Bone loss treatment and prevention method

FIELD: medicine.

SUBSTANCE: compound contacts with 15-lipoxygenase and tested for activity for 15-lipoxygenase inhibition.

EFFECT: extended range of medical products for treatment of diseases associated with bone loss.

7 cl, 6 ex, 4 tbl, 9 dwg

The invention relates generally to inhibitors of 15-lipoxygenase and their use for the treatment and prevention of bone loss. Specifically, the invention relates to inhibitors of 15-lipoxygenase to reduce bone loss and/or gain education reticulated structure of bone tissue.

Lipoxygenase are enzymes containing iron, not included in the heme, which are present in plants and animals, and catalyze the oxidation of certain polyunsaturated fatty acids, for example, included in the composition of lipids and lipoproteins. There are several different lipoxygenase, each of which differs in oxidative activity. Lipoxygenase mammals indicate the position in the molecule arachidonic acid, in which oxidation occurs. 5-lipoxygenase converts arachidonic acid into 5-hydroperoxidation acid (5-HPETE). This represents the first phase of metabolism, leading to the formation of 5-hydroxyeicosatetraenoic acid (5-GAT) and leukotrienes (LT). Similarly, 12 - and 15-lipoxygenase transform arachidonic acid to 12 - and 15-HPETE respectively. The biochemical recovery through 12-HPETE leads to the formation of 12-GAITÉ, while 15 GAITÉ is the predecessor of the class of compounds known as lipoxin.

Each is th number of biological impacts associated with products, with lipoxygenase activity, many of which are mediators of various painful conditions. LT C4 and D4 are powerful constrictors of human bronchial smooth muscle; LT B4 and 5-GAT detected in synovial fluid of patients suffering from rheumatoid arthritis are strong chemotactic factors for inflammatory cells such as polymorphonuclear leukocytes (Green and Lambeth, Tetrahedron, 39, s, 1983); identified high levels of 12 GAITÉ in epidermal tissue of patients suffering from psoriasis; it was found that lipoxin stimulate the release present in lysosome enzyme and ion superoxide from neutrophils. So, lipoxygenase play an important role in the biosynthesis of mediators of asthma, allergies, arthritis, psoriasis, and inflammation, and inhibitors of these enzymes interrupt the path of the biosynthesis involved in the development of these illnesses.

Human 15-lipoxygenase (15-LO) catalyzes the formation of 15-S-hydroxyeicosatetraenoic acid (15-S-GETA) from arachidonic acid (Kuhn and Borngraber, Lipoxygenases and Their Metabolites, published by Plenum Press, new York, 1999). In mice synthesis 15-8 GAITÉ is under the influence of 12/15-lipoxygenase (Alox15), which is a murine homolog of the human 15-LO. Murine 12/15-LO converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic and 15-S-GAITÉ the inratio 3:1 and, in addition, it can convert linoleic acid to 13-hydroxyoctadecadienoic acid (13-Godet-check against delivery).

Previously it was found that 15-lipoxygenase is involved in the pathogenesis of several diseases, including atherosclerosis (Harats, etc., Arterioscler. Thromb. Vase. Biol., cc. 2100-2105, 2000), asthma (Shannon and others, Am.Rev.Respir. Dis., 147, cc.1024-1028, 1993), cancer (Shureiqi, etc., JNCI, 92, cc.1136-1142, 2000) and glomerulonephritis (Montero and Badr, Exp. Neph., 8, cc.14-19, 2000). Identified numerous classes of compounds that have the ability of inhibiting 15-lipoxygenase, in particular phenols, hydroxamic acids and acetylenic fatty acids (for review see Kuhn and Borngraber, Lipoxygenases and Their Metabolites, published by Plenum Press, new York, 1999). These agents have different spectrum of inhibitory activity. For example, it was found that nordihydroguaiaretic acid is an inhibitor of 5-and 15-lipoxygenase, found that naftiliaki acid inhibit 5-, 12-and 15-lipoxygenase (US 4605669), and published evidence that benzofluoranthenes inhibitor of 15-lipoxygenase, PD 146176 has a relatively specific action on the 15-lipoxygenase (Sendobry, etc., Br. J. Pharm., 120, cc.1199-1206, 1997). In experiments on mice with a specially created by deletion of the gene Alox15 (Alox15 -/-) it was found that 15-lipoxygenase is involved in the development of atherosclerosis. Initially it was found that such mice with deficiency of Alox15 have a small fenotipiceski the mi differences, including increased activity present in the 5-LO (Sun and Funk, J. Biol. Chem., 271, cc.24055-24062, 1996). However, the violation of Alox15 leads to a significant reduction of atherosclerotic injuries have a tendency to atherosclerosis in mice with deficiency of apoE (apoE -/-) (Cyrus and others, J. Clin Invest., 103, cc.1597-1604, 1999).

Although there is evidence that 5-LO is involved in the pathogenesis of some diseases, the biological function of murine or human 15-lipoxygenase is not fully identified and described, in clinical experiments revealed the ability of inhibitors of 15-lipoxygenase to humans. In particular, hitherto not described the possibility of using inhibitors of 15-lipoxygenase for the treatment of osteoporosis and/or osteoarthritis in humans.

Osteoporosis is caused by a decrease in the density of the mineral component of bone mineral bone density (BMD)in Mature bone and leads to fractures after minor trauma. The disease is widespread and causes considerable economic damage. The most common fractures occur in the spine, the distal radial bone and the hip joint. Estimates suggest that one third of women over the age of 65 years may have spinal fractures caused, in particular, osteoporosis. In addition, in the elderly in TRUSTe there is a likelihood of fractures of the hip joint in approximately one in three women and one in six men.

It is established that there are two different phases of bone loss. One is the slow associated with age, a process that occurs in people of both sexes and begins at the age of approximately 35 years. This phase occurs with approximately the same speed in people of both sexes and leads to the loss of approximately equal amounts of cortical bone and cancellous (spongy or similar to the grating layer) bone. The cortical layer of bone predominates in the extension skeleton, while the spongy substance is concentrated in the axial skeleton, especially the spine, and at the ends of long bones. Osteoporosis is caused by age-related bone loss, called osteoporosis type II.

Another type of bone loss occurs rapidly, it is manifested in women in the postmenopausal period and is caused by a deficiency of estrogen. This phase is disproportionate loss in cancellous bone. Osteoporosis is caused by the depletion of estrogen, called osteoporosis type I. Main clinical consequences of osteoporosis type I fractures are the spine, hip and forearm. The area of the skeleton, characterized by such damage, contain large amounts of trabecular bone. For osteoporosis type is I, as a rule, characterized by a high level of metabolism of the bone tissue. The bone resorption is increased, but corrective bone formation is inadequate. It was found that osteoporosis is associated with corticosteroids, immobilization or prolonged stay in bed, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, mental anorexia, primary and secondary amenorrhea, immunosuppression, associated with the introduction of the graft, and oophorectomy.

It is assumed that the mechanism of bone loss in osteoporosis is associated with an imbalance of the update process of the skeleton. This process is called remodeling of bone tissue. It occurs in a discrete number of "pockets" of activity. These pockets are formed spontaneously within the bone matrix on a specific surface of bone tissue and represent the area of bone resorption. The osteoclasts (the cells that dissolve bone tissue or responsible for the resorption of bone by resorption part of the bone, as a rule, a certain constant size. Then after this process is complete resorption appear osteoblasts (osteoplastic cells), which fill the cavity left after the osteoclasts, and new bone tissue.

In the body healthy is the first adult individual osteoclasts and osteoblasts function so that bone formation and bone resorption are balanced. However, when osteoporosis develops an imbalance in the remodeling process of bone tissue, resulting in replacement of bone occurs at a slower rate than its loss. Although this imbalance to some extent arises in the majority of individuals in old age, he manifested much more serious and occurs at a young age in postmenopausal osteoporosis after oophorectomy or iatrogenic (due to the use of drugs) States, such as States, caused by the use of corticosteroids or immunosuppressants.

To increase bone mass in people suffering from osteoporosis, various approaches have been proposed, including the introduction of androgens, salts of fluorine and parathyroid hormone and modified versions of parathyroid hormone. It was also proposed to apply for preservation of existing bone tissue individually or in combination with each other bisphosphonates, calcitonin, calcium, 1,25-dihydroxyvitamin D₃and/or estrogens.

The present invention is based on the discovery that inhibitors of 15-lipoxygenase have the ability to increase the education of a mesh structure of bone tissue and/or to enhance the deposition of bone tissue and hasten the th healing of fractures. Such molecules can be entered individually or in combination with additional agents, which have the ability to inhibit bone resorption and/or increase bone formation, in particular, with anabolics.

Thus, one of the objects of the invention is a method of reducing bone loss in an individual. The method consists in the fact that the individual is administered pharmaceutically effective amount of an inhibitor of 15-lipoxygenase.

Another object of the invention is a method of increasing the mineral density of bone tissue, namely, that the individual is administered pharmaceutically effective amount of an inhibitor of 15-lipoxygenase.

Another object of the invention is a method for diagnosing predisposition of the individual to the loss of bone tissue, which consists in the fact that carry out the detection of polymorphism of human chromosome 17, first detection of gene polymorphism and 15-lipoxygenase.

The next object of the invention is a method of selecting compounds with the ability to reduce bone loss or increase the density of the mineral component of bone tissue, which consists in the fact that inhibitors of 15-lipoxygenase is brought into contact with human mesenchymal stem cells and determine cell differentiation glue is OK, involved in the formation of bone tissue.

These and other objects of the present invention are explained in more detail in the following detailed description of the invention and the accompanying drawings. In addition, in the present description fully incorporated by reference various references in more detail certain procedures or compositions.

In the implementation in practice of the present invention can be applied, unless otherwise indicated, standard methods of protein chemistry, biochemistry, methods of recombinant DNA and pharmacology, known to specialists in this field. Such methods are described in detail in the literature (see, for example, I.E. Creighton, Proteins: Structures and Molecular Properties (published by W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (published by Worth Publishers, Inc., the latest edition); Sambrook and other, Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods In Enzymology (edited by S. Colowick and N. Kaplan, published by Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pennsylvania: published by Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd ed.. (published by Plenum Press), volume a and b (1992).

All publications, patents and patent applications, cited above or below in the present description, fully incorporated into the present description by reference.

In the description of the present invention uses the following concepts, values of which are indicated below.

The concept of "loss of bone tissue the mean ratio imbalance of bone formation and bone resorption, leading to a smaller than necessary, the amount of bone in a patient. Bone loss can occur as a result of osteoporosis, osteotomy, periodontitis or be a loose prosthesis. Bone loss may be due to secondary osteoporosis, which includes induced glucocorticoid osteoporosis induced hyperthyroidism osteoporosis induced by immobilization osteoporosis, heparin-induced osteoporosis or induced immunosuppressive osteoporosis. You can monitor bone loss, for example, by the following measurements of the density of the mineral component of bone tissue.

The concept of "effective amount" or "pharmaceutically effective amount" refers to the amount of agent that does not cause toxic effects, but is sufficient to provide the desired biological result. This result can be reduction and/or alleviation of the signs, symptoms or causes of disease, or any other desired alteration of a biological system. For example, an "effective amount" from the point of view of therapeutic use refers to the amount of the composition containing the active substance required to provide a clinically meaningful increase in speed is azulene fractures treatment; reversal of bone loss in osteoporosis; reversion of injuries or disorders of the cartilage; prevent or delay the start of osteoporosis; stimulation and/or enhancing bone formation when nekrashevych fractures and osteogenesis under tension; increase and/or accelerate the growth of bone tissue used as prosthetic devices; and restore the defects of the teeth. The person skilled in the art can determine an appropriate "effective" amount in each case using standard experiments.

The concept of "the increased deposition of bone tissue" means that the accumulation of bone tissue in an individual, which is administered inhibitors of 15-lipoxygenase, proposed in the invention is increased compared with the accumulation of bone tissue from the individual, with which comparison is made and which is not administered inhibitor of 15-lipoxygenase. This increased deposition of bone tissue, as a rule, determined in accordance with the present invention by measuring mineral bone density (BMD). For example, an increase in the deposition of bone tissue can be determined on the model using animals such as undergone oophorectomy mice, dogs and other Animal injected with the test compound and measured mineral bone density (BMD) in the bones, which as a rule is, depleted in osteoporosis type I or type II, such as bone extension and/or axial skeleton, especially the spine including vertebrae, and end parts of the long bones such as the femur, middle Department and the distal radial bone. In the art there are several methods for the determination of the IPC. For example, it is possible to carry out measurement of BMD using dual absorptiometry energy x-rays or quantitative computed tomography and the like (see examples). Similarly, using methods well-known in this field, you can determine the increase in bone formation. For example, you can perform dynamic speed measurement of bone formation (KOS) labeled with tetracycline samples of the trabecular bone of the lumbar spine and distal metaphysis of the femur using quantitative computed morphometry (see, for example. Ling and others, Endocrinology 140: cc.5780-5788, 1999. Alternatively, you can evaluate markers of bone formation, such as alkaline phosphatase activity and levels of osteocalcin serum, to determine indirectly whether the formation of bone tissue (see Looker and others, Osteoporosis International, 11(6): cc.467-480, 2000).

The concept of "increased bone formation" means that the number of clicks is wusasa bone tissue from the individual, which is administered inhibitors of 15-lipoxygenase, proposed in the invention is increased compared with the rate of bone formation in an individual, which does not impose an inhibitor of 15-lipoxygenase. This increased bone formation are determined according to the present invention using, for example, quantitative computed morphometry, as well as using the above other markers of bone formation.

The term "inhibitor of 15-lipoxygenase" refers to a compound that inhibits 15-lipoxygenase and is characterized by the value of the IC₅₀less than 1 μm, preferably less than 100 nm. The values of the IC₅₀you can define standard methods. One particular method is a colorimetric analysis, according to which the estimated inhibitor pre-incubated for about 10 min with 15-lipoxygenase, then add the substrate, representing linoleic acid, and incubated for 10 min. the Product, that is, 13-HPODE, evaluate quantitatively using the method based on the combination of recovery hydroxyprolisilane lipid and oxidation of N-benzoylacetonate blue in the presence of hemin at pH 5. The uptake of oxidized methylene blue is directly proportional to the number of 13-HPODE, formed with the help of 15-lipoxygenase, and can edit the plunge in the presence of inhibitor and without it. This final analysis are described in more detail in Bruce J. Auerbach, John S. Kiely and Joseph A. Cornicelli, A Spectrophotometric Microtiter-Based Assay for the Detection of Hydroperoxy Derivatives of Linoleic Acid, Analytical Biochemistry, 201, cc.375-380, 1992. Other analysis methods include methods in which the initial rate of the enzymatic reaction is measured spectrophotometrically by measuring at 234 nm the number of formed conjugated diene.

In the context of the present description of the concept of "treat" or "treatment" are used interchangeably and are used to denote the slow development of symptoms of bone loss and/or decrease the severity of such symptoms that develop or development which is expected. In addition, the concepts include preventing additional symptoms, relief or prevention of causing the symptoms of metabolic factors and/or stimulating growth of bone tissue.

"Pharmaceutically acceptable" or "pharmacologically acceptable" refers to a product that is not undesirable neither biological, nor from a different point of view, i.e. the product, which you can enter the individual without causing any undesirable biological activity or harmful interactions with all components of the composition in which it is included.

"Physiological pH or the pH value in the range of physiological values, oznacza the t value of pH in the range of about 7.2 to 8.0 inclusive, more preferably in the range of from about 7.2 to about 7.6, inclusive.

In the context of the present description, the term "patient" refers to mammals and animals that are not relevant to mammals. Examples of mammals include, but are not limited to) any representative of the class of mammals: human, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and Guinea pigs, etc. are Examples of animals that are not relevant to mammals, include (but not limited to, birds, fish, etc. The term does not denote a particular age or gender.

In the context of the present description "polymorphism" refers to the presence in the population of two or more genetically determined alternative sequences or alleles. Polymorphic marker or the site is a locus in which there is divergence. Preferred markers are at least two alleles, each of which occurs in the population with a frequency of more than 1%, more preferably more than 10 or 20%. The polymorphism can be a replacement, Ann is a resource center, the repetition or deletion of one or more bases. A polymorphic locus may represent only one pair of bases. Polymorphic markers include polymorphisms of the lengths of restriction fragments, variabelnoe the number of tandem repeats (WCTP), hypervariable sites, minisatellites, dinucleotide repeats, trinucleotide repetitions, tetranucleotide repeats, simple sequence repeats, and insertion elements.

Single nucleotide polymorphism (SNP) occurs at a polymorphic site occupied by a single nucleotide, which is the site of allelic variations of the sequences. Usually website before and after it are highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 population representatives). Single nucleotide polymorphism, usually occurs as a result of the replacement of one nucleotide in other polymorphic site. The transition is the replacement of one purine bases to another purine or one pyrimidine base to another pyrimidine base. Transverse is a replacement of a purine bases and pyrimidine base, or Vice versa. Single nucleotide polymorphism may occur as a result of nucleotide deletions or insertions nucleotide sequence that is Chida in allele, which are not available in allele, with which comparison is made.

In the context of the present description, the term "cell-precursor" refers to a cell that has not yet fully differentiated or progressed to the stage (path) differentiation and which, as a rule, not expresses markers or has no inherent functions of Mature fully differentiated cell.

In the context of the present description, the term "mesenchymal cells" or "mesenchymal stem cells" refers to multipotent precursor cells that have the ability to repeatedly divide and offspring which forms a skeletal tissues, including cartilage, bone, tendon, ligament, bone marrow stroma and connective tissue (see A. Caplan, J. Orthop.Res. 9: cc.641-650, 1991).

In the context of the present description the term "osteogenic cells" refers to osteoblasts and cells-the precursors of osteoblasts.

In the context of the present description the term "quantitative trait locus" (KPL) denotes the measure of the phenotypic trait, such as mineral bone density, which is continuously distributed and can be influenced by several genes. KPL is a site on the chromosome alleles which affect the quantitative trait.

Connections proposed in the present invention, the pre is represent connections which have the ability to inhibit or decrease the activity of 15-lipoxygenase. In this context, the inhibition and reduction of enzyme activity means a decrease in the level of measured activity in comparison with the control experiment in which the enzyme, the cell or the individual is not subjected to the processing of the test compound. In specific embodiments of the invention, the inhibition or reduction of the measured activity is at least 10%. Specialist in this field should be obvious that for specific applications it may be preferable to decrease or inhibition measured activity of at least 20, 50, 75, 90, or 100%, or any integer of percentage between 10 and 100%. Typically, the inhibitors of 15-lipoxygenase proposed in the present invention, characterized by the value of the IC₅₀less than 1 μm, preferably less than 100 nm.

The present invention is based, in particular, on the discovery of the fact that 15-lipoxygenase is an important modulator of bone mass. In particular, genomic scanning 100 microsatellite markers in mice allowed us to identify the loci of sensitivity to violations associated with bone loss, which are located on chromosomes 1, 2, 4 and 11. Differences in the expression of genes, especially genes encoded on XP is mosome 11, determine significant differences in the expression of the gene encoding 15-lipoxygenase, which indicates a correlation between expression of 15-lipoxygenase and decrease in mineral bone density.

Full bone mass is the main criterion for the risk of osteoporotic fracture. On the basis of family studies and studies conducted on twins, it was found that the mineral bone density (BMD) regulate genetic factors (see review by Stewart and Ralston, J. Endocr., 166: cc.235-245, 2000). In the claimed invention was applied to the model using the mouse genes for the identification of loci controlling the IPC. To identify regions of the genome that regulate mineral density of bone tissue, we applied the method includes two stages. First used a computer method and microsatellite markers to scan the database of single nucleotide polymorphism (SNP) of the animal, resulting in the predicted region of the chromosome that are involved in the accumulation and maintenance of skeletal mass (example 1). Then applied the model using animals for identification and evaluation of mutations in genes involved in the accumulation and maintenance of skeletal mass (example 2), and to establish the fact that the violation of gene 15-lipoxygenase leads to an increase in IPC (example 4), which then suggests, what gene 15-lipoxygenase is involved in the regulation of bone mass.

Compounds that inhibit the activity of 15-lipoxygenase and prevent bone resorption or stimulate the formation of bone tissue, are the most preferred for the treatment of osteoporosis. Compounds that inhibit the activity of 15-lipoxygenase, can be used in the treatment of osteoporosis or osteoarthritis, based on the inhibition of bone resorption by osteoclasts or stimulation of differentiation leading to the formation of osteoblasts, and formation of new bone. Example 3 demonstrated the ability of inhibitors of 15-lipoxygenase to stimulate differentiation of human mesenchymal stem cells into osteoblasts in vitro. Example 4 demonstrated that the total disruption of the gene 15-lipoxygenase in vivo leads to an increase in BMD. In example 5, on the model of in vivo demonstrated the ability of inhibitors of 15-lipoxygenase to stimulate the formation of bone tissue.

According to the present invention, the inhibitor of 15-lipoxygenase can be applied for the preparation of a medicinal product which contains an effective amount of the indicated inhibitor of 15-lipoxygenase, designed to reduce loss of bone tissue in a mammal. Thus, in the present invention proposed sposobnosti loss of bone tissue in a mammal, which is that the individual is administered an effective amount of an inhibitor of 15-lipoxygenase. Preferably, the bone loss associated with osteoporosis, osteoarthritis, a disease Tuck or diseases of the periodontium. More preferably, the bone loss associated with osteoporosis.

In addition, according to the present invention, the inhibitor of 15-lipoxygenase can be applied for the preparation of medicines intended to increase the mineral density of bone tissue in a mammal, which contains an effective amount of the indicated inhibitor of 15-lipoxygenase. Thus, in the present invention a method of increasing the mineral density of bone tissue in a mammal, consisting in that the mammal is administered an effective amount of an inhibitor of 15-lipoxygenase.

According to the present invention, the inhibitor of 15-lipoxygenase can be applied for the preparation of a medicinal product containing an effective amount of an inhibitor of 15-lipoxygenase, which is intended for the treatment of osteoporosis in a mammal. Thus, in the present invention, a method of treating osteoporosis in a mammal, consisting in that the mammal is administered an effective amount of an inhibitor of 15-lipoxygenase.

In addition, the inhibitor wipuchanin can be used for preparation of medicines designed to stimulate differentiation of lines of mesenchymal stem cells in human osteoblasts, which contains the number of the specified inhibitor of 15-lipoxygenase, is effective for increasing the number of osteoblasts in the human body. Thus, in the present invention proposed a method of promoting differentiation of a line of mesenchymal stem cells in human osteoblasts, namely, that the person enter the amount of the inhibitor of 15-lipoxygenase, is effective for increasing the number of osteoblasts in the human body. Preferably the inhibitor of 15-lipoxygenase is characterized by the value of the IC₅₀less than 1 micron.

Treatment with inhibitors of 15-lipoxygenase can be used for healing bone fractures and osteectomy, including both bonded and non bonded fractures. The types of fractures that can be treated using the methods proposed in the invention include fractures caused by trauma and osteoporosis, such as fractures of the hip, femur neck, wrist, vertebrae, vertebral column, ribs, sternum, larynx and trachea, radial/ulnar bone, the tibia, the patella, clavicle, pelvis, humerus, lower legs, fingers and toes, face, and ankles. By using the methods suggested in this is sobienie, you can increase the speed of healing, and to stimulate splicing in the case of fractures, which would otherwise remain neradivymi. Prophylactic treatment of the patient for whom it is established that he has the risk of fractures, may also reduce the risk of fractures in this patient.

Therefore, the inhibitor of 15-lipoxygenase can be applied for the preparation of a medicinal product which contains an effective amount of an inhibitor of 15-lipoxygenase, designed to reduce the incidence of fractures in the mammal. Thus, in the present invention, a method for reducing the incidence of fractures in a mammal, consisting in that the mammal is administered an effective amount of an inhibitor of 15-lipoxygenase.

In addition, the inhibitor of 15-lipoxygenase can be applied for the preparation of a medicinal product which contains an effective amount of an inhibitor of 15-lipoxygenase, destined for the healing of fractures in a mammal. Thus, in the present invention, a method for healing of fractures in a mammal, consisting in that the mammal is administered an effective amount of an inhibitor of 15-lipoxygenase.

Preferably the mammal specified in the description of the methods and applications of PR is dstanley a person. More preferably the above-described inhibitor of 15-lipoxygenase is characterized by the value of the IC₅₀less than 1 micron.

There are several inhibitors of 15-lipoxygenase, which can be used according to the methods proposed in the present invention. Such inhibitors are synthetic organic molecules, herbal extracts and other natural products, as well as antibodies to 15-lipoxygenase. Representative examples do not limit the invention described in Cornicelli JA, Trivedi BK., 15-lipoxygenase and its inhibition: a novel therapeutic target for vascular disease [review] [113 references]. Current Pharmaceutical Design; 5(1): cc.11-20, 1999 (described various derivatives of caffeic acid, a simple propargilovyh esters, category and benzothiadiazole); Cornicelli JA. 15-lipoxygenase inhibitors as antiatherosclerotic agents. Idrugs, 1(2): cc.206-213, 1998; Fleischer R., P. Frohberg, Buge A., P. Nuhn, Wiese M. QSAR analysis of substituted 2-phenylhydrazonoacetamides acting as inhibitors of 15-lipoxygenase. Quant Struct - Act Relat; 19(2): cc.162-172, 2000; Kuhn H. Inhibitors of 12/15-lipoxygenase are potential anti-atherosclerotic drugs. Curr Opin Anti-Inflammatory Immunomodulatory Invest Drugs; 1(3): cc.227-237, 1999; Mogul R., Johansen, E., Holman T.R. Oleyl sulfate reveals allosteric inhibition of soybean lipoxygenase-1 and human 15-lipoxygenase. Biochemistry; 39(16): cc.4801-4807, 2000; Sexton, K., Roark W.H., R. Sorenson, Cornicelli J., Sekerke C., Welch K., Thiourea inhibitors of 15 - lipoxygenase. Abstracts of Papers American Chemical Society; 218(1-2), 1999: MEDIO; B.D. Tait, R.D. Dyer, Auerbach B.J., Bornemeier D., Guilds-Zamarka L. Oxender M. and other Catechol based inhibitors of 15-lipoxygenase. Bioorganic & Medicinal Chemistry Letters, vol. 6(1): cc.93-96, 1996; Moreau R. A., Agnew J., K.B. Hicks, M.J. Powell, Modulatio of lipoxygenase activity by bacterial hopanoids. JouPHKl of Natural Products, including; 60(4): cc.397-398, 1997; 219th National Meeting of the American Chemical Society (2000), Poster BIOL-15. authors: E-N-Jonsson and T-R-Holman. University of California, Santa Cruz, CA (described inhibitors of 15-lipoxygenase isolated from marine sponges; and Lyckander I.M., Malterud K.E. Lipophilic flavonoids from Orthosiphon spicatus as inhibitors of 15-lipoxygenase. Acta Pharm Nord; 4(3): cc.159-166, 1992. In addition, according to the methods proposed in the present invention, it is possible to use derivatives of thiourea and benzamide described in U.S. No. 6268387 to name Conner and others (representative of which is 3-amino-N-(3,4-dichlorophenyl)-4-methoxybenzamide, i.e. compound 3), which are inhibitors of 15-lipoxygenase, and 2-phenylbenzo[d]isoclinal-3-one (ebselen), 6,11-dihydro-5-thia-11-azabenzo[and]fluorin (indicated in the present description as compound 2), and phenylacetylene derivative, representative of which is 3-(2-Oct-1-initfini)acrylic acid i.e. the connection 4. You can also apply the compounds described in WO 01/96298 (incorporated in the present description by reference), including the connection 6, i.e. isobutyl ether [[[5-(5,6-debtor-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]carbamino acid.

Other inhibitors of 15-lipoxygenase include the following compounds:

1)

described in the materials 222-th National Meeting of the American Chemical Society, Chicago, stillings, USA, August 26-30, 2001, poster, MEDI 270.

2)PD 148104

described in articles 218-th ACS (new Orleans), 1999, MEDI 200.

3) PD 146176 developed by Parke-Davis (now Pfizer)

4) And 78773 (firm Abbott), described in WO 92/1682

5) QA-208-199 (Novartis)

described in the materials of the 6th Int Conf Prostaglandins (Florence), 1986, 328 Associated with this object the present invention relates to methods of joint treatment using inhibitors of 15-lipoxygenase to increase bone formation and other active substances, such as bisphosphonates, estrogen, IMRE (selective receptor modulators estrogen, calcitonin, or with the use of anabolic steroids. Examples of bisphosphonates include alendronate, ibandronate, pamidronate, etidronate, and risedronate. Examples of IMRE are raloxifene, digitalocean and lasofoxifene. Calcitonin include human and salmon calcitonin. As anabolics can be applied parathyroid hormone (PTH), for example, hPTH(1-34), PTH(1-84), and the protein associated with parathyroid hormone (Rtrr), and its analogues. Specific PTHrP analogs described in "Mono - and Bicyclic Analogs of Parathyroid Hormone-Related Protein. 1. Synthesis and Biological Studies," Michael Chorev, etc. Biochemistry, 36:. 3293-3299, 1997, and "Cyclic analogs of PTH and PTHrP,in WO 96/40193 and U.S. No. 5589452 and WO 97/07815. Another active ingredient can be administered simultaneously, before or after administration of the inhibitor of 15-Lipki is igenity and you can enter it using another method of introduction. Preferably first administered inhibitor of 15-lipoxygenase. The duration of such introduction can be anything, but typically it ranges from 6 to 24 months. After this treatment provide treatment with antiresorptive agent, such as bisphosphonates, IMRE, calcitonin or using hormonesensitive therapy.

So preferably it is possible to apply a composition containing the above-described inhibitor of 15-lipoxygenase and additional active substance. Thus, in the above-described method is preferably used for additional active substance. More preferably the specified active ingredient, which is used or applied according to the method proposed in the invention is an active regulator resorption of calcium from bone. Most preferably the specified active substance is a substance selected from a range, including estrogen, calcitonin, bisphosphonate and IGF.

In a preferred embodiment of the invention the inhibitor of 15-lipoxygenase used in the methods or uses described in the present description, choose from a range that includes synthetic organic molecules, natural products and antibodies to 15-lipoxygenase. In the most preferred embodiment of the invention the inhibitor of 15-Lee is oxygenase choose from a number, includes 3-(2-non-1-initfini)propionic acid, 6,11-dihydro-5-thia-11-azabenzo[and]fluoran, 3-amino-N-(3,4-dichlorophenyl)-4-methoxybenzamide, TRANS-3-(2-Oct-1-initfini)acrylic acid and isobutyl ether [[[5-(5,6-debtor-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]carbamino acid.

According to one embodiments of the invention the method proposed in the invention, is that exercised by the introduction of a therapeutically effective amount of antisense oligonucleotide having a sequence that has the ability to specifically bind to any sequence of the molecule in the genomic DNA or mRNA that encodes a 15-lipoxygenase, resulting prevents transcription or translation of mRNA 15-lipoxygenase. The term "antisense" refers to a composition that contains a nucleotide sequence complementary to the "sense" chain-specific nucleotide sequence. After introduction into the cell of the complementary nucleotides are combined with endogenous sequences produced by cells, resulting in formation of duplexes and blocking either transcription or translation (see, for example, Antisense Therapeutics, Humana Press Inc., Ed. by S. Agrawal, 1996, Totawa N.J.; Alama and other Pharmacol. Res. 36: cc.171-178, 1997; Crooke S.T. Adv. Pharmacol. 40: cc.1-49, 1997; and Lavrosky and other Biochem. Mol. Med. 62(1): cc.1122, 1997). Antisense sequences can be any nucleotide sequence, including DNA, RNA, or any mimetics or analogs of the nucleic acids (see, for example, Rossi and other Antisense Res. Dev. 1: cc.285-288, 1991; Pardridge, etc. Proc. Nat. Acad. Sci. 92: cc.5592-5596, 1995; Nielsen and Haaima Chem. Soc. Rev. 96: cc.73-78, 1997; Lee and others, Biochemistry 37: cc.900-1010, 1998). Introduction antisense sequences can be done in various ways, for example, by introducing into the cell using recombinant vector.

Generally, it is preferable to use antisense oligonucleotides, consisting of about 15-25 nucleotides, as they are easy to synthesize and they have the ability to have the desired inhibitory effect. For this purpose, can be used as molecular analogs of antisense oligonucleotides, which may provide additional advantages in terms of stability, distribution or reduce the toxicity of the pharmaceutical product. In addition to antimuslim oligonucleotides can be attached group having the chemical reactivity, such as associated with iron ethylenediaminetetraacetic acid (add-Fe), resulting in cleavage of the RNA at the site of hybridization. These and other methods of inhibition of in vitro translation of genes using antisense sequences are well known in this region is t (see, for example, Marcus-Sakura, Anal. Biochem. 172: p.289, 1988).

Although many inhibitors of 15-lipoxygenase are known and they are specified in the present description, it should be understood that the methods proposed in the invention, it is possible to use many other inhibitors. It is understood that in presented in this description of the methods can be applied as currently known inhibitors of 15-lipoxygenase, and those that will be revealed later. Presented in this description of the analysis methods and other methods known to the person skilled in the art, make it easy to identify and obtain other inhibitors of 15-lipoxygenase, which can be used to prevent or treat bone loss.

As illustrated in the examples, genetic anomalies or aldelo were found in chromosome 11 and the position of the allele corresponds to the position of a gene 15-lipoxygenase. Thus, inhibition or induction of 15-lipoxygenase can be applied in different situations. It is possible that by intracellular inhibition of 15-lipoxygenase can decrease metabolism of bone tissue and thereby increase mineral bone density and bone quality. Thus, inhibition of 15-lipoxygenase can be used in a method of treating or preventing excessive bone resorption, which PR is coming in osteoporosis and osteoarthritis.

Inhibitors of 15-lipoxygenase proposed in the present invention can be applied also to stimulate growth osteoplastic (osteogenic) cells or their precursors or for induction of differentiation of precursors osteoplastic cells either in vitro, or ex vivo. More specifically inhibitors of 15-lipoxygenase can be used to stimulate a population of cells, including mesenchymal cells in the bone marrow, thereby increasing the number of osteogenic cells in the cell population. In a preferred variant of the method of hematopoietic cells are removed from the population of cells either before or after treatment with inhibitors of 15-lipoxygenase. Using such methods can be propagated osteogenic cells. Reproduced osteogenic cells can be administered by infusion (or re-infusion) vertebral animal in need. For example, mesenchymal stem cells from the body of the individual can be exposed to ex vivo of the compounds proposed in the invention, and the resulting osteogenic cells can be administered by infusion or directly into the desired area of the body of the individual, where further proliferation and/or differentiation of osteogenic cells can occur without immune rejection. In an alternative embodiment, the population of cells subjected to the action of the inhibitor is in the 15-lipoxygenase, may constitute an immortalized osteoblast or osteogenic cells of a human embryo. If such cells are administered by infusion or implantation into the body of vertebrate animal, it may be expedient to provide immune protection of these neautrogena cells or immune suppression (preferably local) recipient to improve the efficiency of transplantation and recovery of bone or cartilage.

The effectiveness of treatment can be assessed by monitoring the effectiveness of and methods presented above in the present description, for the individual, which is to measure the level of 15-lipoxygenase in the body of the individual, who is also the object of the present invention.

You can also use various methods to identify classes of inhibitors of 15-lipoxygenase, which may prevent bone loss and/or stimulate the formation of bone tissue. One of the methods that can be used to identify compounds that inhibit the activity of 15-lipoxygenase, is that the cells, tissues, or preferably an extract of cells or other product containing 15-lipoxygenase, is brought into contact with several known concentrations of the test compound in the buffer, without affecting the activity of 15-lipoxy is easy. The level of activity of 15-lipoxygenase for each concentration of test compounds was measured by quantitative evaluation of the resulting enzymatic reaction product, and using standard methods to determine the values of the IC₅₀(concentration of test compound at which detectable activity of the sample preparation is reduced by half from its initial or reference value). The specialist in this area other known methods of determining the concentration of compounds proposed in the invention, in which there is inhibition of 15-lipoxygenase, and, as it follows from the above descriptions can be applied for this purpose. Specific methods of analysis that can be used to identify inhibitors of 15-lipoxygenase, is described in US no 626887B1 and 5958950 (based analysis using rabbit reticulocytes) and US No. 4623661 (analysis based on the use of radioactivematerials arachidonic acid in cell line RBL-1).

For example, as a rule, the antagonists can be identified, if known, the structure of the ligand. Currently, thanks to the development of highly automated methods of analysis using cells, giving a physiological response, it is possible to carry out the testing of potential analogues of ligands. In particular, with the assistance presented in this op is sanija methods of screening to identify new agonists and antagonists.

According to another method for identification of compounds, three-dimensional structure complementary to the active site of 15-lipoxygenase, you can apply science-based creation of medicines on the basis of structural studies of molecular forms chemokines, and other effectors or analogs. Such compounds can be identified by various methods, including molecular-mechanical calculations, molecular dynamics calculations, molecular dynamics calculations with constraints, where the constraints are determined on the basis of NMR spectroscopy, distance geometry, where the distance matrix is partially determined using the methods of NMR spectroscopy, x-ray diffraction or neutron diffraction. When using all these methods, the structure can be determined on the basis of comparison of data obtained in the presence of any ligand, which is known to possess the ability to interact with a 15-lipoxygenase, or without it.

Such computer programs include, but are not limited to) AMBER (which can be obtained from the University of California (University of California, San Francisco), CHARMM (Chemistry at HARvard Molecular Mechanics, which can be obtained from Harvard University (Harvard University)), MM2, SYBYL (firm Trypos Inc.), CHEMX (Chemical company Design, MACROMODEL, GRID (company Molecular Discovery Ltd) and Insight II (firm Accelryl). Podrazumevao the Xia, these programs can be used to assess the chemical interaction between the two molecules, either isolated or surrounded by solvent molecules such as water molecules, or for calculations based on the approximate accounting for the effects of solvation of the interacting molecules. The relative orientation of the two molecules can be determined manually by visual observation or other computer programs that generate many possible orientations. Examples of computer programs (but not limited to) DOCK and AutoDOCK. With the help of computer programs each orientation can be tested in respect of its degree of complementarity. This way you can create new connections, having the ability of inhibiting 15-lipoxygenase.

Another method of identifying compounds capable of inhibiting 15-lipoxygenase, is the use of techniques such as spectroscopy in the UV-rays or visible spectrum, polarimetry, digital spectroscopy of crystals (CA) or digital Raman spectroscopy (DRS), infrared spectroscopy or Raman spectroscopy (Raman spectroscopy), spectroscopy, nuclear magnetic resonance, fluorescence spectroscopy, IHVR, gel electrophoresis, capillary GE the e-electrophoresis, dialysis, refractometry, conductometry, microscopy-based nuclear interaction, polarography, dielectrometry, calorimetry, measurement of solubility, EPR (electron paramagnetic resonance)or mass spectroscopy. These methods can be used directly for direct evaluation of the interaction of compounds with 15-lipoxygenase or indirectly, when a certain agent, with spectroscopic properties, used as a probe to assess the ability of other compounds to contact 15-lipoxygenase; for example, through substitution or quenching of fluorescence.

Thus, in the present invention, a method of identifying compounds that increase the mineral density of bone tissue, which consists in the fact that the connection is brought into contact with 15-lipoxygenase and determine inhibited if the connection activity of 15-lipoxygenase. Preferably, in addition, the method was provided by the testing of connections with functional analysis to assess the activity of a compound in relation to bone formation. More specifically, functional analysis is that the connection is brought into contact with human mesenchymal stem cells and determine cell differentiation in osteoplastic cells. In another more preferred is the embodiment of the invention functional analysis is what compound is administered to an animal, except man, and measure the index of bone formation. In the most preferred embodiment of the invention the measured index is the mineral density of bone tissue. In another most preferred embodiment of the invention, the index represents a biomechanical parameter of bone tissue.

In another most preferred embodiment of the invention functional analysis is that the connection is brought into contact with human mesenchymal stem cells and evaluate differentiation in osteoplastic cells by analysis of alkaline phosphatase activity, analysis of the content of calcium, analysis of total DNA preparations or combinations thereof.

In yet another embodiment of the invention, a method of identifying compounds that have the potential to increase the mineral density of bone tissue, where the method is that the inhibitor of 15-lipoxygenase is brought into contact with human mesenchymal stem cells and assess the differentiation of cells into cells that form bone tissue. Preferably the assessment of cell differentiation is carried out by analysis of alkaline phosphatase activity, analysis of the content of calcium, analysis of total DNA of the drug is or their combinations

Identified or created thus inhibitors of 15-lipoxygenase can then be tested for their ability to prevent bone loss and/or stimulate the formation of bone tissue. In one of the embodiments of the invention described above apply computer methods. In another embodiment, the method of the connection test from the point of view of their ability to influence the known targets associated with bone loss, such as estrogen receptors, the receptor of tumor necrosis factor, receptor integrin, etc. According to another variant of the method, compounds are tested for their ability to differentiate stem cells into cells of the bone tissue.

According to the methods presented in the present description, pharmaceutical compositions containing the above-described molecules, used in combination with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients. Such carriers are liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc. to the person skilled in the art also known suitable carriers for the compositions not the liquid type. In the compositions proposed in the present invention can be used is ü pharmaceutically acceptable salt, which include, for example, salts of mineral acids, such as hydrochloride, hydrobromide, phosphates, sulfates and the like; and organic acid salts such as acetates, propionate, malonate, benzoate etc. Detailed description of pharmaceutically acceptable excipients and salts can be found in Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pennsylvania, published by Mack Publishing Company, 1990).

In addition, the composition of such media may be present auxiliary substances such as wetting or emulsifying agents, biological tabularasa substances, surfactants, etc. Biological buffer can represent virtually any solution, which is pharmacologically acceptable and which provide the desired pH value of the composition, i.e. the pH value in a physiologically acceptable range. Examples of buffer solutions are saline, phosphate buffered saline, Tris buffered saline, buffered saline solution Hanks, etc.

Depending on the intended route of administration of the pharmaceutical composition can be a solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, predpochtite the flax in standard doses, designed for a single administration of a precise dose. The composition should contain an effective amount of required medicines in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, etc.

For solid compositions of a standard non-toxic solid carriers are, for example, a pharmaceutical purity of mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. of the Input liquid pharmaceutical compositions can be prepared, for example, by dissolving, dispersing, etc. specified in the present description of the active substance and optional pharmaceutical adjuvants in excipient, such as water, saline, aqueous dextrose, glycerol, ethanol and the like, getting a solution or suspension. If necessary intended for the introduction of the pharmaceutical composition may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, sautereau agents for regulating the pH value and the like, for example sodium acetate, sorbitanoleat, triethanolaminato sodium, triethanolamine etc. methods Used in practice recip is of such medicines are known or they are obvious to experts in this field (see, for example, the above-cited reference).

Intended for oral administration, the composition typically is in the form of tablets, capsules, soft gel capsules, or may be in the form of aqueous or nonaqueous solution, suspension or syrup. The preferred forms for oral administration are tablets and capsules. Tablets and capsules for oral administration, as a rule, contain one or more standard carriers such as lactose and corn starch. As a rule, add the oil, such as magnesium stearate. If you use a liquid suspension, the active ingredient can be combined with emulsifying and suspendresume agents. If necessary, you can also add corrigentov, dyes and/or sweeteners. Other optional components which can be included in the oral composition include, but are not limited to, preservatives, suspendresume agents, thickeners, etc.

Parenteral compositions can be prepared in a standard form or in the form of liquid solutions or suspensions, solid forms suitable for solubilization or suspension prior to injection, or as emulsions. Preferably using methods known in this field, prepare a sterile suspension for injection using suitable media, dispel the dominant or wetting agents and suspendida agents. A sterile composition for injection can also be a sterile solution or suspension for injection in non-toxic acceptable for injecting the diluent or solvent. To an acceptable vehicles and solvents that can be used include water, ringer's solution and isotonic sodium chloride solution. In addition, as solvents or suspendida environments, as a rule, apply sterile fatty oils, fatty acid esters, or polyols. In addition, parenteral administration can be accomplished using the system with a slow release or slow release so as to maintain a constant level of dose of the medication.

Alternatively, the pharmaceutical compositions proposed in the invention can be typed into the form of suppositories for rectal administration. They can be prepared by mixing the active substance with suitable not causing irritation excipient, which is in solid state at room temperature but becomes liquid at a temperature in the rectum and therefore melt in the rectum to release the drug. These materials include coconut oil, beeswax and polyethylene glycols.

Pharmaceutical compositions offered by the e in the invention, you can also enter by using a nasal spray or by inhalation. Such compositions are obtained by methods well known in the field of preparation of pharmaceutical compositions and can be prepared as solutions in saline, using benzyl alcohol or other acceptable preservatives, stimulants absorption to increase the biological availability of propellants, such as fluorinated hydrocarbons or nitrogen, and/or other standard solubilizing or dispersing agents.

The preferred compositions for topical drug application are ointments and creams. Ointments are semi-solid preparations, typically based on petrolatum or other petroleum derivatives. Creams containing the selected active substance, as is well known in this field, are viscous liquid or semisolid emulsions of the type oil-in-water type water-in-oil. Fundamentals of creams can be washed off with water and, as a rule, they contain an oil phase, an emulsifier and the aqueous phase. The oil phase, which is sometimes referred to as the "internal" phase, as a rule, consists of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase, as a rule, although it is not required exceeds the amount of the oil phase and the AK rule, contains moisturizer. The emulsifier in the composition in the form of a cream, as a rule, is a nonionic, anionic, cationogenic or amphoteric surfactant. As a concrete basis intended for preparation of ointments or creams, as it should be obvious to experts in this field, you should use such a product, which provides an optimal administration of a medicinal product. As with other carriers or fillers, the basis for the preparation of ointments should be inert, stable, does not cause irritation and sensitization.

Compositions for buccal injection are tablets, lozenges, gels, etc. In an alternative embodiment, buccal introduction can be carried out using well-known specialists in the field of systems through the mucous membrane. Connections proposed in the invention can be administered through the skin or mucous tissue using standard systems transdermal injection of a medicinal product, i.e. transdermal "plaques"in which the active ingredient, as a rule, contained in the laminated structure that serves as the device for injection of a medicinal product which attach to the surface of the body. In such a structure, the composition of the medicinal product, as a rule, sod is Ritsa in the layer, or "reservoir", located under the top protective layer. Laminated device may contain a single reservoir or it may contain multiple reservoirs. In one of the embodiments of the invention, the reservoir is a polymeric matrix of a pharmaceutically acceptable providing the contact adhesive used to attach the system to the skin during injection of the drug. Examples of providing suitable skin contact adhesive are (but not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylates, polyurethanes and the like In an alternative variant containing a drug reservoir and providing a skin contact adhesive are separate and have different structure layers, and the adhesive, which is under the tank, in this case can represent the above-described polymer matrix or may be a containing liquid or gel reservoir, or may have any other shape. The protective layer in such layered structures, which serves as the upper surface of the device, acts as the principal structural element of the laminated structure and attached to the device main part flexibility. The material chosen for the protective layer, should be almost impervious to de the existing substances and other present products.

The patient should enter a pharmaceutically or therapeutically effective amount of the composition. The precise effective amount should vary from individual to individual and must depend on the species, age, size of the individual and his state of health, the nature and seriousness of the subject to treatment condition, the physician's recommendation and a drug or combination of drugs selected for treatment. The effective amount in a particular situation can be selected using the standard experiments. For the purpose of the present invention, a therapeutic amount typically ranges from about 0.05 to about 40 mg/kg body weight, more preferably from about 0.5 to about 20 mg/kg, in the form of at least one dose. For large mammals shows the daily dose can be from about 1 to 100 mg, which is administered once or several times per day, more preferably from about 10 to 50 mg. Individual, you can enter the number of doses required to reduce and/or alleviate the signs, symptoms or causes of the considered infringement or for any other desired changes in the biological system.

The introduction of polynucleotides, such as the introduction of antisense oligonucleotides 15-lipoxygenase can be done using any description is Noah above compositions or using recombinant expression vectors using as carriers of viruses or particles or without them. Such methods are known in the art (see, for example, U.S.№6214804; 6147055; 5703055; 5589466; 5580859; Slater and others, J._Allergy Clin. Immunol. 102: cc.469-475, 1998). For example, the introduction of the polynucleotide sequences can be accomplished using various viral vectors, including retroviral and adeno-associated viral vectors (see, for example. Miller A.D., Blood 76: s, 1990; and Uckert and Walther, Pharmacol. Ther. 63: cc.323-347, 1994). Vectors that can be used for antisense gene therapy include, but are not limited to, adenoviruses, herpes viruses, vaccinia virus, or preferably the RNA-type viruses, such as retroviruses. Other mechanisms for introduction of genes that can be used for introducing polynucleotide sequences into target cells include colloidal dispersion and systems based on liposomes, artificial viral envelope and other systems available to the person skilled in the art (see, for example, Rossi, J.J., Br. Med. Bull. 51: cc.217-225, 1995; Morris and others, Nucl. Acids Res. 25: cc.2730-2736, 1997; and Boado, etc., J. Pharm. Sci. 87: cc.1308-1315, 1998). For example, as systems introduction it is possible to use complexes of macromolecules, nanocapsules, microspheres, granules and based on lipid systems, including emulsions of the type oil-in-water micelles, mixed micelles, and liposomes.

As indicated above, the pharmaceutical compositions can contain one or more active substances that have capable the TEW effectively regulate calcium homeostasis. Additional active ingredient can be represented as (but not limited to, estrogen, calcitonin, bisphosphonate (eg, alendronate, residronate, zoledronate and ibandronate), vitamin D₃or equivalent, androgen, salt, fluoride, parathyroid hormone or its analogue, or IGF, agents that alter the regulation of transcription of the naturally occurring regulators of hormones that are involved in the metabolism of bone tissue, and combinations thereof. Such additional active substance can be entered to the individual prior to, simultaneously with or after administration of the inhibitor of 15-lipoxygenase proposed in the present invention.

Another object of the present invention based on the discovery of the correlation between gene polymorphism of 15-lipoxygenase and mineral density of bone tissue, and it was found that the presence of certain polymorphisms correlated with reduced mineral bone density. Another object of the invention is that the genotype correlates with susceptibility to osteoporosis. An advantage of the invention is that by screening for the presence of a particular genotype is possible to identify individuals who, apparently, have this genetic predisposition. Thus, the next object of the invention is a method terapeuticas the CSO treatment namely, that carry out screening for predisposition of the individual to the loss of bone tissue and, if the predisposition is identified, then such an individual is treated to slow or reduce or prevent bone loss.

According to the method for the diagnosis and prediction proposed in the present invention, identify changes, including deletions, insertions, and point mutations in coding and non-coding regions of the locus of 15-lipoxygenase wild type. For example, in mice it is preferable to detect changes on chromosome 11, whereas in humans revealed changes on chromosome 17. In addition, the method can be carried out by identifying the locus of 15-lipoxygenase wild-type and confirm the absence of predisposition to disorders which cause bone loss, which are called by the locus of 15-lipoxygenase. The presence of such mutations in individuals may be accompanied by symptoms of bone loss, and not be accompanied them. In addition, there may be differences in the reaction to the drug or prognosis of symptoms in individuals who carry mutations in the locus of 15-lipoxygenase, compared to those who do not have such mutations.

Thus, one of the objects of the invention is a method of diagnosis, namely, that determine totip gene 15-lipoxygenase. Generally, according to the method determines whether the individual is homozygous or heterozygous in respect of the gene polymorphism of 15-lipoxygenase. Generally, the method proposed in the invention, is carried out by analysis of in vitro cells of the individual to determine the genotype of the specified individual in the gene locus 15-lipoxygenase.

Diagnostic methods that can be applied, include, but are not limited to) analysis of microradios, fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, analysis by the method of southern blotting, single-stranded conformational analysis (SSCA), analysis of the protection from RNase, analysis using allele-specific oligonucleotide (ASO)analysis method the dot-blot and PCR-SSCP, which are known in this field.

Predisposition to the disease, accompanied by loss of bone tissue can be assessed by testing any tissue of the individual, such as a sick person, in relation to mutations of the gene 15-lipoxygenase. For example, an individual carrying a congenital mutation of 15-lipoxygenase in the germ line may be predisposed to the development of diseases involving bone loss. This can be determined by testing DNA from any tissue of the body of the individual. Most just take blood samples and extracted DNA is C blood cells. In addition, it is possible to perform prenatal diagnosis by testing cells of the embryo, placental cells or amniotic cells against mutations of 15-lipoxygenase. By using any of the set forth in this description of the methods can detect the change of allele 15-lipoxygenase wild type, due to, for example, by point mutation or deletion.

There are several methods that can be used to detect variations in the DNA sequence. The variation of the sequence can be detected by direct DNA sequencing or by sequencing performed manually or automated fluorescent sequencing. Another approach is single-stranded conformational analysis (SSCA), which is sequenced fragments with shifted mobility on gels for SSCA to determine the true nature of variations in the DNA sequence. Other approaches based on the detection area with an unpaired bases for the two complementary strands of DNA include pulsating denaturing gradient gel electrophoresis (CDGE), analysis of heteroduplexes (ON) and chemical cleavage sites erroneous nucleotides (CMC). If the mutation is known, for rapid screening of a large number of samples in respect of this mutation, you can use a specific allele of the methods of the detection, such as hybridization of allele-specific oligonucleotides (ASO) (see, for example, Saiki and others, Proc. Nad. Acad. Sci. USA 86: cc.6230-6234, 1989).

Detection of point mutations may be accomplished by molecular cloning of the allele(s) 15-lipoxygenase and sequencing the allele(s) using known in the field of methods. Alternatively, the sequences of genes can amplify using known methods directly using preparations of genomic DNA from tissue. Then you can determine the DNA sequence of the amplified sequences. The presence of the allele can be followed by single-stranded conformation analysis (SSCA), denaturing gradient gel electrophoresis (DGGE or CDGE), analysis of protection from RNase, using allele-specific oligonucleotides (ASO), allele-specific PCR analysis lengthening of one nucleotide known in this field.

According to a preferred method of the alleles identified using the microarray method. According to this method, thousands of different oligonucleotide probes or genes can be synthesized (U.S. No. 5412087 name McGall and others) or to put the series in the form of drops (U.S. No. 6110426 name Shalon and others) on silicon or glass chip. Designed for analysis of nucleic acid, typically fluorescently mark and hybridize the t with the probes on the chip. As other labels can serve as³²P and Biotin. Using a series of such microradio drops can also explore the interaction of nucleic acid-protein. Using this method to detect the presence of mutations in the gene 15-lipoxygenase.

The presence of an altered (or a mutant) gene 15-lipoxygenase correlates with an increased risk of diseases associated with bone loss. For detection of gene mutation 15-lipoxygenase prepare the biological sample and analyze the difference between the sequence to be analyzed allele 15-lipoxygenase and consistency of allele 15-lipoxygenase wild type. Then the mutant alleles can be subjected to sequencing to identify specific mutations of a specific mutant allele. After that mutations of 15-lipoxygenase, specifically on chromosome 11 and mouse chromosome 17 and human use in the methods of diagnosis and prediction proposed in the present invention.

Thus, an object of the present invention is also a method for diagnosing predisposition to bone loss in an individual, namely, that reveal polymorphisms of the human chromosome 17, where the polymorphism is a criterion of susceptibility to bone loss. In a preferred embodiment, izopet is ment for discovery using genotyping. In a more preferred embodiment of the invention genotyping involves the use of microsatellite markers or one or more single nucleotide polymorphisms.

Another object of the present invention is a method for diagnosing predisposition to bone loss in an individual, namely, that the individual identify gene polymorphism 15-lipoxygenase.

In the present invention proposed inhibitors of 15-lipoxygenase for use as therapeutic active substances for the prevention of bone loss. The object of the present invention are also compounds identified using the methods described above. In addition, the proposed pharmaceutical composition, intended in particular for the prevention of bone loss, which contains the above compound and a carrier. A kit for diagnosing susceptibility to loss of bone tissue that contains at least one oligonucleotide probe designed to detect polymorphism of 15-lipoxygenase-related bone loss.

Finally, the objects of the invention are also compounds, methods of application, composition and set mostly in the present description, primarily in the preceding examples.

In figure 1 proil utrirovanno the comparison is based on the SNP genotyping polirovannyj DNA samples obtained microsatellite genotyping of DNA samples for the detection of polymorphisms, associated with the mineral density of bone tissue. The statistical significance of each difference of occurrence of the allele was calculated using z-test, and build a dependency graph of the logarithm of the odds ratios (LOD) for all chromosomes. The dashed line corresponds to the LOD value of 3.3, which was chosen as the limit values to determine statistically significant differences in the genomes.

Figure 2 - quantitative assessment of the Alox15 gene expression in the mouse kidney, obtained on the basis of the analysis of total RNA kidney, where gene expression was analyzed using microradio.

Figure 3 - quantitative assessment of the ALox15 gene expression in primary mouse osteoblasts depending on the increasing concentrations of IL-4, introduced in vitro.

Figure 4 - position of the polymorphisms single nucleotide (SNP)identified in the gene ALox15 mice.

Figure 5 - quantification of alkaline phosphatase activity in human mesenchymal stem cells (hMSC) when exposed to inhibitors of 15-lipoxygenase, representing a compound 1 or compound 2, in comparison with the effects of the solvent (DMSO).

Figure 6 - quantification of alkaline phosphatase activity in hMSC using inhibitors of 15-lipoxygenase compound 3 or compound 2 as a control DMSO or 1,25-vitamin D .

Figure 7 - a quantitative assessment of the content of total calcium in hMSC using inhibitors of 15-lipoxygenase compound 3 or compound 2 as a control DMSO or 1,25-vitamin D₃.

On Fig - quantification of alkaline phosphatase activity in hMSC after cultivating for 9 days in the presence of inhibitors of 5-LO Zileuton, AA-861 and Rev-5901 in comparison with the same solvent (DMSO)used as control. No reaction is a characteristic difference from the cases of inhibitors of 15-lipoxygenase.

Figure 9 - a quantitative assessment of the content of total calcium in hMSC under cultivation for 16 days in the presence of inhibitors of 5-LO Zileuton, AA-861 and Rev-5901 in comparison with the same solvent (DMSO) or 1,25-vitamin D₃as controls. In contrast to the cases of inhibitors of 15-lipoxygenase reaction is absent.

The following describes specific embodiments of the present invention. Examples are given only for illustration and is in no way intended to limit the scope of the present invention.

Connection #	Structure	IC50for 15-LO
1		1-3 microns
2		~200 nm
3		~10 nm
4		1-3 microns
5		Negative control
6		25-83 nm

Compound 1, which represents a 3-(2-non-1-initfini)propionic acid, described in U.S. No. 5972980.

Compound 2 represents 6,11-dihydo-5-thia-11-Aza-benzo[a]fluoren described in WO 97/12613.

Compound 3 represents 3-amino-N-(3,4-dihydrogen)-4-methoxybenzamide described in WO 99/32433.

Compound 4, which represents TRANS-3-(2-Oct-1-initfini)acrylic acid, described in U.S. No. 4713486.

Connection 5, a Zilueton described in U.S. No. 4873259.

The connection 6, which represents an isobutyl ether [[[5-(5,6-debtor-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]carbamino acid, described in WO 01/96298.

Were taken effort to ensure the accuracy specified in the description of the numerical values (for example, quantities, temperatures and so on), but, of course, it should be borne in mind that a valid some experimental errors and deviations.

Example 1

Model polymorphism genotyping single nucleotide (SNP).

Microsatellites (also referred to as polymorphisms single tandem repeat or single polymorphisms in the sequence length) are the most studied category of genetic markers. They include small series of tandem repeats of small sequences (di-, tri-, Tetra-nucleotide repeats), which have a high degree of length polymorphism, which provides high level information. In the human genome is a little more than 5,000 microsatellites that are easy to topiramate using methods based on PCR (Dib and others, Nature 380: cc.152, 1996).

As a rule, we use the method described Grupe and others, Science 292: cc.1915-1918, 2001. To identify genetic factors that regulate the IPC, was carried out by scanning the genome for 1000 representatives of the F2 generation of intercross C57BL/6 × DBA/2 (firm Jackson Labs, Bar Harbor, stmin) 16 weeks of age. The F2 generation was not sex-linked normal distribution of the IPC (Grupe and others, Science, 292, cc.1915-1918, 2001). With extreme phenotype of the offspring of the F2 with the highest (n=145) and lowest (n=149) IPC (within 15% from the top and bottom which she estimates) were subjected to complete genome scan to detect connection of the IPC by genotyping individual DNA samples with 100 microsatellite markers. In addition, by taking equal quantities of DNA from representatives of the F2 offspring of high and low magnitude of the IPC received two pools. For 109 SNP found in the database mSNP, was measured using allele-specific kinetic PCR frequency of occurrence of alleles in the samples included in the pools. For each marker was assessed by the difference in the frequency of occurrence of alleles in the two extreme pools. If the token is not associated with the IPC, its expected frequency is 50% for both extreme pools. The statistical significance of each difference in the frequency of occurrence of alleles was calculated using z-test, and build a dependency graph of the logarithm of the odds ratios (LOD) (figure 1). For the four regions located on chromosomes 1, 2, 4, and 11, using methods based on the use of microsatellites and SNP genotyping, there was a statistically significant linkage (LOD score>3,3).

Thus, using the method of SNP genotyping was identified genes-candidates that can lead to the IPC.

Example 2

Models with animals

To identify the gene within the identified region on chromosome 11, which regulates the IPC was performed using microradio analysis of differences in gene expression in mice of the strain DBA/2, C57BL6/J, Jackson Labs, Bar Harbor, main) and congrego strain mice (D2.B6chr11, polucen the th from Oregon Health Sciences University). Congenia mouse line D2.B6chr11 bore section from sm to sm chromosome 11 C57BL/6J, introgressions the basis of genomic line DBA2/j Microrate containing genes of the mouse genome, hybridized with labeled crnc obtained from whole kidneys, cultured primary osteoblasts and primary chondrocytes. Hybridization was carried out with crnc obtained from individual mice lines DBA/2J, C57BL/6J and congenic mice B6.D2chr11. Carried out the selection of mRNA (2 row(A)+), synthesis crnc and hybridization. Differential expression was assessed by pairwise comparisons mice lines DBA/2J and C57BL/6 and by the pairwise comparison of mice DBA/2J and Korennoy line B6.D2chr11.

For further characterization of selected differentiation expressed genes encoded within the interval sm-cm on chromosome 11. It was found a significant difference in the expression of individual gene (ALox15, located at 40 cm on chromosome 11) two of the studied lines (figure 2). For line in DBA/2J were characterized by elevated levels of expression of ALox15 compared with line D2.B6chr11 and mice C57BL/6J, and lower levels of IPC compared to the two other lines. Thus, increased expression of ALox15 in mice of the DBA/2J is associated with decreased mineral bone density.

Differential expression of ALox15 mRNA in osteoblasts was evaluated by PCR R is real-time. Cells were obtained from mice lines DBA/2J, C57BL/6J and D2.B6chr11 (figure 3). Total RNA was treated with Dnazol I to remove impurities genomic DNA. RNA was kept at 65°C for 10 min to inactivate Gnkazy I. All PCR was carried out in a volume of 100 μl. RNA (100 ng) was subjected to RT-PCR using DNA polymerase rTh (2 units) (firm Perkin Elmer) in a reaction mixture containing 5×EZ buffer, Mn(SLA) (3 mm), ethidiumbromid (1 µg/µl), dNTP (200 μm), direct primer (200 nm) and reverse primer (200 nm). cDNA was obtained by incubation of the samples for 30 min at 60°C. After this was carried out by 60 cycles of PCR (95°C, 20 s; 58°C, 20) and then incubated for 20 min at 72°C. Fluorescence at each cycle is associated with the number of the resulting product and it was measured using a kinetic thermoacetica and analyzed using available software (Germer and Higuchi, Genome Research, 9, cc.72-78, 1999). In addition to kidney cells osteoblasts mice of the DBA/2J were also produced elevated levels of ALox15 mRNA compared with mice C57BL/6J. RNA ALox15 were induced with IL-4 in osteoblasts C57BL/6 to a greater extent than in mice of the DBA/2 (figure 3).

To identify the molecular basis statusbarisvisible difference in the expression of ALoxl5 was performed using methods known in this field, sequencing of genomic DNA gene ALox15 mice lines DBA/2J and C57BL6/j Were found 14 poly is artizov DNA sequences characterizing the differences between the two strains (presented in chart form in figure 4 and table 1 below). Gene Ensembi 15LO designated as ENSMUSG000000 18924. Gene sequence extending from position 70929619 to 70937482 mouse genome represented in Seq ID No. 1. The first SNP in table 1, with the position 70938498 in the sequence of the mouse genome, localized at position 541 sequence placed in Genbank under registration number № U04332, which is represented in Seq ID No. 2.

Example 3

The ability of inhibitors of 15-lipoxygenase to increase bone formation

To determine the ability of inhibitors of 15-lipoxygenase to stimulate bone formation and accumulation of bone tissue in vitro experiments evaluated the ability of inhibitors of 15-LO increase the level of differentiation of stem cells in osteoplastic cells (osteoblasts). In particular, by measuring two markers of differentiation into osteoblasts, the cellular activity of alkaline phosphatase and calcium in culture was evaluated the ability of three inhibitors, namely compounds 1-3-(2-non-1-initfini)propionic acid), compounds 2 (6,11-dihydro-5-thia-11-azabenzo [and]fluoren) and compound 3 (3-amino-N-(3,4-dichlorophenyl)-4-methoxybenzamide)obtained according to the methods described in the literature, to stimulate differentiation of human mesenchymal stem cells into osteoblasts.

In General, the method consisted in the fact that human mesenchymal cells (hMSC line Poietics No. of RT-2501, firm Bio Whittaker) stump Aravali 12-hole covered with collagen tablets with a density of 3100 cells on 1 cm ²in 1 ml of culture medium (firm Clonetics, catalogue number RT-4105), supplemented inducers of osteoblast (100 nm Dex, 0.05 mm L-ascorbic acid-2-phosphate, 10 mm betapacewithout). To each culture were added individually three inhibitor of 15-LO except the control wells, which added to 0.1% DMSO (negative control) or 1 nm 1,25-vitamin D₃(positive control). Used the following concentrations of inhibitors: compounds 1 - 3 and 30 microns; for connection of 2 - 0,1, 0,2, 0,3 or 1.0 μm for compound 3 - 3, 10 or 30 μm. For each dose level were prepared from 4 up to 8 independent replicative cultures. At the end of the experiment, the grown cells were collected by scraping from the hole and made the appropriate environment for further analysis or activity of alkaline phosphatase (firm Sigma kit No. 104-LL), or maintenance of cellular calcium (firm Sigma kit No. 587-M). Culture, designed for the analysis of alkaline phosphatase, collected by scraping cells and resuspendable in 250 μl of Tris buffered 0.1%Triton X-100 and then subjected to analysis either in fresh form or after thawing of the frozen cultures. Separately selected crop for analysis of the content of total calcium and resuspendable in 250 μl of 0.5m HCL. The results of these analyses were standardized in relation to the detention of total cellular DNA, standardize thus differences in cell proliferation. At the same time by scraping cells were collected separately replicative culture, designed for the analysis of alkaline phosphatase and calcium, and resuspendable them in 250 ál of balanced salt solution Hanks and evaluated the number of cells with DNA (kit DNeasy Tissue Kit, the company Qiagen, catalog number 69506).

Quantification of differentiation into osteoblasts in vitro.

For a quantitative evaluation of the activity of alkaline phosphatase inhibitors were prepared in 0.1% DMSO and were added to the cultures at day 1 and after that within 14-16 days every 2-3 days. Conducted three different series of experiments.

In the first series of experiments as inhibitors used compound 1 or compound 2 (0.2 or 1 μm) and the solvent (DMSO) as negative control, and for each dose level was used in four independent replicative culture. The tablets were cultured for 14 days. Standardized DNA alkaline phosphatase activity presented graphically in figure 5.

In the second series of experiments used eight replicative cultures and connection 3 (3, 10 and 30 μm) or compound 2 (0,1, 0,3, 1,0 µm) as inhibitors of 15-LO. Inhibitors were added on either day 1 or day 11. As a negative control using the Ali DMSO, and 1,25-vitamin D3 as a positive control for the induction of differentiation into osteoblasts. Standardized alkaline phosphatase activity and calcium content presented in the form of plots 6 and 7, respectively. Compound 2 had the highest efficiency when added on day 1 and compound 3 had the greatest efficiency when adding it to day 11.

To control the specificity of inhibition of 15-LO in relation to induction of differentiation into osteoclasts in vitro, in the third series of experiments investigated the 8 samples of standard inhibitors of lipoxygenase, which was found to have greater specificity in respect of the enzyme 5-LO than in respect of the enzyme 15-LO. Every 2-3 days to prepare fresh preparations of Zileuton (1, 10, 50 μm, which was obtained from the firm Roche), representing the connection 5, AA-861 (1, 10, 50 µm, the company Sigma, No. A) and Rev-5901 (15 μm, the company Sigma, No. R5523) and tested using human cells-the precursors of osteoblasts. It was found that inhibitors of 5-LO lost the ability to induce alkaline phosphatase activity at day 9 or the deposition of calcium in the culture after culturing for 16 days, which is illustrated in Fig and 9, respectively.

The results of the quantitative evaluation of markers of differentiation in osteoblasts in cultures of hMSC, stimulating avannah in relation to differentiation in osteoblasts, conducted by determining alkaline phosphatase activity and calcium content in culture have demonstrated that specific inhibitors of 15-lipoxygenase stimulate the differentiation of human osteoplastic cells in vitro.

In addition, conducted an assessment of the ability of inhibitors of 15-lipoxygenase to stimulate bone formation and deposition of bone tissue using a count of clonogenic assays, which were determined count of clonogenic potential of progenitor cells of bone marrow cells. Cells osteoblasts and osteoclasts were treated with inhibitors of 15-lipoxygenase. According to one of the methods cells and inhibitors were incubated in vitro. In another method, the mammal is administered the inhibitor, was isolated precursor cells of bone marrow cells and were sown. In both methods was measured by colony forming units formed from these cells in the bone marrow. It is possible to carry out the screening of compounds for their ability to increase the mineral density of bone tissue, if it is determined that they increase the count of clonogenic potential of bone marrow in respect of osteoblasts or reduce count of clonogenic potential of bone marrow in respect of osteoclasts.

Example 4

Violation of gene 15-lipoxygenase leads to increased mineral bone density of the femur

Original is but it has been revealed, that in mice with deficiency of 15-LO have small phenotypic changes, but then on the model using the same mice, it was found that they are partially protected from atherosclerotic lesions (Sun and Funk, J. Biol. Chem., 271, cc.24055-24062, 1996; Cyrus and others, J. Clin. Invest, 103, cc.1597-1604, 1999). However, mice with a deficit of 15-LO did not measure BMD. To investigate the impact of genetic disorders 15-LO on the mineral density of bone tissue, mice with deficiency ("knock-out") 15-LO (15-LO-KO) compared with baseline mice from the point of view of mineral density of bone tissue. Measurement of mineral density of bone tissue was performed by the method of double absorbtiometry energy x-rays (DEXA). All analyses were performed using a densitometer type Lunar PIXImus, Lunar Corp., Madison, steveascension). Densitometric analyses of the whole body were performed on shot mice by the age of 4 months, when they ended the accumulation of masses of Mature bone. Global window was a picture of the whole body with the exception of vault of skull, lower jaw and teeth. After killing the right femur was carefully allocated and before performing DEXA scans were purified from tissues attached to it. Found no significant differences in mineral density of bone tissue of the whole body of mice 15-LO-KO and the original line of C57BL/6 mice(49,8+/minus 0.6 mg/cm ²compared to 49,5+/-0,4 mg/cm²). However, it is important that the BMD of the femur was significantly greater in mice with deficiency of 15-LO in comparison with the original line C57BL/6 (54,0+/-1,2 mg/cm²compared with 49.7+/a-0.7 mg/cm²). These results demonstrate that a violation of 15-LO leads to increased mineral bone density of the femur.

Example 5

Inhibitors of 15-lipoxygenase stimulate bone formation in vivo

The ability of inhibitors of 15-lipoxygenase to stimulate bone formation in vivo was assessed by measuring the ability of inhibitors to improve the characteristics of the bone tissue in the simulation of osteoporosis in mice. Constitutive expression of the transgene interleukin-4 under the control of the promoter of the gene Lck (Lck-IL4) in mice C57BL/6 led to the phenotype of bone tissue, simulating severe osteoporosis (Lewis and others, Proc. Natl. Acad. Sci., 90, cc.11618-11622, 1993), and these mice were used to demonstrate the effect of treatment with inhibitors of 15-LO in bone mass.

Four groups, which carried out the experiment, presented in table 2. During weaning from breast milk to mice wild-type C57BL/6 or transgenic lines Lck-IL4 was administered daily twice a day along with food within 84 days of 150 mg/kg compound 2 PD 146176 (food 5001: 23% protein, 10% fat, of 0.95% calcium and 0.67% phosphorus; firm PMI Feeds, Inc., The St. Louis, stricture). Mice twice daily at approximately 12-hour intervals were given access to half of the daily diet and daily watched the food intake. They had access to water without restrictions. Control groups were given the same food that does not contain the compound 2. Animals in all groups during the experiment consumed food in the same way.

Table 2. The experimental group, which conducted the study of inhibitor in vivo. After weaning (at about the age of 28 days), the mice within 84 days were given feed containing inhibitor of 15-LO (compound 2), or without it. The number of females (F) and males (M) in the groups was approximately equal.

The effects of overexpression IL4 inhibition of 15-LO in total body BMD of the femur and its geometry and biomechanical characteristics of the femur are presented in table 3. Overexpression of IL4 led to a significant decrease in BMD of the whole body, body weight and hematocrit, and increased percentage of body fat. Overexpression of IL4 has also led to the decrease in BMD of the femur, cortical area, moment of inertia and cortical thickness compared with mice C57BL/6 wild-type. It is established that the increase in the area of bone marrow of mice Lck-IL4 correlated with decreased hematocrit and associated anemia. In addition, overexpression of IL4 had a substantial impact on the biomechanical characteristics of the femur, including load reduction, leading to fracture, bone stiffness and strength of bone (table 3). In mice which were administered inhibitor of 15-LO (compound 2), revealed a partial reversal of the destructive effects on the bone tissue of the whole body and femur (table 3). The processed drug mice revealed a significant increase in BMD of the whole body hematocrit, BMD of the femur and cortical thickness compared with the control mice bearing the transgene, Lck-IL4. It is important that the treated inhibitor of 15-LO mice revealed a significant increase in the biomechanical characteristics of the femur, including the load, leading to fracture, stiffness and strength. This is of particular importance for osteoporo is a, as the level of bone strength and load, leading to fracture, are important determinants for patients, which increases the likelihood of fractures. In General, the results obtained on the model of osteoporosis with animals, suggest that inhibition of 15-LO in vivo leads to higher levels of the IPC and higher strength of the bone.

Table 3. The effects of overexpression IL4 and inhibitor of 15-LO on the parameters of the whole body and femur. The parameters of the whole body and femur were measured in mice bearing the transgene, Lck-IL4, which causes overexpression of IL4, and compared with mice C57BL/6, used as controls. These parameters were also measured in mice line Lck-IL4, which was administered inhibitor of 15-LO, representing a compound 2 and compared with the parameters of untreated mice of Lck-IL4, used as controls, the concentration indicates not detected differences between groups, N.D. indicates that the p value is not valid.

Animals

All mice used in experiments in vivo, were grown in identical conditions in veterinary Institute of Portland, stuitje, line, originally by the scientists from the Jacksonian laboratory (the Jackson Laboratory (Bar Harbor, Maine). From the baseline obtained from the Jacksonian laboratory, was supported by no more than three generations of mice. After weaning, the mice were kept in groups (9-10 animals per cage) under a light cycle of 12 h light/dark (6:00 to 18:00 h) at 21±2°C. All procedures were approved by the state Committee on the content and use of animals stuitje (VA Institutional Animal Care and Use Committee and were conducted according to the guidelines on the content and use of animals in research, approved by the National Institute of health.

Bone densitometry

Measurement of the mineral content of bone tissue was performed using a dual absorptiometry energy x-rays (DEXA). All analyses were performed using a densitometer type Lunar PIXImus, Lunar Corp., Madison, Wisconsin). Densitometric analyses of the whole body were performed on shot mice by the age of 4 months, when they ended the accumulation of masses of Mature bone. Global window was a picture of the whole body with the exception of vault of skull, lower jaw and teeth. After killing the right femur was carefully allocated and before performing DEXA scans were purified from tissues attached to it.

Processing samples

Adult mice (age 16 weeks) were subjected to euthanasia p is the inhalation of CO ₂and weighed to the nearest 0.1, Immediately after the killing took blood from the heart and a small aliquot of whole blood was saved for determination of hematocrit. Immediately allocated lumbar vertebrae and both femoral bones were wrapped in sterile gauze soaked in phosphate buffered saline, and stored in a frozen state at a temperature not less than about -20°C for further analysis.

The geometry of the stem of the femur

Geometrical parameters of the cross section of the middle part of the diaphysis of the femur were determined using a portable x-ray micro (model 1074, firm-CT skyscan, Antwerp, Belgium). Scanning was carried out at intervals of 40 mm, and slice located in the middle part of the femur, analyzed the total area (FCSA), cortical area (Ct Ar) and the area of modular channel (MA Ar). In addition, based on the number of pixels in the cortical region of the digital image, to calculate the radius from the centroid of the cross section to the outer fibrous layer in the anteroposterior plane, the static moment of inertia of sectional area (Ixx) in the plane of bending and average cortical thickness (Ct Th).

The study of biomechanical parameters

Thigh investigated in relation to fracture by bending the application efforts in the PEX points using precision instruments to study the characteristics of the materials (Instron model 4442, Canton, iminnesota). The load causing fracture, and hardness were determined using the system software. Then, using data from previously performed measurements of the cross-sectional area was calculated bending strength.

Data analysis

All data were analyzed using bi-directional analysis of variance (ANOVA) using the software package JMP (SAS Institute).

Example 6

The ability of inhibitors of 15-lipoxygenase to increase bone formation in vivo

To determine the ability of inhibitors of 15-lipoxygenase to stimulate bone formation and accumulation of bone tissue was obtained according to the method described in WO 0196298, isobutyl ether [[[5-(5,b-debtor-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]carbamino acid, i.e. the compound 6, and tested according to the standard method of analysis osteopenia to undergo oophorectomy (Ovx) rats with estrogen deficiency.

Rats aged 3 months were subjected oophorectomy (Ovx) and was administered orally using a stomach probe 1 mg/kg/day of compound 6 or 0.1 ág/kg/day of 1,25-dihydroxyvitamin D₃(positive control) and abbreviation in the table .D) once daily, starting from the second week after oophorectomy, and continued for 7 weeks. The dose was increased to 2 mg/kg/day and continued to okonchan the I 11-week period. Rats in the control groups, both groups of animals imitators (rats that were not subjected oophorectomy), and Ovx-group was injected only media. Mineral bone density of the spine and right femur were evaluated using the software package High Resolution using a densitometer bone type QDR-4500, Hologic, Valtan, iminnesota). Animals were subjected to scanning, placing them in the supine position, so that the right thigh is perpendicular to the body and tibial bone was perpendicular to the thigh. Mineral bone density (g minerals/cm²in the tibia and spine when using compounds shown in table 4. In animals treated with the inhibitor of 15-LO revealed the increase in BMD in the spine in the time period >3 weeks and in the proximal part of the femur in the time period >7 weeks. The results are shown in table 4.

Values in parentheses represent p values compared to OVX-control at the same time, N.O.=no data.

Example 7

Gene manipulation in order to reduce gene expression of 15-lipoxygenase in laboratory mice leads to an increase in mass and bone strength.

For studying the relationship between the expression of 15-lipoxygenase and education to the things of fabric created a genetically heterogeneous population of F ₂two strains predecessors (lines), B6.129S2-Alox15^tm1Fun(deficit Alox15 or 15LOKO) and DBA/2 (D2), which are received from the Jacksonian laboratory (Bar Harbor, stmin). Mouse lines B6.129S2-Alox15^tm1Fun(15LOKO) were homozygous for a targeted mutation in the gene Alox15 and therefore they could not happen expression arachidonate under the influence of 15-lipoxygenase. In contrast, in mice lines D2 detected high levels of Alox15. Mice generation F₁line 15LOKO-D2 were bred in the laboratory, where they spent the experiences of the parental lines obtained from Jacksonian laboratory, and subjected to intercross, receiving a total of 292 individuals of generation F₂of mice 15LOKO-D2. After weaning, the mice were kept in groups of 2-5 animals per cage) under a light cycle of 12 h light/dark (6:00 to 18:00 h) at 21±2°C, while the animals had free access to feed and water for laboratory rodent diet 5001: 23% protein, 10% fat, of 0.95% calcium and 0.67% phosphorus; firm PMI Feeds, Inc., St. Louis, stricture).

All mice which performed the experiments were age 4 months, when the accumulation of bone mass (28). Measurement of mineral density of bone tissue produced using a densitometer with a narrow beam type Hologic QDR 1500 (firm Hologic, Waltham, iminnesota), which was daily calibrated using a hydroxyapatite phantom of pojasnitj the nd Department of human spine. The analysis was performed using the software to analyze the entire body of the mouse (version 3.2), kindly provided by the manufacturer (company Hologic, Waltham, iminnesota). Densitometric analysis was performed on shot mice. Feeding was stopped on the night before the examination (in order to eliminate the effect of undigested feed for rodents on the evaluation of mineral density of bone tissue, which may distort the results), while mice were anestesiologi by inhalation soporano. Animals were weighed to the nearest 0.1 g and then was subjected to scan to determine bone density. Mice were subjected to euthanasia by inhalation of CO₂and in aseptic conditions were isolated spleen and femur and then immediately frozen for further analysis. All procedures were approved by the state Committee on the content and use of animals stuitje (VA Institutional Animal Care and Use Committee and were conducted according to the guidelines on the content and use of animals in research, approved by the National Institute of health.

The structure of the femur (the area of the middle part of the cortical bone area and cortical thickness) was measured using table microtopographical x-ray scanner (model Sky Scan 1074, aartselaar, Belgium). The left femur was investigated in relation to fracture by bending the application efforts at three points using precision instruments to study the characteristics of the materials (Instron model 4442, Canton, iminnesota). Attach the extensometer (model 2630-113, the company Instron Corp.) and used system software (series IX for Windows 95, firm Instron Corp.) to move the actuator with strain-rate of 0.5%/C up until there was no fracture. Recorded data on the load and the displacement (measured by extensometer) and using the system software calculates the load, leading to fracture (F), and strength (k, defined on the basis of dependence of the linear component of the load from moving).

Genomic DNA was isolated from the spleens of individual mice according to the method of vysalivaniya. Mice were genotypically according to the Protocol polymerase chain reaction (PCR), developed Jacksonian laboratory (http://aretha.jax.org/pub-cgi/protocols/protocols.sh?objtype=protocol&protocol_id=304 developed to create with different sizes of products ALox15 gene wild type (266 base pairs) and the mutant gene (172 base pairs). Amplification was carried out in thermoacetica type Perkin-Elmer 9700 (Branchburg, new Jersey). PCR products were separated on a 4-percentage agarose gels and carried out visas is the alization by staining with ethidium bromide.

Population generation F₂15LOKO-D2 consisted of approximately equal numbers of males (n=141) and females (n=151) and, as illustrated in table 5, the prevalence of genotype ALox15 is consistent with the criterion of the expectations of hardy-Weinberg equilibrium at 70 (24%) homozygous mice with deficiency (absence of allele 15-LO), 153 (52%) heterozygous mice (one allele 1 5-LO from the line D2) and 69 (24%) homozygous mice of D2 mice (both alleles 15-LO from the D2 line). Although there was no difference in body weight in mice generation F₂, homozygous deficiency ALox15, was found a large quantity BMD of the whole body compared to homozygous mice D2 or heterozygous mice (p=0.006 by the results of analysis of variance (ANOVA)). In addition, mice of F₂, homozygous deficiency ALox15, revealed increased cortical bone tissue stem hip (cortical area and cortical thickness) and a significantly higher structural characteristics, as evidenced by obtained by measuring higher pressures, resulting in fracture and strength (table 6).

(IPC=IPC whole body; the horse Ct=cortical area of the stem of the femur; thickness Ct=cortical thickness of the stem of the femur).

Thus, in the description presented new ways of treating or preventing bone loss, increasing the mineral density of bone tissue and increasing the formation and deposition of bone tissue. Although the application is described in detail preferred embodiments of the invention, it should be understood that it can be made obvious variations without deviating from the essence and scope of the invention defined by the attached claims.

1. A method for identifying compounds that have the potential to increase the mineral density of bone tissue, which consists in the fact that the connection is brought into contact with 15-lipoxygenase and determine whether the connection activity against inhibition of 15-lipoxygenase.

2. The method according to claim 1, characterized in that the connection test using functional analysis, which allows to determine the effectiveness of the connection with respect to the formation of bone tissue.

3. The method according to claim 2, in which functional analysis is that the connection is brought into contact with human mesenchymal stem cells and determine di is ferentinou cells in osteoplastic cells.

4. The method according to claim 2, in which functional analysis lies in the fact that the compound is administered to an animal, except man, and measure the index of bone formation.

5. The method according to claim 4, in which the measured index is the mineral density of bone tissue.

6. The method according to claim 4, in which the index represents a biomechanical parameter of the bone.

7. The method according to claim 3, in which to determine the differentiation of cells carry out the analysis of alkaline phosphatase, an analysis of the calcium content, the analysis of total DNA of the drug or a combination of both.