELD OF THE INVENTION

The present invention relates to calcifying disorders or calcification in skeletal, vascular and skin disorders and more particularly to compounds and a method to treat or prevent calcifying disorders. The invention further relates to a member of the SPARC family or a modified form thereof, more particularly to Smoc2 and fragments, homologues and derivatives thereof, for use to treat or prevent calcifying disorders. The invention also provides a screening assay for identification of agents useful in the treatment or prevention of calcifying disorders. BACKGROUND

Calcification of tissues is an important process in development, health and disease. In skeletal development and growth, calcification of the extracellular matrix produced by hypertrophic chondrocytes and osteoblasts is essential. The deposition of calcium containing crystals confers mechanical strength to the bone thereby establishing its structural function. Calcium deposition in the skeleton serves as the main calcium storage in the body that can be called upon for the different biochemical processes at the cellular and molecular level in which calcium is essential. Calcification of the skeleton and its renewal remains equally important during adult life in the skeletal remodeling cycle. Impaired calcification of the bone matrix, for instance due to Calcium or Vitamin D deficiency, will weaken the bone and contribute to osteoporosis and fractures.

However, abnormal or excessive calcification also contributes to the signs, symptoms and progression of different diseases. These include skeletal disorders characterized by calcium crystal deposition in the joints and in tendons, vascular calcification contributing to ischemic vascular disease or as a part of the vascular syndrome in patients with chronic kidney failure, as well as calcifications in the skin as seen in patients with systemic sclerosis.

Chondrocalcinosis is a term used to describe the presence of calcium-containing crystals in the articular cartilage. However, abnormal calcium deposition is not limited to the articular cartilage but can also be found in the synovium, ligaments and tendons, and even in the periarticular soft tissues. Chrondocalcinosis is a common feature, increasing with age, found in about 4% of the adult population at the time of death and radiographically in up to 50% of people in their ninth decade. Calcium pyrophosphate dihydrate (CPPD) crystals can cause severe symptoms when they trigger acute inflammatory attacks. CPPD occurs both isolated and in a familial pattern. Phagocytosis of the crystals by neutrophils results in the activation and amplification of an inflammatory cascade leading to symptoms. These may become apparent as acute attacks but also develop in a more chronic disease of multiple joints with a rheumatoid arthritis-like appearance. Currently there is no way to remove CPPD crystals from the joint.

Alternatively, basic calcium phosphate (BCP) crystals may form in the joint but also in the skin and arteries. Formation of BCP crystals in the joint has an intriguing relationship with osteoarthritis or degenerative joint disease. The crystals are often found in osteoarthritic joints and have been hypothesized to contribute to progression of the disease and its associated loss of function and disability. BCP crystal associated joint problems include a rapidly progressive destructive arthritis of large joints, in particular the shoulder (Milwaulkee shoulder syndrome) or contribute to joint pain and loss of function by affecting the tendons, e.g. the rotator cuff in the shoulder leading to chronic calcifying tendinitis. Again few specific treatments are available including Shockwave therapy and most patients are treated with different antiinflammatory strategies including intra-articular injection of corticosteroids.

In patients with systemic sclerosis (SSc), a rare but life-threathening systemic disease affecting skin and inner organs, subcutaneous calcification is often a prominent feature contributing to skin ulceration and triggering acute inflammation. Most of the patients involved have so-called limited SSc and a number show the CREST syndrome (calcinosis, raynaud's phenomenon, esophageal dysmotility, sclerodactily and telangiectasia). More rarely, skin calcification is also found in other autoimmune connective tissue diseases.

Arterial calcifications are associated with increased cardiovascular risk. Their prevalence rises with aging and is associated with the occurrence of different cardiovascular risk factors. The calcium deposits in the vessel wall are part of the atherosclerosis process and appears very actively regulated from early disease phases onwards.

The prevalence of arterial calcification is highly dependent on the population studied and the imaging technique used. However, simple chest X-ray suggests a prevalence of aorta calcification of about 2% in middle to old aged population (mean age 47 years; 30-89 years range). Aorta calcification enhances its stiffness and can worsen systolic hypertension and left ventricular hypertrophy, coronary insufficiency, ischemia and congestive heart failure. Risk factors for aortic calcification include common cardiovascular risk factors but the calcification is also an independent predictor of cardiovascular risk. Mechanisms suggested include changes in the mechanical properties of the atherosclerotic lesion and increased inflammation in response to calcification, which may increase the risk of plaque rupture.

Coronary artery calcification (CAC) in people over 40 years old, may already reach a prevalence of over 20%. The presence of CAC in asymptomatic individuals has been shown to be associated with an increased risk for cardiovascular end-points. Calcification of the abdominal aorta is positively correlated with age, systolic blood pressure and aortic stiffness. In patients with type 2 diabetes, femoral artery calcification is an independent predictor of cardiovascular morbidity and mortality. Overall, both type 2 diabetes and chronic kidney failure patients appear to be particularly at risk for calcification-associated increased cardiovascular morbidity and mortality. Smoc2 (SPARC-related modular calcium-binding protein 2) is a secreted calcium- binding protein, belonging to the BM-40/SPARC/osteonectin family of secreted matricellular proteins. The proteins classified as members of the BM-40 super family are modular, extracellular proteins, localized in the Extracellular Matrix (ECM) of various tissues including bone. All members of the BM-40 family contain an extracellular calcium-binding (EC) domain consisting of two canonical a-helix-loop- second a-helix (EF-hand) motifs, both containing two cysteine residues, a follistatin- like (FS) domain containing characteristically 10 cysteine-residues, and an acidic N- terminal domain. Two EF-hand calcium-binding motifs present in the EC domain, bind one calcium ion each and are calcium saturated under normal conditions. The affinity for extracellular ligands such as collagens is enhanced by calcium binding. Matricellular proteins are thought to influence many cellular functions like growth factor signaling, cell migration, cell adhesion and proliferation.

Smoc2 has been discovered as a member of the BM-40 family although it has a unique domain composition. In the other BM-40 family members, the EC and FS domains always appear as a pair whereas in Smoc2 these two domains are separated by two thyroglobuline-like domains and a SMOC-specific domain that has a high proportion of aromatic amino acids (Fig. 1 ). mRNA expression for Smoc2 shows a broad tissue distribution. Smoc2 is diffusely present in both epithelia (for example epidermis) and mesenchymal tissues (for example cartilage). Studies suggest that Smoc2 could mediate interactions between integrins and the ECM and thereby contributing to cell-cycle progression by maintaining integrin-linked kinase activity during the G1 phase of the cell cycle. In addition to its role in cell cycle progression, Smoc2 mediates the mitogenic and angiogenic effects of VEGF, PDGF and FGF. Studies suggest that Smoc2 is required in growth factor-induced DNA synthesis and that Smoc2 can stimulate endothelial cell proliferation, migration and angiogenic activity both in vitro and in vivo.

There is a clear need in the art for new compounds for treatment and screening tools for identification of agents useful for treatment and prevention of calcifying disorders. This invention provides for the use of Smoc2 and variants thereof in treatment and prevention of calcifying disorders. No link between Smoc2 and calcifying disorders has been previously described in the prior art.

SUMMARY OF THE INVENTION

The present invention solves the problems of the related art by providing the use of SPARC/BM40 family members to inhibit calcifying disorders such as vascular diseases characterized by increased calcification, including coronary and aortic calcification, or vascular calcification in association with chronic kidney disease; skeletal disorders characterized by calcium deposition in the joint and tendons such as calcium containing crystal disease in the joint, including basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) disease; and skin disorders such as calcinosis cutis, a manifestation of scleroderma and dermatomyositis. More particularly, the invention provides the use of Smoc2 and fragments, homologues and derivatives thereof to inhibit calcification in calcifying disorders. Numbered statements of the invention are as follows:

1. A compound for use in a method of treatment or prevention of calcifying disorders in a subject in need thereof, wherein said compound is a member of the SPARC family or fragments or derivatives thereof.

2. The compound according to statement 1 , wherein said compound is Smoc2 or fragments or derivatives thereof.

3. The compound according to statement 1 or 2, wherein said compound has an amino acid sequence selected from the group consisting of SEQ ID No1 , SEQ ID No2, SEQ ID No3, and SEQ ID No4. 4. The compound of any one of the previous statements 1 to 3, wherein said calcifying disorder is a vascular disease, a skeletal disorder, a joint disorder or a skin disorder.

5. A pharmaceutical composition comprising an effective amount of the compound according to any one of the previous statements 1 to 4 as an active ingredient in admixture with at least one pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to statement 5, for use in treatment or prevention of a calcifying disorder in a subject in need thereof.

7. The pharmaceutical composition according to statement 6, wherein said calcifying disorder is a vascular disease, a skeletal disorder, a joint disorder or a skin disorder.

8. A method of treating or inhibiting the development of a calcifying disorder in a subject, which method comprises administering to the subject in need of such treatment a therapeutically effective amount of a compound according to any one of the previous statements 1 to 4 or a pharmaceutical composition according to any one of the previous statements 5 to 7.

9. A method of treating or inhibiting the development of a calcifying disorder in a subject in need thereof, the method comprising administering an agent which increases expression or activity of Smoc2.

10. A screening method for identifying a pharmaceutically active agent for use in a method of treatment or prevention of a calcifying disorder in a subject, said method comprising the steps of: a) contacting a cell that expresses Smoc2 with a candidate agent; and

b) detecting whether that agent increases the expression or activity of Smoc2 in the cell; wherein a compound that increases the expression or activity of Smoc2 in the cell is suitable for use in a method of treating or preventing said calcifying disorder.

1 1. The screening method according to statement 10, further comprising the step: c) measuring mineralization in the cell in the presence of said agent; wherein a compound that increases the expression or activity of Smoc2 in the cell and that decreases mineralization, is suitable for use in a method of treating or preventing said calcifying disorder. BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein: Fig. 1. is a picture showing the domain structure of Smoc2. The follistatin-like (FS) domain and the extracellular calcium-binding (EC) domain are separated by two thyroglobulin-like (TY) domains and the SMOC-specific domain.

Fig. 2. is a graph showing ALP activity of Smoc2 overexpressing cells. Smoc2+ cells show less ALP activity as compared to 3.1 at D21 . Statistically significant differences versus D1 3.1 (internal control condition) are indicated as *: p<0.05; versus Smoc2+ at D1 as If: p<0.05; versus D21 3.1 as #: p<0.05).

Fig. 3. Are graphs showing gene expression of osteoblastic markers. mRNA levels were normalized to S29 (reference gene). Gene expression of (A) Runx2, (B) Osx, (C) Opn, (D) Ocn was assessed in cells overexpressing Smoc2 or not. Statistically significant differences versus D1 3.1 (internal control condition) are indicated as *: p<0.05, versus Smoc2+ cells at D1 as #: p<0.05, and versus D21 3.1 cells as 1f:p<0.05.

Fig. 4. are graphs showing gene expression of genes involved in inorganic phosphate/pyrophosphate balance. mRNA levels were normalized to S29 (reference gene). Gene expression of (A) Ank, (B) Npp1 and (C) Pit1 was assessed in cells overexpressing Smoc2 or not. Statistically significant differences versus D1 3.1 (internal control condition) are indicated as *: p<0.05, versus Smoc2+ cells at D1 as #: p<0.05, and versus. D21 3.1 cells as 1f:p<0.05.

Fig. 5. is a graph showing western blot densitometry analysis of phosporylated ERK1/2 and p38MAPK in MC3T3-E1 cells overexpressing Smoc2 or not. Total forms of the corresponding proteins are used as internal controls to compensate for loading differences. Activation of ERK1/2 (A) and p38MAPK (B) is calculated upon area under curve (AUC) values. Statistically significant differences versus D1 3.1 (internal control condition) are indicated as *: p<0.05, versus Smoc2+ cells at D1 as #: p<0.05, and versus D21 3.1 cells as 1f:p<0.05. Fig. 6. are graphs showing ALP activity of cells overexpressing Smoc2 lacking CA (A) or FS domain (B). No difference in activity was seen at D1. At D21 , more mineralization was seen in differentiated FS and all CA mutants compared to WT. ALP activity was not different between WT and FS, except for FS3 (B). Statistically significant differences versus D1 Smoc2 WT (internal control condition) are indicated as *: p<0.05; versus mutants at D1 as If: p<0.05; versus D21 Smoc2 WT as #: p<0.05).

Fig. 7. are graphs showing gene expression of a marker of proliferation and of osteoblastic markers. mRNA levels were normalized to S29 (reference gene). Gene expression of (A) Runx2, (B) Osx, (C) Opn, (D) Ocn, was assessed in cells overexpressing Smoc2 mutants lacking either the CA or the FS domain or in cells overexpressing Smoc2 WT. Statistically significant differences versus D1 Smoc2 WT (internal control condition) are indicated as *: p<0.05; versus mutants at D1 as If: p<0.05; versus D21 Smoc2 WT as #: p<0.05).

Fig. 8. are graphs showing gene expression of genes involved in inorganic phosphate/pyrophosphate balance. mRNA levels were normalized to S29 (reference gene). Gene expression of (A) Ank, (B) Npp1 and (C) Pit1 was assessed in cells overexpressing Smoc2 mutants lacking either the CA domain or the FS domain or not. Statistically significant differences versus D1 Smoc2 WT (internal control condition) are indicated as *: p<0.05; versus mutants at D1 as If: p<0.05; versus D21 Smoc2 WT as #: p<0.05) in cells overexpressing Smoc2 or not. Fig. 9. is a graph showing ALP activity of cells overexpressing Smoc2 in the presence or absence of extracellular calcium supplementation. ALP activity showed no difference at D1 , while more ALP activity was observed in Smoc2 WT + Ca compared to Smoc2 WT at D21. Statistically significant differences versus D1 Smoc2 WT (internal control condition) are indicated as *: p<0.05; versus Smoc2 WT + Ca at D1 as If: p<0.05; versus D21 Smoc2 WT as #: p<0.05).

Fig. 10. are graphs showing Alizarin Red staining and ALP activity of hPDCs. Smoc2+-treated cells show less Alizarin Red staining compared to 3.1 , ACaBD ("Ca") being intermediate, as demonstrated by quantification (B). Similar results were obtained for ALP activity (A). Statistically significant differences versus D1 3.1 (internal control condition) are indicated as *: p<0.05; versus Smoc2+ at D1 as If: p<0.05; versus D21 3.1 as #: p<0.05). Fig. 11. is a graph showing Alizarin Red staining of HUVECs. Smoc2+-treated cells show less Alizarin staining compared to 3.1 , ACaBD ("Ca") being intermediate as demonstrated by quantification. Statistically significant differences versus D1 3.1 (internal control condition) are indicated as *: p<0.05; versus Smoc2+ at D1 as If: p<0.05; versus D21 3.1 as #: p<0.05).

Fig. 12. Alignment between the human and murine Smoc2 protein sequence.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims and equivalents thereof.

Several documents are cited throughout the text of this specification. Each of the documents herein (including any manufacturer's specifications, instructions etc.) is hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.

The following terms are provided solely to aid in the understanding of the invention.

One aspect of the invention relates to a member of the SPARC family or fragments or derivatives thereof as a compound for use in a method of treatment or prevention of calcifying disorders in a subject in need thereof. In one embodiment of the invention, said member of the SPARC family is selected from the group consisting of Srnod and Smoc2. In another specific embodiment of the invention, said SPARC family member is Smoc2. In another specific embodiment of the invention, the member of the SPARC family is a Smoc2 fragment or Smoc2 derivative.

In another embodiment of the invention, Smoc2 has an amino acid sequence selected from the group consisting of SEQ ID No1 , SEQ ID No2, SEQ ID No3, and SEQ ID No4. In a particular embodiment, Smoc2 has an amino acid sequence according to SEQ ID No1. In another particular embodiment, Smoc2 has an amino acid sequence according to SEQ ID No2. In another particular embodiment, Smoc2 has an amino acid sequence according to SEQ ID No3. In another particular embodiment, Smoc2 has an amino acid sequence according to SEQ ID No4.

Another aspect of the invention relates to the use of the above described compound in a method of treatment or prevention of calcifying disorders in a subject in need thereof, wherein said calcifying disorder is selected from the group consisting of a vascular disease, a skeletal disorder, a joint disorder and a skin disorder.

Another aspect of the invention relates to a pharmaceutical composition comprising an effective amount of the above-described compound as an active ingredient in admixture with at least one pharmaceutically acceptable carrier. In one embodiment, said pharmaceutical composition is used in treatment or prevention of a calcifying disorder in a subject in need thereof. In a particular embodiment, said calcifying disorder is selected from the group consisting of a vascular disease, a skeletal disorder, a joint disorder and a skin disorder.

Another aspect of the invention relates to a method of treating or inhibiting the development of a calcifying disorder in a subject in need thereof, comprising administering to the subject in need of such treatment a therapeutically effective amount of the compound or the pharmaceutical composition according to the embodiments described above.

Another aspect of the invention relates to a method to induce a local overexpression of Smoc2, fragments, derivatives or homologues thereof for the treatment or prevention of calcifying disorders. One embodiment of the invention relates to the a method of treating or inhibiting the development of a calcifying disorder in a subject in need thereof, wherein the method comprises administering an agent which increases expression or activity of Smoc2, fragments, derivatives or homologues thereof. In another aspect of the invention, Smoc2, fragments, derivatives or homologues thereof are administered through the use of nucleotide sequences encoding said Smoc2, fragments, derivatives or homologues thereof, whether in an expression vector or not and thus relating to gene therapy methods. Where the administration is through gene-therapy, the amount of naked DNA or viral vectors is adjusted to ensure the local production of the relevant dosage of Smoc2. Thus, one embodiment of the present invention provides for methods of treatment or prevention of calcifying disorders in a subject in need thereof, preferably a mammal, using gene transfer. More particularly the invention provides for methods of treatment or prevention of calcifying disorders of a subject in need thereof, comprising administering to the subject a nucleic acid which is capable of increasing expression of Smoc2, fragments, derivatives or homologues thereof in the subject upon administration thereto. According to one embodiment of this aspect of the invention, an increase in expression of Smoc2, fragments, derivatives or homologues thereof is achieved by administering a nucleic acid comprising a sequence encoding said Smoc2, fragments, derivatives or homologues thereof. The present application envisages different methods of gene transfer which ensure appropriate expression of Smoc2, fragments, derivatives or homologues thereof, such as, but not limited to methods wherein one or more nucleic acids capable of increasing expression of Smoc2, fragments, derivatives or homologues thereof are administered by electroporation and methods wherein the nucleic acid is administered via an adenoviral vector.

Another aspect of the invention provides for a screening method for identifying a pharmaceutically active agent for use in a method of treatment or prevention of a calcifying disorder in a subject, said method comprising contacting a cell that expresses Smoc2 with a candidate agent, and detecting whether that agent increases the expression or activity of Smoc2 in the cell, wherein a compound that increases the expression or activity of Smoc2 in the cell is suitable for use in a method of treating or preventing said calcifying disorder. In one embodiment, the screening method for identifying a pharmaceutically active agent for use in a method of treatment or prevention of a calcifying disorder in a subject, comprises contacting a cell that expresses Smoc2 with a candidate agent, detecting whether that agent increases the expression or activity of Smoc2 in the cell, and measuring mineralization in the cell in the presence of said agent, wherein a compound that increases the expression or activity of Smoc2 in the cell and that decreases mineralization, is suitable for use in a method of treating or preventing said calcifying disorder.

Definitions

"SPARC family of proteins" represents a diverse group of proteins that modulate cell interaction with the extracellular milieu. The eight members of the SPARC protein family are modular in nature. Each shares a follistatin-like domain and extracellular calcium binding EF hand motif. In addition, each family member is secreted into the extracellular space. Some of the shared activities of this family include regulation of extracellular matrix assembly and deposition, counter-adhesion, effects on extracellular protease activity, and modulation of growth factor/cytokine signaling pathways. The SPARC family of proteins consists of SPARC (osteonectin, BM-40) (NCBI access number human: NP_003109.1 , NCBI access number mouse: NP_033268.1 ), Hevin (SPARC-like (SPL) 1 , SC1 , MAST 9, RAGS-1 , QR1 , ECM 2) (NCBI access number human: NP_004675.3, NCBI access number mouse: NP_034227.3), secreted modular calcium binding protein (SMOC) 1 (NCBI acces numbers human: NP_001030024.1 , NP_071420.1 , NCBI access numbers mouse: NP_001 139689.1 , NP_07171 1 .2) and 2 (SRG) (NCBI access numbers human: NP_071421 .1 , NP_001 159884.1 , NCBI access number mouse: NP_071710.2), testican 1 , 2, and 3 (SPARC/osteonectin, CWCV, and Kazal-like domains proteoglycans, SPOCK) (Testican-1 (SPOCK1 , isoform 1 -5) NCBI access number human NP_004589.1 and NCBI access numbers mouse: NP_033288.2, NP_001 159938.1 , NP_001 159935.1 , NP_001 159936.1 , NP_001 159937.1 ; Testican- 2 (SPOCK2, isoform 1 , 2) NCBI access numbers human: NP_001 127906.1 , NP_055582.1 and NCBI access number mouse: NP_443720.1 ; Testican-3 (SPOCK3, isoform 1-1 1 ) access NCBI numbers human: NP_001035249.1 , NP_058646.2, NP_001 191281.1 , NP_001 191282.1 , NP_001 191283.1 , NP_001 191284.1 , NP_001 191285.1 , NP_001 191287.1 , NP_001238896.1 and NCBI access numbers mouse: NP_001239549.1 , NP_001239550.1 ), and follistatin like protein 1 (fstl-1 , TSC- 36/Flik, follistatin related protein (FRP), TGF-β inducible protein) (NCBI access number human: NP_009016.1 , NCBI access number mouse: NP_032073.2). Based on sequence homologies of the EC domains, the family members can be separated into four distinct phylogenetic groups: 1 ) SPARC and Hevin; 2) SMOC 1 and 2; 3) testican 1 , 2 and 3; and 4) Follistatin-like protein (Fstl)-1 (TSC-36/Flik).

The term "Smoc2" as used herein refers to the protein with a 446 amino acid long sequence also named Smooth Muscle Associated Protein 2, in a particular embodiment according to SEQ ID No 1 and accession number AAH28420. Smoc2 should be understood as including all naturally-occurring Smoc2 isoforms, but also variants thereof such as amino-terminal and/or carboxy-terminal truncated forms, variants including one or more internal deletions or mutations of one or more amino- acids, provided that the said variants have at least 75%, preferably at least 80% and more preferably at least 90%, most preferably 95% similarity with the aforesaid isoforms.

Smoc2 of the present invention is Smoc2 originating from mammals, preferably from humans. Smoc2 administered to one species is preferably originating from the same species (e.g. use of human Smoc2 for treating humans), although the administration of Smoc2 from other species is not excluded (e.g. administration of murine, porcine, bovine or primate Smoc2 to humans). In a particular embodiment, the term refers to the human protein. The term thus also encompasses its homologues from other species than human, including the ones that have not been identified or sequenced yet. In another embodiment, the term refers to the murine Smoc2 protein with an amino acid sequence according to SEQ ID No 3. The alignment between human and murine Smoc2 protein sequence is shown in Fig. 12.

The term "homologue" as used herein with reference to Smoc2 refers to molecules having at least 50%, more preferably at least 70%, yet more preferably 80%, still more preferably 90%, again more preferable 95% and most preferably at least 98% amino acid sequence identity with Smoc2.

The term "fragment" as used herein with reference to Smoc2 refers to molecules which contain the active portion of Smoc2, i.e. the portion which is functionally capable of treating calcifying disorders, and which may have lost a number of non- essential (with respect to the inhibition or reduction of calcifying disorders) properties of the parent Smoc2. Whether a fragment is functionally capable of treating calcifying disorders can be determined by detecting whether that fragment increases the expression or activity of Smoc2 in the cell, and/or by measuring mineralization in the cell in the presence of said fragment, wherein a fragment that increases the expression or activity of Smoc2 in the cell and that decreases mineralization, is functionally capable for use in a method of treating or preventing said calcifying disorder. In a particular embodiment, the term refers to a fragment according to SEQ ID No 2, wherein the follistatin-like binding domain (amino acids 39 to 84) from human Smoc2 (SEQ ID No 1 ) is deleted. In another embodiment, the term refers to a fragment according to SEQ ID No 4, wherein the follistatin-like binding domain (amino acids 39 to 84) from murine Smoc2 (SEQ ID No 3) is deleted.

The term "derivative" as used herein with reference to Smoc2 refers to molecules which contain at least the active portion of Smoc2 (as defined hereinabove) and a complementary portion which differs from that present in the wild-type Smoc2, for instance by further manipulations such as introducing mutations. Derivatives of Smoc2 thus include molecules having essentially the same biological activity as Smoc2 and having at least 80% amino acid sequence identity with Smoc2, more particularly at least 90%, most particularly between 95 and 99% sequence identity at the amino acid level with Smoc2. Similarly, derivatives of a fragment of a protein refer to biologically active fragments having a modified amino acid sequence compared to the native sequence of the protein (i.e. an amino acid sequence identity with the relevant fragment of between 80-99%).

The term "sequence identity" of two sequences as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. Preferably, said sequence identity is higher than 70%-80%, preferably, 81 %-85%, more preferably 86%-90%, especially preferably 91 %-95%, most preferably 96%-100%, more specifically is 100%. As used herein, the term "biologically active" in reference to a protein, protein fragment, derivative or homologue thereof is defined as an ability of the nucleic acid or amino acid sequence to mimic a known biological function elicited by the wild type form of the protein. Unless specified, when referring to a homologue, derivative or fragment of Smoc2, a biologically active protein is a protein having at least 50% of the activity of Smoc2 (in inhibiting or reducing calcifying disorders) under comparable conditions in vitro.

A "disorder" or "disease" as used herein is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question.

"Calcifying disorders" as used herein are disorders characterized by abnormal or excessive calcification or the accumulation of calcium salts in body tissues. Non limiting examples of calcifying disorders to be treated herein are vascular diseases, including coronary and aortic calcification, or vascular calcification in association with chronic kidney disease; skeletal disorders characterized by calcium deposition in the joint and tendons such as calcium containing crystal disease in the joint, including basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) disease; and skin disorders such as calcinosis cutis, a manifestation of scleroderma and dermatomyositis. As used herein, a "treating" or "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

The term "therapeutically effective amount" is meant an amount of Smoc2, a fragment, derivative or homologue thereof, which produces the desired therapeutic effect in a subject. For example, in reference to a disease or disorder, it is the amount that reduces to some extent one or more symptoms of the disease or disorder, and preferably returns to normal, either partially or completely, the physiological or biochemical parameters associated or causative of the disease or disorder. Preferably, according to one aspect of the present invention, the therapeutically effective amount is the amount of Smoc2 that will lead to an inhibition or reduction of calcifying disorders.

The term "screening" used herein, refers to a process of testing one or a plurality of compounds (including a library of compounds) for some activity. A "screen" is a test system for screening. Screens can be primary, i.e., an initial selection process, or secondary, e.g., to confirm that a compound selected in a primary screen (such as a binding assay) functions as desired (such as in a signal transduction assay). Screening permits the more rapid elimination of irrelevant or non-functional compounds, and thus selection of more relevant compounds for further testing and development. The term "subject" as used herein, e.g. in connection with therapeutic methods, refers to a human or non-human animal subject. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The subject to be treated with the inhibitory compound will normally be a mammal. In a specific embodiment, the subject is a human. Generally, the subject can be of any age. The subject may be suffering from a calcifying disorder. The subject may be in any stage of the disease, i.e. the disease may be in an early stage wherein the subject shows only the first pathological signs that are normally associated with said condition, or the subject may be in a later stage of advanced calcification in the calcifying disorder. Amino acids are referred to herein with their full name, their three-letter abbreviation or their one-letter abbreviation.

EXAMPLES EXAMPLE 1. Overexpression of wild-type and mutants Smoc2 : influence on mineralization in an in vitro model of osteogenesis.

Methodology :

The osteogenic cell line MC3T3-E1 was differentiated in fully functional osteoblasts by seeding the cells at the density of 2600 cells/cm², and culturing them for 21 days in aMEM supplemented with 10% FBS, 1 % sodium pyruvate, 1 % antibiotics- antimycotic, 10 mM β-glycerophosphate and 50 pg/ml ascorbic acid-2-phosphate. We created three stable clonal populations overexpressing wild-type (WT) Smoc2 (pcDNA3.1 -Smoc2, named Smoc2+ here) or mutants forms of the gene lacking a calcium-binding domain (ACaBD) or lacking the follistatin-like domain (AFSLD), by selecting the cells with 1 mg/ml of G418. Three clonal populations knocked-down for Smoc2 (ShSmoc2, using a short hairpin micro RNA (shmiRNA) process) were also created, by selecting the cells with 0.5 pg/ml puromycin. These clonal populations were differentiated using the procedure described above, and compared to their respective controls (3.1 stably-transfected cells for overexpression, non-interfering shmiRNA for ShSmoc2). The read-outs included Alizarin red staining and quantification, alkaline phosphatase (ALP) activity, gene expression (quantitative realtime PCR) and signaling pathways linked to mineralization.

Results :

Knocking-down Smoc2 expression did not lead to any modification in the differentiation process. Overexpressing Smoc2 led to severe modifications of this process.

After 21 days of differentiation, Smoc2+ cells exhibited a strongly reduced alizarin red staining and ALP activity as compared to 3.1 control cells (Fig. 2). The expression of genes representative of the osteoblastic phenotype (Runx2, Osterix (Osx), Osteopontin (Opn), Osteocalcin (Ocn)) was strongly reduced in Smoc2+ cells (Fig. 3). Interestingly, the expression of genes involved in the inorganic phosphate (Pi)/inorganic pyrophosphate (PPi) balance was strongly affected in Smoc2+ cells (Fig. 4). Indeed, the mRNA expression of PPi transporter ANK (major contributor of extracellular (e)PPi level, exports PPi from cells to the extracellular milieu) was strongly up-regulated in Smoc2+ cells (4-fold). However, the expression of nucleotide pyrophosphatase phosphodiesterase (NPP-1 ), responsible for maximum 50% of ePPi level, remained unchanged (and was barely detectable). The mRNA expression of the Pi transporter (PiT-1 ) was decreased by approximately 8-fold in Smoc2+ cells compared to controls. ALP activity, as mentioned above, was also reduced in Smoc2+ cells. Altogether, these data underline the critical effect of SMOC2 on the ePi/ePPi balance, as it is clearly oriented towards ePPi excess, thereby inhibiting calcification.

The study of the signaling pathways involved was concordant with the gene expression. On one hand, ERK1/2 activation, linked to Pi, is lower in Smoc2+ cells compared to 3.1. On the other hand, p38MAPK phosphorylation, related to PPi, was stronger in Smoc2+ cells (Fig. 5). To gain further insight in the mechanisms underlying these effects, we analyzed the influence of the mutants ACaBD and AFSLD, in comparison to WT Smoc2+ cells. At day 21 , Alizarin red staining was only increased by 1 .4-fold in one out of three clonal populations of AFSLD compared to WT, while it was enhanced by 2-fold in all ACaBD clones. The ALP activity followed the same trend, as it was not different between WT and AFSLD clones, while it was increased by 2- and 2.9-fold in two out of three ACaBD clones (Fig. 6A, B).

The expression of gene representative of the osteoblastic phenotype (Runx2, Osterix, Osteopontin, Osteocalcin) was not different between WT and AFSLD clones at day 21 , while being systematically significantly higher in ACaBD clones (Fig. 7). Genes involved in the Pi/PPi were differentially altered in the mutants. Ank expression was the same at day 21 in WT and mutants, although it was 2.5-fold higher before the initiation of differentiation in both AFSLD and ACaBD clones compared to WT. Npp1 level was identical at day 21 in WT and mutants, although it was 2.5-fold lower in both AFSLD and ACaBD clones before differentiation (and also, it was barely detectable). The expression of PiT-1 was not different in AFSLD clones and WT at day 21 , while being 3-fold higher in ACaBD clones (Fig. 8).

A supplementation of the differentiation culture medium with 1 mM of CaCI₂ was tested in Smoc2+ cells. This partially antagonized the influence of SMOC2 on the mineralization and ALP activity (Fig. 9). Altogether, these data suggest a critical role for the CaBD. Indeed, the corresponding mutant and the supplementation of culture medium with CaCI₂ led to a partial reversal of the effect of Smoc2 overexpression. Interestingly, the AFSLD mutants kept their negative influence on the mineralization process, and to a lesser extent, the differentiation. This mutant is shorter by 44 amino acids as compared to WT, and also lacks a domain reported to potentially interact with BMP ligands. Therefore, such a mutant may be of interest in applications concerning excessive/anarchic mineralization in joints, such as hydroxyapatite-induced arthritis.

EXAMPLE 2. Effect of wild-type and calcium-binding domain deleted Smoc2 : influence on mineralization in different in vitro human models of osteogenesis and transdifferentiation.

We aimed at evaluating the influence of Smoc2 in human models of osteogenesis, either direct (human periosteum derived cells (hPDCs)) or indirect (by transdifferentiation, in human umbilical vein endothelial cells (HUVECs)). To that goal, we used supernatants from cultured stable MC3T3-E1 controls (3.1 ), overexpressing Smoc2 (Smoc2+) or Smoc2 lacking calcium-binding domain (ACaBD).

Methodology :

Harvesting supernatants: Two T75 flasks of each 3.1 , Smoc2+ and ACaBD were maintained in culture. One flask was kept under G418 antibiotic pressure, while the other was not. Every two days, supernatants from the flask without antibiotic were harvested, and the antibiotic pressure was re-established in that flask, while being removed from the other one. This workflow allowed us to harvest G418 free supernatants. Differentiating hPDCs: Cells were seeded at the density of 4500 cells/cm², and we cultured them for 21 days in DMEM supplemented with 10% FBS, 1 % sodium pyruvate, 1 % antibiotics-antimycotic, 10 mM β-glycerophosphate, 50 pg/ml ascorbic acid-2-phosphate and 100 nM dexamethasone. We cultured cells into 1 ml of this medium, to which we added 0.5 ml of the supernatants we harvested from MC3T3- E1 , supplemented also with 10 mM β-glycerophosphate, 50 pg/ml ascorbic acid-2- phosphate and 100 nM dexamethasone. We therefore had three different conditions, i.e. hPDCs cultured with supernatants from either 3.1 , or Smoc2+ or ACaBD cells. Differentiating HUVECs: Cells were seeded on gelatin-coated vessels at the density of 4500 cells/cm², and we stimulated them in basal EGM-2 medium (no supplement) in the presence of 200 ng/ml of BMP6, known to trigger the endothelial to mesenchymal transition (Valdimarsdottir G, Circulation, 2002). Afterwards, these cells were cultured for 21 days in DMEM supplemented with 10% FBS, 1 % sodium pyruvate, 1 % antibiotics-antimycotic, 10 mM β-glycerophosphate, 50 pg/ml ascorbic acid-2-phosphate and 100 nM dexamethasone. We cultured cells into 1 ml of this medium, to which we added 0.5 ml of the supernatants we harvested from MC3T3- E1 , supplemented also with 10 mM β-glycerophosphate, 50 pg/ml ascorbic acid-2- phosphate and 100 nM dexamethasone. We therefore had three different conditions, i.e. HUVECs cultured with supernatants from either 3.1 , or Smoc2+ or ACaBD cells.

Results :

After 21 days of differentiation, hPDCs cultured in the presence of Smoc2+ supernatants exhibited a fairly reduced alizarin red staining and ALP activity as compared to 3.1 control cells (Figure 10A, 10B). Moreover, hPDCs in the presence of ACaBD supernatants exhibited a lesser decrease in these markers as compared to WT Smoc2 (Fig. 10A, 10B)

After 21 days of differentiation, HUVECs cultured in the presence of Smoc2+ supernatants exhibited a fairly reduced alizarin red staining as compared to 3.1 control cells (Fig. 1 1 ). Moreover, HUVECs in the presence of ACaBD supernatants exhibited a lesser decrease in this marker as compared to WT Smoc2 (Fig. 1 1 )

SEQ ID No 1 (human Smoc2 446 AA):

1 mllpqlcwlp llagllppvp aqkfsaltfl rvdqdkdkdc sldcagspqk plcasdgrtf

61 Isrcefqrak ckdpqleiay rgnckdvsrc vaerkytqeq arkefqqvfi pecnddgtys

121 qvqchsytgy cwcvtpngrp isgtavahkt prcpgsvnek Ipqregtgkt ddaaapalet

181 qpqgdeedia sryptlwteq vksrqnktnk nsvsscdqeh qsaleeakqp kndnvvipec

241 ahgglykpvq chpstgycwc vlvdtgrpip gtstryeqpk cdntarahpa kardlykgrq

301 Iqgcpgakkh efltsvldal stdmvhaasd pssssgrlse pdpshtleer vvhwyfklld

361 knssgdigkk eikpfkrflr kkskpkkcvk kfveycdvnn dksisvqelm gclgvakedg

421 kadtkkrhtp rghvestsnr qprkqg

SEQ ID No 2 (human Smoc2 with deletion of the FS-domain (amino acids 39 to 84)):

1 mllpqlcwlp llagllppvp aqkfsaltfl rvdqdkdk kdvsrc vaerkytqeq arkefqqvfi pecnddgtys

75 qvqchsytgy cwcvtpngrp isgtavahkt prcpgsvnek Ipqregtgkt ddaaapalet

135 qpqgdeedia sryptlwteq vksrqnktnk nsvsscdqeh qsaleeakqp kndnvvipec

195 ahgglykpvq chpstgycwc vlvdtgrpip gtstryeqpk cdntarahpa kardlykgrq

255 Iqgcpgakkh efltsvldal stdmvhaasd pssssgrlse pdpshtleer vvhwyfklld

315 knssgdigkk eikpfkrflr kkskpkkcvk kfveycdvnn dksisvqelm gclgvakedg

375 kadtkkrhtp rghaestsnr qprkqg

SEQ ID No 3 (murine Smoc2 447 AA):

1 mlppqicwip llaallppvp aqkfsaltfl rvdqdkdrdc sldcpsspqk plcasdgrtf

61 Isrcefqrak ckdpqleiah rgnckdvsrc vaerkytqeq arkefqqvfi pecnddgtys

121 qvqchsytgy cwcvtpngrp isgtavahkt prcpgsinek vpqregagka ddaaapalet

181 qpqgdeedia sryptlwteq vksrqnktnk nsasscdqeh qsaleeakqp kndnvvipec

241 ahgglykpvq chpstgycwc vlvdtgrpip gtstryeqpk cdntarahpa kardlyknrp

301 Iqgcpgakkh efltsvldal stdmvhavsd pssssgrlse pdpshtleer vvhwyfklld

361 knssgdigkk eikpfkrflr kkskpkkcvk kfveycdmnn dksitvqelm gclgvtreeg

421 kantrkrhtp rgnaessssn rqprkqg SEQ ID No 4 (murine Smoc2 with deletion of the FS-domain (amino acids 39 to 84)):

1 mlppqicwip llaallppvp aqkfsaltfl rvdqdkdr kdvsrc vaerkytqeq arkefqqvfi pecnddgtys 75 qvqchsytgy cwcvtpngrp isgtavahkt prcpgsinek vpqregagka ddaaapalet

135 qpqgdeedia sryptlwteq vksrqnktnk nsasscdqeh qsaleeakqp kndnvvipec

195 ahgglykpvq chpstgycwc vlvdtgrpip gtstryeqpk cdntarahpa kardlyknrp

255 Iqgcpgakkh efltsvldal stdmvhavsd pssssgrlse pdpshtleer vvhwyfklld

315 knssgdigkk eikpfkrflr kkskpkkcvk kfveycdmnn dksitvqelm gclgvtreeg

375 kantrkrhtp rgnaessssn rqprkqg

Claims

2. The compound according to claim 1 , wherein said compound is Smoc2 or fragments or derivatives thereof.

3. The compound according to claim 1 or 2, wherein said compound has an amino acid sequence selected from the group consisting of SEQ ID No1 , SEQ ID No2, SEQ ID No3, and SEQ ID No4.

4. The compound of any one of the previous claims 1 to 3, wherein said calcifying disorder is a vascular disease, a skeletal disorder, a joint disorder or a skin disorder.

5. A pharmaceutical composition comprising an effective amount of the compound according to any one of the previous claims 1 to 4 as an active ingredient in admixture with at least one pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, for use in treatment or prevention of a calcifying disorder in a subject in need thereof.

7. The pharmaceutical composition according to claim 6, wherein said calcifying disorder is a vascular disease, a skeletal disorder, a joint disorder or a skin disorder.

8. A method for treating or inhibiting the development of a calcifying disorder in a subject, which method comprises administering to the subject in need of such treatment a therapeutically effective amount of a compound according to any one of the previous claims 1 to 4 or a pharmaceutical composition according to any one of the previous claims 5 to 7.

9. A method for treating or inhibiting the development of a calcifying disorder in a subject in need thereof, the method comprising administering an agent which increases expression or activity of Smoc2.

10. A screening method for identifying a pharmaceutically active agent for use in a method of treatment or prevention of a calcifying disorder in a subject, said method comprising the steps of: a) contacting a cell that expresses Smoc2 with a candidate agent; and b) detecting whether that agent increases the expression or activity of Smoc2 in the cell; wherein a compound that increases the expression or activity of Smoc2 in the cell is suitable for use in a method of treating or preventing said calcifying disorder.

1 1. The screening method according to claim 10, further comprising the step: c) measuring mineralization in the cell in the presence of said agent; wherein a compound that increases the expression or activity of Smoc2 in the cell and that decreases mineralization, is suitable for use in a method of treating or preventing said calcifying disorder.