MXPA00010113A - Compositions and methods for the prevention and treatment of cardiovascular diseases - Google Patents

Abstract

Methods and compositions for the prevention and treatment of cardiovascular disease are described. Administration of osteoprotegerin (OPG) in a pharmaceutical composition prevents and treats atherosclerosis and associated cardiovascular diseases.

Description

COMPOSITIONS AND METHODS FOR THE PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASES FIELD OF THE INVENTION The invention relates to cardio-vascular diseases. More particularly, the invention involves the use of ost eopr ot eger ina (OPG) to treat and prevent cardio-vascular diseases associated with occlusion and calcification of blood vessels, such as atherosclerosis.

BACKGROUND OF THE INVENTION The development and maintenance of the mammalian skeleton involves the regulation and interaction of its component cell types (Erlebacher et al., Cell 8_0, 371-380 (1995), Marks, Acta Med Dent Helv 2_, 141-157 (1997)). The main contributors to the skeletal architecture include the chondrocytes that form the cartilage, the osteoblasts that synthesize and deposit the bone matrix, and the osteoclasts that REF .: 123679 reabsorb the bone. Chondrocytes are derived from mesenchymal cells and function to generate an initial cartilage template required for endochondral bone formation. Osteoblasts, derived from mesenchymal osteoprogenitor cells, are located on the bone surface where they synthesize, transport and accommodate matrix proteins. Osteoclasts are derived from precursors of granulocytes and monocytes, present in the hematopoietic marrow (Roodman, Endrocrine Rev. 17, 308-332 (1996), Mundy, J. Bone Min. Res. 8_, S505-S510 (1993); and Jilka New Eng. J. Med. 332, 305-311 (1995)). After establishing a close adherence to the bone surface, the osteoclasts form resorption zones that are acidified by a specialized structure known as the shirred edge. The shirred edge functions as a secretory conduit where acid protons and proteases are secreted, which decalcify and then digest the bone matrix. During the process of osteoclast-mediated resorption, it is believed that factors are elaborated Proteins that act as signal molecules to initiate bone turnover by osteoblasts. Osteoblasts, in turn, can influence the function of osteoclasts through the expression of soluble or membrane-binding regulators (Takahashi et al., Endocrinology 123, 2600-2602 (1988)). The coupling between the functions of osteoblasts and osteoclasts is critical for modeling, remodeling and skeletal repair (Mundy, J. Cell Biochem.5_3, 296-300 (1993), Mundy et al., Bone 17_, 71S-75S F (1995). )). Postmenopausal osteoporosis, the most common bone disease in the developed world, has been found causally associated with the loss of estrogen (for a review, see Pacifici, J. Bone Min. Res. 11, 1043-1051 (1996) ). Postmenopausal bone loss can be attributed to the loss of regulatory control exercised by estrogen in the production of cytokines and other factors that regulate the development of osteoclasts. The displacement resulting from the balance of osteoclast and osteoblast activity favors a net loss of bone mass that finally it leads to osteoporosis. Osteoporosis in human populations has been found to be associated with an increased incidence of arterial calcification, a component of many atherosclerotic lesions (Parhami and Demer, Curr Opin. Lipidology 8, 312-314 (1997), Banks et al., Eur. J. Clin. Invest. 24_, 813-817 (1994), Parhami et al., Arterioscler, Thromb. Vasc. Biol. _17_, 680-687 (1997)). Common factors can be based on the pathogenesis of these two diseases. Indeed, some arterial deposits of calcium minerals appear to be identical to those of completely formed lamellar bone, including the trabeculae, lagoons and islets of the marrow (Haust and Geer, Am. J. Pathol., 60, 329-346 (1970). ); Buntmg, J. Exp. Med. 8_, 365-376 (1906)). In addition, it has been shown that calcified arteries express several bone matrix proteins, including Type I collagen, matrix GLA protein, osteocal tissue, osteonectin, and the bone-protein gene of bone type. 2 (Bostrom et al., J. Clin, Invest, 9J, 1800-1809 (1993), O'Bpen, et al Circulation 92, 2163-2168 (1995); Giachelli et al. J. Clin. Invest. 92_, 1686-1696 1993); Bostrom et al. Am. J. Cardiol. 75_, 88B-91B (1995)). These findings have led to the speculation that arterial calcification is an organized and regulated process with cellular and molecular mechanisms similar to those of organized bone formation (Demer, Ciculation 92_, 2029-2032 (1995); Parhami et al. J. Atheroscler, Thromb. 3_, 90-94 (1996)). Osteoproteger ina (OPG), a recently identified member of the tumor necrosis factor receptor superfamily, is a secreted factor that inhibits the development of osteoclasts both in vitro and in vivo (Simonet et al., Cell 8_9, 309 -319 (1997); PCT Application No. US96 / 20621 (W097 / 23614) which is incorporated herein by reference). Transgenic mice that overexpress OPG in the liver, have high levels of OPG protein in their systemic circulation, and exhibit a marked increase in bone density (os t eope t r os i s). In normal mouse embryos, OPG has been located within the cartilage rudiments of developing bones, as well as in the small intestine and the muscular wall of the aorta and in several main arteries. Given the strong correlation between the presence of osteoporosis and the onset of conditions that could lead to cardiovascular disease, particularly the disease characterized by arterial calcification, and the similarities in the processes for calcium deposition in bone and along the interior of the walls of the arteries, an object of the invention is to develop pharmaceutical compositions and methods for the concurrent prevention and treatment of osteoporosis and cardiovascular disease. The development of a single therapeutic product for the prevention and treatment of both conditions would greatly improve the longevity and quality of life of the affected patients, reducing the risk of tearing and possibly fatal fractures of the bones and, at the same time, preventing or slowing conditions that could lead to hypertension, ischemia, heart attacks, and stroke. Surprisingly, it has been discovered that the loss of OPG in an animal lacking OPG, It results in the calcification of the aorta and renal arteries, which are sites of endogenous expression of OPG in normal animals. These findings involve OPG in the regulation of pathological calcification of the arteries, so that when circulating OPG is absent or present at low levels, the accumulation of calcium deposits in the walls of the arteries is accelerated to a large degree. The presence of normal or above normal levels of OPG (such as in transgenic mice expressing OPG) is not associated with vascular calcification.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions for the treatment or prevention of cardiovascular diseases. The methods comprise the administration of a therapeutically effective amount of OPG, wherein the amount is sufficient to treat or prevent cardiovascular disease. The present invention is concerned also to OPG compositions useful for the treatment or prevention of cardiovascular disease. The OPG compositions are typically pharmaceutically acceptable mixtures suitable for a variety of administration routes.

DESCRIPTION OF THE FIGURES Figure 1. Hybridization analysis In situ expression of OPG on frozen sections of rat embryonic heart E17 (Panels A and B) and adult rat renal artery (Panels C and D). In the light microscope the presence of OPG mRNA is observed as dark grains on the aorta, and the background dyeing is Methyl green (A). In dark field microscopy of the same specimen, the strong expression of OPG mRNA is observed, on the edges and the aorta (B). A somewhat weaker signal is present in the renal artery of the adult rat in light and dark field microscopy (C and D, respectively).

Figure 2. Analysis of hybrid idl zac ion In situ expression of OPG in sections, fixed with formalin, rat embryo E20.5. In light microscopy, the presence of OPG RNA is observed, as dark grains on the aorta, the background dyeing is hemalauno (Panels A, B and D). In dark field microscopy of the same specimen, the strong expression of OPG mRNA is observed above the aorta. (Panels C and E) rat embryo E20.5, increase of 1 / 2X, section stained with hematoxylin and eosin (A); 4X, H and E (B); 4X (C); 10X, H and E (D); 10X (E).

Figure 3. Arterial calcification in OPG mice _ _ males OPG_ "# 26 has intimal calcification and proliferation in the descending aorta (Panel A) and in the renal artery (Panel B). The OPG_ "# 38 mouse has pronounced calcification in the aortic bulb (Panel C). Massive subintimal proliferation could be the consequence of a dissection of the aortic wall and subsequent bleeding into the space between the layers. of the aortic wall.Aneurysm formation and dissection of the aortic wall is a common complication of Severe arteriosclerosis. There is severe calcification as well as intimal and median proliferation in the renal artery (Panel D).

Figure 4. Arterial Calcification in OPG_ ~ female mice. Wild type mouse aorta # 82 is shown as a negative control (Panel A). The OPG "_ # 86 mouse has several calcified lesions in the abdominal aorta (Panel B.) The OPG mouse" _ # 77 has several calcified lesions in the abdominal aorta (Panel D) and in several smaller branches (Panel C).

DETAILED DESCRIPTION OF THE INVENTION OPG_ ~ homozygous mice deficient in OPG exhibited severe osteoporosis when analyzed by whole body X-rays and by histology. The characterization of bone structure of OPG-deficient mice is described in the jointly owned co-pending US patent, Serial No. 08 / 943,687 which is incorporated herein by reference. HE .."to--". .- found unexpectedly that OPG-deficient mice, homocs, both male and female, also exhibited a noticeable calcification and intimate proliferation, in the aorta and in the renal artery. These arterial changes were not observed in OPG ~ + heterozygous mice, deficient in OPG, in normal OPG + + mice, or in transgenic mice that exhibited elevated OPG levels in the circulation. OPG + mice "show bone loss at 6 months of age. Taken together, these observations indicated a role for OPG in the prevention or reduction of arterial calcification and in the reduction of the risk of atherosclerosis.

OPG POLYPEPTIDES The OPG polypeptides of the invention include human OPG or a derivative, truncation, or chemically modified form thereof, having at least one of the biological activities of OPG. The amino acid sequence of human OPG is shown in SEQ ID NO: 1 and SEQ ID NO: 2. A derivative of OPG refers to a polypeptide having an addition, deletion, insertion or substitution of one or more amino acids, such that the resulting polypeptide has at least one of the biological activities of OPG. The biological activities of OPG include, but are not limited to, activities involving bone metabolism. In one embodiment, OPG polypeptides have an anti-reabsorbant activity in bone. In another embodiment OPG polypeptides have activity in the reduction or elimination of calcification of arterial walls. The OPG polypeptides will be mature OPG polypeptides having the 21 amino acid amino terminal leader sequence removed. The polypeptides include residues 22-401 as shown in SEQ ID NO: 1 and derivatives thereof having deletions or truncations at the carboxy terminus, of part or all of amino acid residues 180-401 of OPG; one or more amino acid changes at residues 180-401; deletion of part or all of the cysteine-rich domain of OPG, particularly the deletion of the domain rich in distal cysteine (carboxy terminal); and one or more amino acid changes in a cysteine-rich domain, particularly in the distal cysteine-rich domain (carboxy terminal). In one embodiment, OPG has up to about 216 amino acids deleted from the carboxy terminus. In another modality OPG has up to approximately 10 amino acids eliminated from The term "mature amino" (wherein the term "mature amino" is found in residue 22) and, optionally, has up to about 216 amino acids deleted from the carboxy terminus. Additional OPG polypeptides, comprised by the invention, include the following: fusion [22-180] human-Fc, fusion [22-201] -Fc human, fusion [22-401] human-Fc, fusion [22-185] human-Fc and fusion [22-194] human-Fc. These polypeptides are produce in mammalian host cells, such as CHO or 293 cells. Additional OPG polypeptides, encompassed by the invention, which are expressed in prokaryotic host cells, include the following: human [22-25,491] human, fusion met Fc- [22-401] human (the Fc region is fused to the amino terminus of the full length OPG coding sequence) the fusion [22-401] human-Fc fusion (the Fc region fused at the carboxy terminus to the full-length OPG sequence), the fusion metal Fc- [22-201] human, the fusion [22-201] human-Fc, met-Fc- [22-194] human, met [22-194] human-Fc, human [27-401], met [22-185] human, met [22-189] human, met [22-194] human, met [22-194] human (P25A), met [22-194] human (P26A), met [27-] 185] human, met [27-189] human, met [27-194] human, me ta g-gl and- ser- (hi s) 6 [22-401] human, met-lys [22-401] human , met- (lys) 3- [22-401] human, met [22-401] human-Fc (P25A), met [22-401] human (P25A), met [22-401] human (P26A), met [22-401] human (P26D). It is understood that the above OPG polypeptides, produced in prokaryotic host cells, have a methionine residue at the amino terminus, if that residue is not indicated. In specific examples, the OPG-Fc fusion polypeptides were produced using a 227 amino acid region of human IgG-α1, where it was used and has the sequence shown in Ellison et al. (Nuc.Aids Res. 10, 4071-4079 (1982)). Without ! £ _-------., ---- a a ----.--. - However, variants of the Fc region of human IgG can also be used. The analysis of the biological activity of the OPG truncations of the carboxy teminal, fused to the Fc region of the human IgG, indicates that a portion of the OPG of approximately 164 amino acids is required for the activity. This region encompasses amino acids 22-185, preferably those which are in SEQ ID NO: 1 and comprises four cysteine-rich domains, characteristic of the cysteine-rich domains of the extracellular domains of the tumor necrosis factor receptor (TNFR). ). Using the homology between the cysteine-rich domains of the OPG and the members of the TNFR family, a three-dimensional model of the OPG was generated, based on known crystal structures of the extracellular domain of TNFR-I (see 097/23614). This model was used to identify those residues within OPG that could be important for biological activity. We identified the cysteine residues that are involved in the maintenance of the structure of the four domains rich in cysteine. In the model, the following disulfide bonds were identified: Domain 1: from cys41 to cys54, from cys 44 to cys62, tyr23 and his 66 can act to stabilize the structure for this domain; Domain 2: from cys65 to cys80, from cys83 to cys98, from cys87 to cysl05; Domain 3: from cysl07 to cysllβ, from cysl24 to cysl42; Domain 4: from cys 145 to cys 160, from cysl66 to cysl85. Residues that were in close approximation to TNFβ were also identified as shown in Figures 11 and 12A-12B of W097 / 23614. In this model it is assumed that the OPG is linked to a corresponding ligand; TNFβ was used as a model ligand to simulate the interaction of OPG with its ligand. Based on this modeling, the following residues in the OPG can be important for the binding of the ligand: glu34, lys43, from pro66 to gln91 (in particular, pro66, his68, tyr69, tyr70, thr 71, asp72, ser73, his76, ser77, asp78, glu79, leu81, tyr82, pro85, val86, lys88, glu90 and gln91), glul53 and serl55. Alterations in this amino acid residue, either singly or in combination, can alter activity of the OPG. For example, changes in specific cysteine residues may alter the structure of individual cysteine-rich domains, while changes in residues important for ligand binding may affect the physical interactions of OPG with the ligand. Structural models can help identify analogs that have more desirable properties, such as improved biological activity, greater stability or easier formulation. Modifications of the OPG polypeptides are encompassed by the invention and include post-translational modifications (eg, N-linked or O-linked carbohydrate chains, processing of the N terminal or C terminal ends), the binding of chemical moieties to the amino acid skeleton, chemical modifications of N-linked or O-linked carbohydrate chains, and the addition of an N-terminal methionine residue as a result of expression in the prokaryotic host cell. The polypeptides can also be modified with a marker detectable, such as an enzymatic, fluorescent, isotopic or affinity marker, to allow detection and isolation of the protein. Further modifications of the OPG include chimeric or fusion proteins of the OPG, wherein the OPG is fused to a heterologous amino acid sequence. The heterologous sequence can be any sequence that allows the resulting fusion protein to retain OPG activity. Heterologous sequences include, for example, fusions of the immunoglobulin, such as Fc fusions, which may aid in the purification of the protein. A heterologous sequence that promotes the association of monomers of OPG to form dimers, trimers and other higher multimeric forms is preferred. In one embodiment, a chimeric OPG protein comprises a fusion of a truncated OPG polypeptide, an Fc region or human IgG. Truncations of OPG can occur in the amino or carboxy terms, or both, and are preferably truncations of up to about 216 amino acids from the carboxy terminus at residue 401. Fusion to an Fc region can occur between the carboxy terminus of an Fc and the amino terminus of a truncated OPG polypeptide, or alternatively between the amy rite term of an Fc region and the carboxy terminus of a truncated OPG polypeptide. Examples of truncated OPG polypeptides fused to an Fc region include residues 22-185, 22-189, 22-194 or 22-201 such as those shown in SEQ ID NO: 1 or variants thereof. The polypeptides of the invention are isolated and purified from other polypeptides present in tissues, cell lines and transformed host cells expressing OPG, or purified from components found in cell cultures containing the secreted protein. In one embodiment the polypeptide is free from association with other human proteins, such as the expression product of a bacterial host cell. Also provided by the invention are chemically modified derivatives of the OPG that can provide additional advantages "such as increased stability and circulation time of the polypeptide, or reduced immunogenicity (see U.S. Patent 4,179,337.) The chemical portions for derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol copolymers. propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like Polypeptides can be modified at random positions within the molecule, or at predetermined positions, within the molecule, and can include one, two, three or more chemical moieties attached. can be of any molecular weight and can be branched or unbranched For polyethylene glycol, the preferred molecular weight is about lkDa and about 100kDa (the term "about" indicates that in polyethylene glycol preparations, some molecules will weigh more, or something less, than the molecular weight established) r ease in handling and manufacturing. Other sizes can be used, depending on the therapeutic profile desired (eg, the duration of the desired sustained release, the effects, if any, on biological activity, ease of handling, degree of lack of antigenicity, and other known effects of polyethylene glycol for a therapeutic or analogous protein ). The polyethylene glycol molecules (or other chemical moieties) should be bound to the protein with consideration of effects on the functional or antigenic domains of the protein. There are a number of binding methods available to those skilled in the art, for example EP 401 384 incorporated herein by reference (coupling of PEG to G-CSF), see also Malik et al., Exp. Hematol. 2_0_: 1028-1035 (1992) (r epor t of binding of polyethylene glycol to GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently linked through amino acid residues through a reactive group such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule can be attached. The amino acid residues that have a free amino group may include lysine residues and amino acid residues of the N-terminus; those having a free carboxyl group can include residues of aspartic acid, glutamic acid residues and the terminal amino acid residue C. Sulfhydryl groups can also be used as a reactive group to join the polyethylene molecule (s) ileglycol Preferred for therapeutic purposes is the binding to an amino group, such as the linkage at the N-terminus or the lysine group. The invention also provides OPG modified chemically and selectively at the amino terminus. Using polyethylene glycol as an illustration of the compositions herein, one can select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules relative to the protein (or peptide) molecules in the reaction mixture, the type of binding reaction of polyethylene glycol 1, which is to be carried out, and the method for obtaining the polyethylene glycol-bound protein at the N-terminal, selected.

- ¥ The method to obtain the N-terminal polyethylene glycol-linked preparation (ie, separate this portion from other polyethylene glycol-linked portions, if necessary) may be by purification of the material bound to polyethylene glycol at the N-terminal, from a population of protein molecules linked to polyethylene glycol. Selective chemical modification at the N-terminus can be carried out by reductive alkylation which exploits the differential reactivity of different types of primary amino groups (lysine versus N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, the substantially selective derivation of the protein at the N-terminus with a carbonyl-containing carbonyl group is achieved. The invention also provides an OPG multimer comprising OPG monomers. OPG appears to be active as a multimer (e.g., dimer, trimer or a higher number of monomers). Preferably, the OPG multimers are dimers or trimers. The multimers of OPG may comprise monomers having the amino acid sequence of the OPG sufficient to promote the formation of the multimer or may comprise monomers having heterologous sequences such as an Fc region of the antibody. The carboxy terminal deletion analysis of the OPG suggests that at least a portion of the 186-401 region is involved in association of the OPG polypeptides. The replacement of part or all of the region of amino acids 186-401 of the OPG, with an amino acid sequence capable of self-association is also comprised by the invention. Alternatively, the OPG polypeptides or derivatives thereof can be modified to form dimers or multimers by site-directed mutagenesis, to create undamaged cysteine residues, for the formation of disulfide bonds between chains, by photochemical crosslinking, such as exposure in ultraviolet light or by chemical cross-linking with bi-functional linker molecules such as bifunctional polyethylene glycol and the like. In one embodiment, the OPG multimers are formed by the covalent bonding of OPG monomers lacking part or all of region 186-401 in such a manner that such an association of OPG monomers occurs to a large extent through modification with the linking group. The OPG multimers can be prepared by various chemical crosslinking processes. The OPG monomers can be chemically linked in any form that retains or enhances the biological activity of the OPG. A variety of chemical crosslinkers can be used depending on what properties are desired for the protein dimer. For example, r e t c i culators may be short and relatively rigid or longer and more flexible, may be biologically reversible and may provide a reduced immunogenicity or a longer pharmacokinetic half-life. The OPG molecules are linked through the amino terminus through a two-step procedure where the OPG is chemically modified at the amino terminus to introduce a protected thiol, which, after purification, is deprotected and used as a binding point for site-specific conjugation, through a variety of crosslinkers with a second OPG molecule. Amino terminal cross-links include, but are not limited to, a disulfide bond, thioether bonds using aliphatic, bi-functional, short-chain crosslinkers, and thioester bonds for polyethylene glycol, bi-functional, variable-length crosslinkers (" bells "of PEG). Also understood by the synthesis by PEG bells, OPG dimers, is a by-product of that synthesis, called "monocampana". An OPG monocampane consists of a monomer coupled to a bifunctional linear PEG with a free polymeric term. Alternatively, OPG can be cross-linked directly through a variety of homobi functional 1 crosslinking techniques, specific for amine, which include reagents such as: dianhydride dietary lent r iaminpent acte ti co (DTPA), p-benzoquinone (pBQ) or sub-ratio of bi s (sul fo succinimidi lo) (BS3) as well as others known in the art. It is also possible treat OPO with thiolate, directly with reagents such as iminot iolane, in the presence of a variety of thiol-specific, bi-functional crosslinkers, such as PEG bismaleimide, and achieve dimerization and / or bells, in a process of one step The OPG multimers can also be formed by linking OPG monomers with peptides of variable length. The peptides are selected to have an amino acid sequence and a composition such that they act as flexible binders between the OPG monomers. Peptide binders can bind monomers in a head-to-head manner (N-terminal to N-terminal, or C-terminal to C-terminal) or in a head-to-tail manner (N-terminal to C-terminal). The peptide binders will preferably have a length of about 15-60 amino acids. Also included is a method for the purification of OPG from natural sources and from transfected host cells. The purification process can employ one or more purification steps of proteins, standards, in proper order to obtain the purified protein. Chromatography steps may include ion exchange, gel filtration, hydrophobic interaction, reverse phase, chromat oenf oque, and affinity chromatography using an anti-OPG antibody or biotin affinity complex ina-tr ept avidin, and the like.

Nucleic acids Nucleic acid molecules encoding OPG polypeptides are also provided. The nucleic acid molecules are selected from the following: a) the nucleic acid sequence shown in SEQ ID NO: 1 or in the complementary strand thereof; b) nucleic acids that hybridize under stringent conditions to the polypeptide encoding the region in SEQ ID NO: 1; and c) the sequence of nucleic acids that have degenerated into the sequences in (a) and (b).

The hybridization con-cliciones are generally of high astringency such as 5x? SC, 50% formamide and 42 ° C or the equivalent that can be easily obtained by adjusting salt and organic solvent concentrations, and temperature. For example, equivalent stringency conditions can also be used by increasing the temperature of the hybridization or wash step (at a range of 50 ° C-65 ° C) and decreasing the salt concentration (up to a range of 1 to 0.2 x SSC). ) omitting at the same time the organic solvent. Hybridization conditions for nucleic acids are described in greater detail in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2a. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The length of the hybridizing nucleic acids of the invention may be variable, since hybridization may occur in part or in the entire coding region of the polypeptide, as shown in SEQ ID NO: 1, and may occur. also in non-coding regions. The hybridizing nucleic acids can have a shorter or longer length than the complementary sequence shown in SEQ ID NO: 1- Truncated or extended nucleic acids that hybridize to SEQ ID NO: 1 may retain one or more of the biological properties of OPG, such as anti-absorber activity in the bone, or protection against arterial calcification. Hybridizing nucleic acids may also include adjacent non-coding regions, which are 5 'and / or 3' for the coding region of OPG. Non-coding regions include regulatory regions involved in the expression of OPG, such as promoters, int ensicator, translation initiation sites, transcription termination sites and the like. Also provided by the invention are derivatives of the nucleic acid sequences shown in SEQ ID NO: 1. As used herein, the derivatives include nucleic acid sequences having the addition, substitution, insertion or deletion of one or more residues, such that the resulting sequences encode polypeptides having one or more residues of amino acids that have been added, deleted, inserted or substituted and the resulting polypeptide has the activity of OPG, such as the anti- reabsorbing activity in bone, or 1 aH. protection against arterial calcification. Nucleic acid derivatives can be natural, such as by splice variation or polymorphism, or can be constructed using site-directed mutagenesis techniques, available to one skilled in the art. An example of a natural variant of OPG is a nucleic acid encoding a change from lys to asn, at residue 3 within the leader sequence (see 097/23614). It is anticipated that the nucleic acid derivatives will encode amino acid changes in the regions of the molecule, which are less likely to interrupt biological activity. In one modality, derivatives of the OPGs include nucleic acids encoding truncated forms of the full-length OPG (the full-length OPG encompasses residues 22 to 401 of SEQ ID NO: 1) having one or more amino acids deleted from the carboxy term. The nucleic acids encoding OPG can have up to about 216 amino acids deleted from the carboxy terminus. Optionally, an antibody Fc region can be extended from the new carboxy terminus to produce a biologically active OPG-Fc fusion polypeptide, or an Fc region can be extended from the amino terminus of the truncated OPG. In preferred embodiments, nucleic acids encoding the OPG have the amino acid sequence of residues 22-185, 22-189, 22-194 or 22-201 (using the numbering in SEQ ID NO: 1) and optionally, encode a Fc region of human IgG. Also included are nucleic acids encoding the truncated forms of OPG that have one or more amino acids removed from the amino terminus. the truncated forms include those lacking part or all of the 21 amino acids comprising the leader sequence. The mature OPG lacks all the 21 amino acids of the leader sequence. Additionally, the invention provides nucleic acids encoding the -_3 ^ - a- ..-- 1-3 -.-- OPG having from 1 to 10 amino acids deleted from the mature amino terminus (at residue 22) and, optionally, having from 1 to 216 amino acids deleted from the carboxy terminus (at residue 401). Optionally, more nucleic acids can encode a methionine residue at the amino terminus. Examples of nucleic acids of the invention include synthetic cDNA, genomic DNA, DNA and RNA. The cDNA is obtained from libraries prepared from mRNA isolated from various tissues expressing OPG. In humans, tissue sources for OPG include the kidney, liver, placenta and heart. The genomic DNA encoding OPG is obtained from genomic libraries that are commercially available from a variety of species. Synthetic DNA is obtained by chemical synthesis of overlapping oligonucleotide fragments, followed by assembly of the fragments to reconstitute part or all of the coding region and flank sequences (see U.S. Patent No. 4,695,623 which describes the chemical synthesis of the genes of interferon).

RNA is obtained most easily by prokaryotic expression vectors that direct high-level mRNA synthesis, such as vectors using the T7 promoters and RNA polymerase.

Vectors and Host Cells The invention also provides expression vectors containing nucleic acid sequences encoding OPG, host cells transformed with those vectors and methods for the production of OPG. A general review of the expression of recombinant proteins can be found in Methods or f Enzymology v. 185, Goeddel, D.V. ed. Academic Press (1990). Host cells for OPG production include prokaryotic host cells, such as E_. coli, yeast, plant, insect and mammalian host cells. E. coli strains such as HB101 or JM101 are convenient for expression. Preferred mammalian host cells include COS, CHOd-, 293, CV-1, 3T3, kidney cells from young hamster (BHK) and others. Mammalian host cells are preferred when post-translational modifications, such as glycosylation and polypeptide processing, are important for OPG activity. Expression in mammals allows the production of secreted polypeptides that can be recovered from the culture medium. The vectors for the expression of OPG contain a minimum of sequences required for the propagation of the vector and for the expression of the cloned insert. These sequences include an origin of replication, a selection marker, a promoter, a ribosome binding site, internal sequences, RNA splice sites, and a transcription termination site. Vectors suitable for expression in the aforementioned host cells are readily available and the nucleic acids of the invention are inserted into the vectors, using standard recombinant DNA techniques. Vectors for the expression of tissue-specific OPG are also included. Those vectors include. promoters that function specifically in the liver, kidney, or other organs, for production in mice, and viral vectors for the expression of OPG in human cells specified as target. Using an appropriate host and vector system, OPG is produced recombinantly by culturing a host cell transformed with an expression vector containing nucleic acid sequences encoding OPG under conditions such that OPG is produced, and isolating the expression product. OPG is produced in the supernatant of transfected mammalian cells or in inclusion bodies of bacterial, transformed host cells. The OPG thus produced can be purified by methods known to a person skilled in the art, as described below. The expression of OPG in mammalian and bacterial host systems is described in W097 / 23614. Expression vectors for mammalian hosts are exemplified by plasmids such as the pDSRa described in O90 / 14363. Vectors of -------, --....

Expression for bacterial host cells are exemplified by the pAMG21 and pAMG22-His plasmids described in 097/23614. It is anticipated that the specific plasmids and host cells described are for illustrative purposes, and that other plasmids and available host cells could also be used to express the polypeptides. The invention also provides for the expression of OPG from endogenous nucleic acids by recombination events in vivo or ex vivo. One strategy involves the activation of an endogenous, normally silent, OPG gene by introducing exogenous regulatory sequences, for example (promoters or int ensifers) capable of directing the expression of OPG from the endogenous gene, or starting of a variant gene thereof, which is present in the host genome or that is generated by the introduction of exogenous sequences. Typically, the exogenous sequences are carried in vectors capable of homologous recombination with the host genome. In addition, regulatory sequences, endogenous or exogenous, capable of direct OPG, can be activated or stimulated to express OPG by exposure to certain activation or stimulation factors, for transcription and / or translation.

OPG Pharmaceutical Compositions The OPG pharmaceutical compositions typically include a therapeutically effective amount of OPG protein product, mixed with one or more physiologically and pharmaceutically acceptable formulation materials. Suitable materials for formulation include, but are not limited to, anti-oxidants, preservatives, colorants, flavors and diluents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffer solutions, carrier vehicles, administration, diluents, excipients and / or pharmaceutical adjuvants. For example, a suitable vehicle may be water for injection, physiological saline or other common materials in compositions for parenteral administration. The regulated, neutral saline solution or saline solution mixed with serum albumin are additionally exemplary vehicles. The primary solvent in a vehicle can be either aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmaceutically acceptable excipients to modify or maintain the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution or odor of the formulation. Similarly, the carrier may still contain other excipients, pharmaceutically acceptable, to modify or maintain the stability, dissolution rate or release rate of the OPG. These excipients are those substances that are usually and customarily employed to formulate dosages for parenteral administration, either in the unit dose or multiple dose form. Once the therapeutic composition has been formulated, it can be stored in sterile vials such as a solution, suspension, gel, emulsion, solid or Dehydrated or lyophilized powder. These formulations can be stored, either in a ready-to-use form or in a form, for example, lyophilized, which requires reconstitution prior to administration. The optimal pharmaceutical formulation will be determined by a person skilled in the art, depending on the route of administration and the desired dosage. See, for example, Remingston's Pharmaceutical Sciences, 18a. edition (1990, Mack Publishing Co., Easton PA 18042) pages 1435-1712, the description of which is incorporated herein by reference. Other affective administration forms are also contemplated, such as parenteral slow release formulations, mists for inhalation, oral active formulations, or suppositories. In one embodiment, the OPG pharmaceutical compositions are formulated for parenteral administration. These parenterally administered therapeutic compositions are typically found in the form of a parenterally acceptable, pyrogen-free aqueous solution containing OPG in a pharmaceutically acceptable vehicle. A preferred vehicle is physiological saline. Sustained release and / or delivery compositions of OPG contain OPG polypeptides modified with water soluble polymers as described above to increase solubility or stability. The compositions may also comprise the incorporation of OPG into liposomes, microemulsions, miscels or vesicles for controlled administration over a prolonged period of time. Specifically, the OPG compositions may comprise incorporation, into polymer matrices, such as hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers. Examples of hydrogels include polyhydroxyalkyl methacrylates (p-HEMA), polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, and various complexes. Examples of biodegradable polymers include polylactic acid (PLA) polyglycolic acid (PGA), copolymers of the PLA and PGA, polyamides and copolymers of polyamides and polyesters. Other controlled release formulations include microcapsules, microspheres, macromolecular complexes and polymeric beads that can be administered by injection. Hyaluronic acid can also be used and this may have the effect of promoting sustained duration in the circulation. These compositions can influence the physical state, stability, release rate in vivo, and the rate of in vivo elimination of the proteins and derivatives of the present. It is also contemplated that certain formulations containing OPG, are administered orally. The OPG that is administered in this form can be encapsulated and can be formulated with or without the carriers commonly used in the preparation of solid dosage forms. The capsule can be designed to release the active portion of the formulation, in the gastrointestinal tract, at the point where the biodi sponsibiity is maximized and where the presystemic degradation is minimized. Excipients may be included additional to facilitate absorption. Diluents, flavors, low-melting waxes, vegetable oils, lubricants, suspending agents, disintegrating agents and binders may also be employed.

Administration of the OPG The OPG polypeptides can be administered parenterally through the subcutaneous, intramuscular, intravenous, transpulmonary or transdermal route. To achieve the desired dose of OPG, repeated injections may be administered daily or less frequently. The frequency of the dosage will depend on the pharmacokinetic parameters of the OPG polypeptide as formulated, and on the route of administration. Regardless of the manner of administration, the specific dose is typically calculated in accordance with body weight or body surface area. The additional adjustment of the calculations necessary to determine the appropriate dosage, for the treatment, involves that each of the ^^ agá ^^^^^^ The aforementioned formulations are prepared routinely by those skilled in the art, especially in light of the dosage information and tests described herein. Appropriate dosages can be determined through the use of established assays for the determination of dosages used in conjunction with the appropriate dose response data. He The final dosage regimen, which is involved in a method of treating a specific condition, will be determined by the doctor treating the case, considering several factors that modify the action of the drugs, For example, the age, condition, body weight, sex and diet of the patient, the severity of any infection, the time of administration and other clinical factors. In one embodiment, the dosage range for a protein of Fc-OPG fusion wherein the carboxy terminus of an Fc region is linked to the amino terminal residue of a truncated OPG polypeptide (e.g., Fc-OPG [22-194]), from about 10 μg / kg to about 10 mg / kg.

It also contemplates in vivo therapy with OPG genes, wherein a nucleic acid sequence encoding the OPG, a derivative thereof or a chimeric OPG protein, is introduced directly into the patient. For example, a nucleic acid sequence encoding an OPG polypeptide is introduced into the target cells through local injection of a nucleic acid construct with or without an appropriate delivery vector, such as an adeno-associated virus vector. Alternative viral vectors include, but are not limited to, retroviruses, adenoviruses, herpes simplex viruses, and papilloma virus vectors. Physical transfer can be achieved in vivo by local injection of the desired nucleic acid construct or other appropriate delivery vector containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), receptor-mediated transfer ( ligand-DNA complex) or bombardment with microparticles (genetic cannon). Atherosclerosis causes the greatest Part of the degenerative arterial disease and the cali fi cation of the arterial wall that typically occurs in clinically significant lesions. Narrowing and occlusion of the artery are the most common features of the disease, although the resistance of the arterial wall can also be compromised by the loss of elastin and collagen. The consequences of arterial occlusion include dissection, aneurysms, ischemia, thrombosis, and acute and chronic heart disease. In many cases surgical and angioplastic treatments are required and, although effective, these treatments are necessarily invasive, do not prevent occlusion in other sites of the arteries, and in some cases may need to be repeated in the original sites (for example, in restenosis). OPG can be used to prevent or treat atherosclerosis and arteriosclerosis of Mockenberg (medium qualifying sclerosis), and other conditions characterized by arterial cali fi cation. OPG can be administered alone or in combination with other drugs for the treatment of atherosclerosis, tg-? is like anti-hypertensive drugs and drugs to reduce cholesterol. Anti-hypertensive drugs include diuretics, a-adrenergic blocking drugs, β-adrenergic blocking drugs, calcium-entry blocking drugs, enzyme inhibitors for angiotensin conversion and vasodilators. . Cholesterol-lowering drugs that reduce low-density lipoprotein (LDL) cholesterol levels include bile acid sequestrants, HMG-CoA reductase inhibitors, fibric acid and nicotinic acid derivatives. OPG can also be administered with anti-absorption agents that may exhibit cardiovascular benefit, such as hormones (estrogens), vitamin D and vitamin D derivatives, and selective estrogen receptor modulators (SERMs), such as raloxifene (EVISTA). In addition, OPG can be administered in conjunction with surgical and angioplastic treatments such as arterial prostheses and balloon angioplasty. The invention will be understood in a more complete with reference to the following examples. These examples should not be considered in any way as limiting the scope of this invention.

EXAMPLE 1 Expression of the OPG analyzed by in situ hybridization It has previously been described, in Simonet et al. supra , the preparation of embryos and tissues for in situ hybridization experiments and for the preparation of radiolabelled oligonucleotide probes to detect OPG mRNA levels. The location of high levels of OPG mRNA in the incipient part of the aorta, in 18.5 day mouse embryos, is shown in Figure 1A-1D. The expression of OPG in the adult rat is also evident in the smooth muscle wall of the renal artery as shown in Figure 2A-2E.

EXAMPLE 2 Preparation of mice deficient in OPG In the jointly owned and co-pending US patent, Serial No. 08 / 943,687, which is incorporated herein by reference, methods for the preparation of OPG-deficient mice are described, which include the construction of vectors to direct the sequences of OPG to the mouse genome and for the introduction of these vectors into mouse embryos.

EXAMPLE 3 Phenotypic analysis of mice deficient in OPG Groups of mice deficient in OPG, homologous (OPG "'"), mice deficient in heterozygous OPG (OPG "+) and control mice (0PG + +) were necropsied at 18 and 7 days, 14 days, 60 days and 180 days after birth, the radiograph was taken before the crude dissection, the serum of the mice was analyzed for --b .---. perform clinical chemistry and complete hematology. The whole body and main organs were weighed and fixed in formalin. A summary of the times that underwent the necropsy is presented in Table 1.

TABLE 1 »The OPG_" 1-26 mouse was the littermate, approximately half the size of a normal mouse, became dying and died a short time before the scheduled sacrifice, exhibited the symptoms of respiratory insufficiency shortly after death. Blood for hematology and serum chemistries was taken immediately after death by heart puncture and A regular necropsy was performed.

? The mouse 1-38 OPG "_ was placed in a cage with the mouse 1-27 OPG" "in preparation for the procedures and died within the last hour before slaughter, no blood could be collected for the analysis. the autopsy was performed as usual and the organs underwent histology.

In the jointly owned and co-pending US patent, Serial No. 08 / 943,687, analyzes and results of bone morphology, histology and density, and parameters of hematology and serum chemistry in OPG-deficient mice have been reported. Two of the three OPG_ "male mice had arterial changes.In the heart of the mouse # 26 a severe severe subendocardial calcinosis was present.Primary proliferation and calififi- cations could be detected in the aorta (Figure 3A) and in the renal artery (Fig. 3B), serum calcium rose to 11 versus 8.8 ± 0.17 in the OPG + + group, and the # 38 OPG_ ~ had an intimate proliferation and a tissue with chronic granulation sub-intima in the initial part of the aorta (Figure 3C) and in the renal artery (Figure 3D). The serum calcium index was not available. Two of the three female OPG mice had calcification and intimal proliferation in the aorta and in the renal artery (Figure 4B, 4C and 4D), serum calcium indices were within the normal range. "Female presented osteoporosis in bone, had normal calcium levels and no arterial change. Although the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications may occur to those skilled in the art. Therefore, it is intended that the api indications cover all those equivalent variations that fall within the scope of the invention as claimed.

LISTING DSWEFECUENCIES (1) GENERAL INFORMATION (i) APPLICANT: Simonet, Scott Sarosi, Idilko (ii) TITLE OF THE INVENTION: COMPOSITIONS AND METHODS THE PREVENTION AND TREATMENT OF CARDIOVASCULAR DISEASES (iii) NUMBER OF SEQUENCES: 2 (iv) ADDRESS FOR CORRESPONDENCE : (A) RECIPIENT: Amgen Inc. (B) STREET: One Amgen Center Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: United States of America (F) ZIP: 91320-1789 (v) ) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: PC Compatible with IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) COMMON DATA OF THE APPLICATION: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) INFORMATION FROM THE LAWYER / MANDATORY: (A) NAME: Winter, Robert B. (C) REFERENCE / FILE NUMBER: A-525 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1355 base pairs (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 94..1S97 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCAC CGGAGCGCTC GCCCAGCCGC 60 CGCTCCAAGC CCCTGAGGTT TCCGGGGACC ACA ATG AAC AAG TTG CTG TGC TGC 114 Met Asn Lys Leu Leu Cys Cys 1 5 GCG CTC GTG TTT CTG GAC ATC TCC ATT AAG TGG ACC ACC GA GA ACG 162 Ala Leu Val Phe Leu Asp lie Ser lie Lys Trp Thr Thr Gln Glu Thr 10 15 20 TTT CCT CCA AAG TAC CTT CAT TAT GAC GAA GAA ACC TCT CAT CAG CTG 210 Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gln Leu TTG TGT GAC AAA TGT CCT CCT GGT ACC TAC CTA AAA CAAC CAC TGT ACA Leu Cvs Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His Cys Thr 40 45 50 55 GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TAC TAC ACA Wing Lys Trp Lys Thr Val Cys Wing Pro Cys Pro Asp His Tyr Tyr Thr 60 65 70 GAC AGC TGG CAC ACC AGT GAC TG TGT CTA TGC AGC CCC GTG TGC n Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys 75 80 85 AAG GAG CTG CAG TAC GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC Lys Glu Leu Gln Tyr - al Lys Gln Glu Cys Asn Arg Thr Kis Asn Arg 90 95 100 GTG TGC GAA TGC AAG GAA GGG CGC TAC CTT GAG ATA GAG TTC TGC TTG 5 Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 105 110 115 AAA CAT AGG AGC TGC CCT GTC TTG GGA GTG GTG CAG GCT GGA ACC Lys Hs Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln Wing Gly Thr 120 125 130 135 0 CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 140 145 150 AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC ACA AAT TGC AGT Asn Glu Thr Ser Ser Lys Wing Pro Cys Arg Lys His Thr Asn Cys Ser 155 160 165 5 Gtc GG ctc CTG CTA ACp AG jyy ^ GGA j GCA ACA GAC AAC Val Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Wing Thr His Asp Asn 170 175 180 ATA TGT TCC GGA AAC AGT GAA TCA ACT CAA AAA TGT GGA ATA GAT GTT lie Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Cys Gly He A = p Val 185 190 195 0 ACC CTG TGT GAG GAG GCA TTC TTC AGG TTT GCT GTT CCT ACA AAG TTT Thr Leu Cys Glu Glu Wing Phe Phe Arg Phe Wing Val Pro Thr Lys Phe 200 205 210 215 ACG CCT AAC TGG CTT AGT GTC TTG GTA GAC AAT TTG CCT GGC ACC AAA Thr Pro A = n Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 5 220 225 230 GTA AAC GCA GAG AGT GTA GAG AGG ATA AAA CGG CAA CAC AGC TCA CAA Val Asn Ala Glu Ser Val Glu Arg He Lys Arg Gln His Ser Ser Gln 235 240 245 GAA CAG ACT TTC CAG CTG CTG AAG TTA TGG AAA CAT CA AAC AAA GCC Glu Gln Thr Phe G n Leu Leu Lys Leu Trp Lys Hrs Gln Asn Lys Wing 0 250 255 260 CAA GAT ATA GTC AAG AAG ATC ATC CAA GAT ATT GAC CTC TGT GAA AAC Gln Asp lie Val Lys Lys He He Gln Asp He Asp Leu Cys Glu Asn 265 270 275 AGC GTG CAG CGG CAC ATT GGA CAT GCT AAC CTC ACC TTC GAG CAG CTT 5 Ser Val Gln Arg His He Gly His Wing Asn Leu Thr Phe Glu Gln Leu 280 285 290 295 CGT AGC TTG ATG GAA AGC TTA CCG GGA AAG AAA GTG GGA GCA GAA GAC Arg Ser Leu Mee Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 300 305 310 0 ATT GAA AAA ACA ATA AAG GCA TGC AAA CCC AGT GAC CAG S ^ £ - g X ™ LÍ? Yes --or -er; 5-n - - í 330 j3: > 345 350 ACT GTC ACT CAO AGT CTA AAG AAG ACC ATC AGG TTC CTT CAC AGC TTC Thr Val Thr Gln Ser Leu Lys Lys Thr He Arg Phe Leu H-s Ser Phe 360 365 370 375 ACA ATG TAC AAA TTG TAT CAG AAG TTA TTT TTA GAA ATG ATA GGT AAC 15 thr Me. Tyr Lys Leu Tyr Gln Lys Leu Phe Leu Glu Met He Gly Asn 380 385 390 CAG GTC CAÁ TCA GTA AAA ATA AGC TGC TTA T AACTGGAAAT GGCCATTGAG Gln Val Gln Ser Val Lys He Ser Cys Leu 395 400 20 CTGTTTCCTC ACAATTGGCG AGATCCCATG GATOATAA (2) INFORMATION FOR SEQ ID NO: 2: (1) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 401 amino acids 25 (B) TYPE: amino acids (D) TOPOLOGY: linear (n) TYPE OF MOLECULE: protein 30 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp He Ser He 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 20 25 30 -3 ^ S Glu Glu Thr Ser Hs Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 40 65 70 75 0 Leu Tyr cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 100 105 110 45 Leu Glu He Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly phe 115 120 125 Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr val Cys Lys Ara 130 135 140. - Cys pro AsP G1y phe phe Ser Asn Glu thr s "Ser Lys Ala Pro Cys 50 145 150 155 160 Arg Lys His Thr Asn Cys Ser Val phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 180 185 190 Gln Lys cys Gly He Asp Val Thr Leu Cys Glu Glu Wing Phe phe Arg 195 200 205 Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220 Asp Asn Leu Pro Gly Thr Lys Val A = n Wing Glu Ser Val Glu Arg He 225 230 235 240 Lys Arg Gln H1S Ser Ser G n Glu Gln Thr Phe G n Leu Leu Lys Leu 245 250 255 Trp Lys His Gln Asn Lys Wing Gln Asp He Val Lys Lys He He Gln 260 265 270 Asp He Asp Leu Cys Glu Asn Ser Val Gln Arg His He Gly His Wing 275 280 285 Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 290 295 300 Lys Lys Val Gly? Glu A p He Glu Lys Thr He Lys Wing Cys Lys 305 310 315 320 Pro Ser Asp Gln He Leu Lys Leu Leu Ser Leu Trp Arg He Lys Asn 325 330 335 Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Wing Leu Lys His Ser 340 345 350 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr 355 360 365 He Arg Phe Leu His Being Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu 370 375 380 Phe Leu Glu Met He Gly Asn Gln Val Gln Ser Val Lys He Ser Cys 385 390 395 400 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (16)

1. A method of treatment or prevention of cardiovascular disease, characterized in that it comprises administering to a patient in need thereof a therapeutically effective amount of osopyrotene (OPG) in a pharmaceutical composition.

2. The method according to claim 1, characterized in that the cardiovascular disease is associated with the rosy s or with the ar t er ioe seleros i s of Monc enbe r g.

3. The method according to the rei indication 1, characterized in that it also comprises administering a therapeutically effective amount of an antihypertensive drug.

4. The method according to claim 1, characterized in that it further comprises administering a therapeutically effective amount of a drug that reduce cholesterol.

5. The method according to claim 1, characterized in that the estrogen is administered before, concurrent with, or after the onset of cardiovascular disease.

6. The method according to claim 1, characterized in that the osteoperceptor is administered in conjunction with the surgical or angioplastic treatment.

7. The method according to claim 1, characterized in that it further comprises administering an antiresorptive agent, selected from the group consisting of estrogen, vitamin D compounds and selective modulators of estrogen receptors.

8. The method according to claim 1, characterized in that the osteoproteger ina is a truncated polypeptide of the OPG.

9. The method according to claim 8, characterized in that the truncated polypeptide has up to about 216 amino acids deleted from the carboxy terminus as shown in SEQ ID NO: 2

10. The method according to claim 1, characterized in that the or eoproteger comprises a chimeric polypeptide comprising a truncated OPG polypeptide, fused to an Fc region of human IgG.

11. The method according to claim 12, characterized in that the carboxy terminus of the Fc region is fused to the amino terminus of the truncated OPG polypeptide.

12. The method according to claim 10, characterized in that the amino terminus of the Fc region is fused to the carboxy terminus of the truncated OPG polypeptide.

13. The method of compliance with sJ - claim 9, characterized in that the truncated OPG polypeptide is a covalently linked multimer.

14. The method according to claim 11 or 12, characterized in that the truncated polypeptide comprises residues 22-185, 22-189, 22-194 or 22-201 as shown in SEQ ID NO: 2.

15. The method according to claim 1, characterized in that the osteoproteger ina comprises residues 22-401 as shown in SEQ ID NO: 2.

16. The method according to claim 1, characterized in that a therapeutically effective amount of a nucleic acid encoding the protein or protein in a pharmaceutical composition is administered to a patient in need thereof.