Method of treating il-1beta-dependent diseases

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to methods of treating type 2 diabetes, insulin resistance, insulin hyposecretion, obesity, hyperglycaemia and hyperinsulinemia, involving administering an effective amount of an anti-IL-1β antibody or its fragment into an individual, as well as to using the anti-IL-1β antibody or its fragment in preparing a composition applicable for treating the above diseases or conditions.

EFFECT: group of inventions is effective in treating type 2 diabetes mellitus, insulin resistance, insulin hyposecretion, obesity, hyperglycaemia and hyperinsulinemia.

67 cl, 13 dwg, 5 tbl, 14 ex

 

In compliance with United States code (35 U. S. C. § 119) the present invention claims priority on provisional application U.S. No. 60/871046, filed December 20, 2006, No. 60/908389, filed on March 27, 2007, and No. 60/911033, filed April 10, 2007, in its entirety is included in this invention is in the form of links.

AREA of TECHNOLOGY

The invention relates to methods of treating and/or prophylaxis of type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and disease States characterized by insulin resistance. Such methods can be used to treat mammals suffering from type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and disease States characterized by insulin resistance, or to prevent their manifestations in subjects at risk.

The LEVEL of TECHNOLOGY

This invention is directed to methods for the treatment and/or prophylaxis of type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and disease States characterized by insulin resistance in mammals. Such methods can be used to treat mammals (e.g., humans) suffering from type 2 diabetes, obesity, hyperglycemia, g�peninsulae, type 1 diabetes, insulin resistance and disease States characterized by insulin resistance, or to prevent their manifestations in subjects at risk.

Diabetes mellitus is a metabolic disorder in humans, and its prevalence among the total population is about 1 percent (Foster, D. W., Harrison's Principles of Internal Medicine, Chap.114, pp.661-678, 10th Ed., McGraw-Hill, New York). The disease manifests itself in the form of a series of hormone-induced metabolic abnormalities that eventually lead to serious, long-lasting and geeky weakness complications affecting multiple organs, including the eyes, kidneys, nerves and blood vessels. From the point of view of the pathology of the disease is characterized by damage (pathological changes) of the basal membrane, which was demonstrated using electron microscopy. Diabetes mellitus can be divided into two sets of clinical symptoms: diabetes mellitus type 1 and diabetes type 2. Type 1 or insulin-dependent diabetes mellitus (IDDM), also called juvenile (juvenile) form of the disease, is a chronic autoimmune disease characterized by extensive loss of beta cells in the islets of Langerhans of the pancreas that produce insulin. As these cells are gradually destroyed�tsya, the amount of secreted insulin decreases, leading, ultimately, to hyperglycemia (abnormally high level of glucose in the blood), when the amount of insulin secreted falls below the normal level required to maintain normal levels of glucose in the blood. Although the exact trigger this immune response is unknown, in patients with IDDM there are high levels of antibodies to the proteins expressed in the beta cells of the pancreas. However, not all patients with high levels of these antibodies is developing IDDM.

Diabetics type 1, usually have very low or not available to measure the level of insulin in blood plasma at elevated glucagon. Regardless of what the exact etiology, most patients with type 1 diabetes, have circulating antibodies to their own cells of the pancreas, including insulin antibodies to the cytoplasm of cells of islets of Langerhans and the enzyme decarboxylase glutamic acid. Immune response directed specifically against the beta cells (cells that produce insulin), leading to type 1 diabetes. Widespread at the present time the treatment of patients suffering from type 1 diabetes is insulin injections and may also include changes in the diet to minimize hyperslices�Yu, resulting from a lack of natural insulin, which, in turn, is the result of damage to the beta cells. Nutrition also change, given the introduction of insulin in order to counteract the hypoglycemic effects of the hormone.

Type 2 diabetes (also called non-insulin-dependent diabetes mellitus (insd), Mature or adult form (maturity onset form, adult onset form) develops when the muscle cells, adipose tissue and liver are not adequately respond to the insulin. This inadequate response (called insulin resistance) may be due to a reduced number of insulin receptors on these cells, and dysfunction of signaling pathways inside the cells, or by both reasons. The beta cells initially compensate for this insulin resistance by producing more insulin. Over time, these cells lose the ability to produce the amount of insulin sufficient to maintain normal glucose levels, indicating a movement towards type 2 diabetes. Type 2 diabetes is caused by a combination of genetic factors and risk factors, including a diet high in fat, lack of physical activity and age (aging). More than 90% of diabetics suffer from type 2 diabetes, and this percentage continues to grow, becoming a major cause of mortality, morbidity and races�ODS on patient care worldwide (Amos, etc., Diabetic Med. 14:S1-85, 1997).

Type 2 diabetes is a complex disease, characterized by impaired metabolism of glucose and lipids. Usually there are violations of many parameters of metabolism, including increased levels of glucose in the blood plasma fasting levels of free fatty acids and triglycerides, and reduce the ratio of high density lipoprotein to low-density lipoprotein (HDL/LDL). As mentioned above, it is believed that one of the fundamental, underlying causes of diabetes is to improve insulin resistance of peripheral tissues, mainly muscle tissue and fat tissue. The causes of type 2 diabetes is not entirely clear. It is believed that the resistance of target tissues to the action of insulin, and decreased insulin secretion ("the failure of β-cells"). The main insulin-reactive tissues that support glucose homeostasis, are liver, in which insulin stimulates glycogen synthesis and inhibits gluconeogenesis; the muscles in which insulin stimulates glucose uptake and glycogen stimulates glucose uptake and inhibits lipolysis. Thus, the effects of the diabetic condition are increased levels of glucose in the blood, which can lead to glucose-madirovalo cellular toxicity and subsequent disease (nephropathy, neuropathy, p�tinopai, etc.). Insulin resistance is rigidly correlated with the development of type 2 diabetes.

Currently, there are various pharmacological approaches to the treatment of type 2 diabetes (Scheen, etc., Diabetes Care, 22(9):1568-1577, 1999). They operate in different ways: 1) sulfonylureas (e.g., glimepiride, glisenti, sulfonylurea, AY31637) significantly stimulated insulin secretion; 2) biguanides (e.g., Metformin) act by stimulating the utilization of glucose by reducing glucose production by the liver and reducing intestinal glucose output; 3) inhibitors of alpha-glucosidase (e.g., acarbose, miglitol) slow the breakdown of carbs and consequently absorption from the intestine and reduce afternoon hyperglycemia; 4) thiazolidinediones (e.g. troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP 221, MK-0767, ciglitazone, aleglitazar (adaglitazone), CLX 0921, darglitazone, MS 92768, VM 152054) enhance the action of insulin, stimulating, thus, glucose utilization in peripheral tissues; 5) glucagon-like peptides, including inhibitors of the dipeptidyl peptidase-4 (DPP4) (e.g., sitagliptin); and 6) insulin stimulates glucose utilization by tissues and inhibits the production of glucose by the liver. The above pharmacological approaches can� to be used separately or in combination therapy. However, each approach has its limitations and has adverse side effects. Over time, a significant percentage of patients suffering from type 2 diabetes, no longer respond to these drugs. Insulin therapy is usually appointed after diet, exercise, and oral administered drugs, is unable to adequately control glucose levels in the blood. The disadvantages of insulin therapy are the need for injection of medications, the potential for hypoglycaemia and weight gain.

IL-1β is a proinflammatory cytokine that is secreted by a number of different cells, including monocytes and macrophages. When you release that is part of the inflammatory process, IL-1β causes a number of biological effects, mainly by inducing the formation of other inflammatory mediators such as corticotropin, platelet factor-4, prostaglandin E2 (PGE2), IL-6 and IL-8. IL-1β induces both local and systemic inflammatory effects through the activation of the receptor of IL-1, detected in almost all cell types. The family of cytokines interleukin-1 (IL-1) is involved in a number of disease States. The members of the family of IL-1 include IL-1α, IL-1β and IL-1Ra. Despite the fact that they share the ability to bind with the receptors of IL-1 (IL-1R1 and IL-1R2), all of these cytokines are �depended, expressed different genes and have different primary amino acid sequence. Moreover, the physiological actions of these cytokines may be different from each other. Experiments indicating the undoubted involvement of IL-1β in diabetes, have been published.

Maedler et, J Clin Invest (2002) 110:851-860 suggested that when type 2 diabetes mellitus chronic hyperglycemia may have a detrimental effect on β-cells of the pancreas, causing weakened insulin secretion, and noticed that IL-1β is a proinflammatory cytokine acting in the course of development of autoimmune type 1 diabetes, and inhibits beta-cell function. In particular, they tested the hypothesis that IL-1β can mediate harmful effects of high glucose levels. Treatment floretina animals suffering from diabetes, normalized levels of plasma glucose and prevented the expression of IL-1β β-cells. It was concluded that this involves an inflammatory process in the pathogenesis of glucotoxicity with type 2 diabetes. They also identified IL-1β/NF-KB the path as a goal for conservation of mass and functions of β-cells in this state.

Donath, etc., J Mol med (2003) 81:455-470 noticed the obvious importance of IL-1β for the path to apoptosis of β-cells in pancreatic islets leading to insulin deficiency and diabetes, and suggested approaches based on protivovospalitel�your therapy aimed at blocking the apoptosis of β-cells in diabetes type 1 and 2.

WO 2004/002512 directed to the use of antagonists of the receptor of IL-1 (IL-1Ra) and/or pyrrolidinedithiocarbamate (PDTC) for the treatment or prophylaxis of type 2 diabetes. However, the usual dose proposed for therapeutic use of the polypeptide of IL-Ra in the treatment of type 2 diabetes (injections every 24 hours), can lead to problems associated with the patient's consent, reducing, thus, the effectiveness of this method of treating and/or limiting its desirability. Thus, the need for effective means for the treatment of type 2 diabetes remained, especially for those who do not want injections.

Larsen and others, New England Journal of Medicine (2007) 356:1517-1526 describes the use of the drug anakinra, which is a recombinant receptor antagonist IL-1 (IL-1Ra), for the treatment of diabetes mellitus type 2. However, the introduction of 100 mg anakinra once a day, daily for 13 weeks, may cause problems with the patient's consent, reducing, thus, the effectiveness of this method of treating and/or limiting its desirability. Thus, the need for effective means for the treatment of type 2 diabetes remained, especially for those who don't want frequent (e.g., daily) injections.

US 2005/0256197 and US 2005/0152850 directed to a method for facilitating control of metaboli�mA (for example, glucose) in a subject (e.g., in a subject with diabetes), comprising decreasing the level of IL-1β in the liquid of the gingival sulcus (gingival crevicular fluid) from the subject so that the level of circulating TNF is decreased, in particular, through the use of anti-inflammatory agent, such as a mouth rinse anti-inflammatory Ketorolac.

Obesity is a chronic disease that is widespread and is not only a social stigma (stigma), but also with decreased life span and numerous medical problems, including adverse psychological development, dermatological disorders such as infections, varicose veins, exercise intolerance, diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia and coronary heart disease (Rissanen and others, British Medical Journal, 301: 835-837, 1990). Obesity is clearly correlated with insulin resistance and diabetes in experimental animals and in humans. Indeed, obesity and insulin resistance, the latter of which is generally accompanied by hyperinsulinemia or hyperglycemia, or both, are the signs of type 2 diabetes. In addition, type 2 diabetes is associated with increased two to four times the risk of coronary artery disease. Despite decades of research with these�raznyh health problems, the etiology of obesity and insulin resistance remains unknown.

Insulin resistance is associated with a number of disease States and conditions and there is approximately 30-40% of individuals not suffering from diabetes. These disease States include, but are not limited to only these: prediabetes and the metabolic syndrome (also called syndrome of insulin resistance). Prediabetes is a condition of abnormal glucose tolerance, characterized by either decreased glucose tolerance (IGT) or reduced levels of fasting glucose (IFG). Patients with pre-diabetes are insulin-registername and are at high risk for further progression and explicit manifestations of type 2 diabetes. Metabolic syndrome comprises a group of interrelated characteristics, including but not limited to only these: hyperinsulinemia, abnormal glucose tolerance, obesity, redistribution of fat in the abdominal region or the upper part of the body, hypertension, defibrinated and a dyslipidemia characterized by high triglycerides, low high density cholesterol and low density particles of low density lipoprotein. Insulin resistance associated with each of these characteristics, suggesting that the metabolic syndrome and resistance to Insa�inu closely related. The detection (diagnosis) metabolic syndrome is a strong risk factor for future development of type 2 diabetes, and accelerated development of atherosclerosis, leading to heart attacks, strokes and peripheral vascular disease. It has been shown that inflammatory cytokines, including IL-1, mediashout inflammation within adipose tissue, which apparently involved in the resistance of adipocytes to insulin (Trayhurn, etc., Br. J. Nutr. 92:347-355, 2004; Wisse, J. Am. Soc. Nephrol. 15:2792-2800, 2004; Fantuzzi, J. Allergy Clin. Immunol. 115:911-919, 2005; Matsuzawa, FEBS Lett. 580:2917-2921, 2006; Greenberg, etc., Eur J. Clin. Invest. 32 Suppl. 3:24-34, 2002; Jager et, Endocrinology 148:241-251, 2007). Adipocytes are cells storing fat and secreting adipokines (i.e., the subpopulation of cytokines), and are a major component of adipose tissue. Macrophages are inflammatory cells and the main producers of inflammatory cytokines, IL-1, TNF-α and IL-6, also exist within adipose tissue, especially inflamed adipose tissue associated with obesity (Kern, etc., Diabetes 52:1779-1785, 2003). Previously it was known that TNF-α and IL-6 reduce the sensitivity of adipocytes to insulin stimulation (i.e. insulin resistance).

In connection with the difficulties of modern methods of treatment, type 2 diabetes and other symptoms such as listed above, you must replace them with new ones or add to the available pharmaceutical approaches�. The present invention includes a method of treating type 2 diabetes. In addition, the present invention also includes a method of treating obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance and disease States characterized by insulin resistance. Disclosed here include, for example, the introduction of antibodies to IL-1β or the fragment of the antibody. The ways in which the ligand IL-1β bind to the antibodies, particularly antibodies with high affinity, have advantages over other potential methods of treatment, such as those that use receptor antagonists of IL-1β (e.g., IL-1Ra, Anakinra, Kineret®). The problem of therapeutic treatments based on receptor antagonists, IL-1β, is the need to link a large number of receptors, which is a difficult task, because such receptors are widely represented in all cells except red blood cells (Dinarello, Curr. Opin. Pharmacol. 4:378-385, 2004). In most immunomediated diseases, such as described in the present invention, the amount of cytokine IL-1β, measured in body fluids or associated with activated cells is relatively small. Therefore, the method of treatment and/or prophylaxis, directly aimed at the ligand IL-1β, is an excellent strategy, especially with the introduction of antibodies to IL-1β,�laduma high affinity.

Disclosure of the INVENTION

This invention is directed to methods for the treatment and/or prophylaxis in a mammal of type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, type 1 diabetes, insulin resistance and/or disease States characterized by insulin resistance. Such methods can be used to treat mammals (e.g., humans) suffering from, or at risk of developing diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, insulin resistance and/or disease States characterized by insulin resistance. The methods can also be used to prevent symptoms (development) of type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, insulin resistance and disease States characterized by insulin resistance, subjects are at risk.

On the one hand, the present invention is a method of treatment for people of diseases or disease States selected from the group including: type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion, type 1 diabetes and insulin resistance. The method comprises administering the antibody to IL-1β or frame�the people. In a preferred embodiment of the invention, the disease or condition is selected from the group including type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance. Preferably, the disease or condition is a type 2 diabetes, obesity, reduced insulin secretion or insulin resistance. More preferably, the disease or condition is a type 2 diabetes mellitus, obesity or insulin resistance. Most preferably, the disease or condition is a type 2 diabetes. According to one embodiment of the invention, the method does not cause amplification (increasing) cardiovascular disease or condition. In certain embodiments of the invention, the antibody or a fragment thereof is used for the treatment of two or more of the above diseases or conditions in a single patient (e.g., human).

According to another aspect of the invention provides a method of treating or prophylaxis of a disease or condition by administering antibodies to IL-1β or the fragment, wherein the disease or condition is a pre-diabetes, dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome or a disease state. According to another aspect, a method of reducing�AET or prevents associated with type 2 diabetes mellitus complication or condition, selected from the group including retinopathy, renal failure, disease of the cardiovascular system and the treatment of wounds in humans; the method comprises administering the antibody to IL-1β or its fragment to the people. In certain embodiments of the invention, the antibody or fragment is used for the treatment of two or more of the above diseases or conditions in a single patient (e.g., human). In another embodiment, the antibody or fragment is used for the treatment of renal failure (e.g., kidney disease), which can be a consequence of the state that are not type 2 diabetes. In another embodiment, the antibody or fragment is used to reduce the level of C-reactive protein (CRP) in a subject with elevated CRP levels.

According to another aspect of the invention, claimed the antibody to IL-1β or fragments of such antibodies for use in the treatment or prevention of the aforementioned diseases or conditions. In addition, in the invention of the claimed antibodies to IL-1β or fragments of such antibodies for use in the treatment or prevention of diseases or conditions selected from the group including type 2 diabetes, obesity, reduced insulin secretion and insulin resistance. In another aspect, the invention includes antibodies to IL-1β or fragments of such antibodies for the ISP�of litvania in the treatment or prevention of type 2 diabetes.

According to another aspect, the invention includes pharmaceutical compositions containing the antibodies to IL-1β or fragments of such antibodies, and optionally at least one pharmaceutically acceptable excipient, for use in the treatment or prevention specified in the invention, diseases or conditions. In addition, the invention includes pharmaceutical compositions containing the antibodies to IL-1β or fragments of antibodies, and optionally at least one pharmaceutically acceptable excipient, for use in the treatment or prevention of diseases or conditions selected from the group including type 2 diabetes, obesity, reduced insulin secretion and insulin resistance. In another aspect the invention includes pharmaceutical compositions containing the antibodies to IL-1β or fragments of such antibodies, and optionally at least one pharmaceutically acceptable excipient, for use in the treatment or prevention of type 2 diabetes.

Antibodies to IL-1β or fragments of such antibodies used in the methods according to the present invention, typically have high affinity binding to IL-1β. In preferred embodiments of the invention, the dissociation constant of binding of the antibody or fragment of an antibody to IL-1β with�is about 10 nm or less, about 5 nm or less, about 1 nm or less, about 500 PM or less, about 250 PM or less, about 100 PM or less, about 50 PM or less or about 25 PM or less. In especially preferred embodiments of the invention, the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 100 PM or less, about 50 PM or less, about 10 PM or less, about 5 PM or less, about 3 PM or less about 1 PM or less, about 0.75 PM or less, about 0.5 PM or less, about 0.3 PM or less, about 0.2 PM or less or about 0.1 PM or less. In especially preferred embodiments of the invention, the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 10 PM or less.

According to another aspect of the invention, the antibody to IL-1β or the fragment of the antibody is a neutralizing antibody. According to another aspect, the antibody to IL-1β or the fragment of the antibody binds to an epitope of IL-1β in such a way that the bound antibody or fragment substantially permits the binding of IL-1β to its receptor I, IL-1 (IL-1RI). According to another aspect, the antibody to IL-1β or the fragment of the antibody binds to IL-1β, but not markedly inhibits the binding of the bound IL-1β with its receptor I, IL-1 (IL-1RI). According to another aspect, the antibody or fragment� antibody does not bind to IL-1α, IL-1R or IL-1Ra to the extent available for the detection/detection. According to another aspect of the present invention, the antibody or antibody fragment binds with an epitope contained in the sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO. 1). According to another aspect of the present invention, the antibody or antibody fragment binds to an epitope containing Glu64 of interleukin IL-1β. According to another aspect of the invention, the antibody or antibody fragment binds to amino acids 1-34 of the N-end of IL-1β. Preferably, the antibody or fragment of antibody is designed for the person (human engineered), humanized or human.

According to another aspect, the invention includes a method of treatment which is manifested in humans the symptoms of the aforementioned diseases or conditions or symptoms risk of developing such diseases or conditions (e.g., type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion, insulin resistance). The method comprises administering the antibody to IL-1β or its fragment to the people in one or more doses.

According to another aspect of the present invention is claimed a method of treating disease in humans or condition selected from the group including: type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and resistance in�uline. The method comprises administering the antibody to IL-1β or its fragment people, and after administration of the initial dose of the antibody to IL-1β or the fragment of the antibody is administered one or more subsequent doses. According to one embodiment of the invention after administration of the initial dose of the antibody or antibody fragment is administered two or more subsequent doses. According to another embodiment of the invention, after administration of the initial dose of the antibody or antibody fragment is administered one or more subsequent doses, and these one or more subsequent doses largest are about the same or less than the initial dose. In another embodiment of the invention after administration of the initial dose of the antibody or antibody fragment is administered one or more subsequent doses, wherein at least one of the subsequent doses in magnitude is greater than the initial dose.

According to one embodiment of the invention is administered two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more subsequent doses of the antibody. In another embodiment of the invention the introduction of the initial dose and each one or more subsequent doses are separated from each other by a period at least about two weeks, at least about t�and weeks at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, or at least about twelve months.

In another embodiment, the antibody or fragment is administered in the form of one or more doses of 5 mg of antibody or fragment per kg or less, 3 mg of antibody or fragment per kg or less, 2 mg of antibody or fragment per kg or less, 1 mg of antibody or fragment per kg or less, 0.75 mg of antibody or fragment per kg or less, 0.5 mg of antibody or fragment per kg or less, 0.3 mg of antibody or fragment per kg or less 0.1 mg of antibody or fragment per kg or less, or 0.03 mg of antibody or fragment per kg or less. Preferably, in each of the above embodiments of the invention, the antibody or fragment is administered in the form of one or more doses of at least 0.01 mg of antibody or fragment per kg, at least 0.03 mg of antibody or fragment per kg, at least 0.05 mg of the antibody or fragment per kg, or at the extreme�th least 0.09 mg of antibody or fragment per kg. of the above dosages are given in mg (antibody or fragment per kg (mass of the individual, which is supposed to treat).

In another embodiment, the initial dose and one or more subsequent doses of antibody or fragment are each from about 0.01 mg/kg to about 10 mg of antibody per kg, from about 0.05 to about 5 mg of antibody per kg, from about 0.05 mg/kg to about 3 mg of antibody per kg, from about 0.1 mg antibody per kg to about 3 mg/kg, from about 0.1 mg/kg to about 1 mg of antibody per kg, from about 0.1 mg/kg to about 0.5 mg of antibody per kg, from about 0.3 mg/kg to about 5 mg of antibody per kg, from about 0.3 mg/kg to about 3 mg of antibody per kg, from about 0.3 mg/kg to about 1 mg of antibody per kg, from about 0.5 mg/kg to about 5 mg of antibody per kg, from about 0.5 mg/kg to about 3 mg of antibody per kg, from about 0.5 mg/kg to about 1 mg of antibody /kg, from about 1 mg/kg to about 5 mg of antibody per kg or from about 1 mg/kg to about 3 mg of antibody per kg. In certain embodiments of the invention is administered two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more subsequent doses of the antibody. The above dosages are given in mg (anti�ate or fragment) in kg (weight of the individual, which is supposed to treat). This applies to all the following dosages.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment in the form of an initial dose of approximately 5 mg of antibody or fragment per kg or less, and a plurality of subsequent doses of the antibody or fragment of about the same or less than the initial dose, with subsequent doses separated by a time interval, at least 2 weeks.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment in the form of an initial dose of approximately 3 mg of antibody or fragment per kg or less, and a plurality of subsequent doses of the antibody or fragment of about the same or less�e, than the initial dose, with subsequent doses separated by a time interval of at least 2 weeks.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment in the form of an initial dose of approximately 1 mg of antibody or fragment per kg or less, and a plurality of subsequent doses of the antibody or fragment of about the same or less than the initial dose, with subsequent doses separated by a time interval, at least 2 weeks.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment in the form of an initial dose of approximately 0.5 mg of the antibody or fragment per kg or less, and a plurality of subsequent doses of antibody or fragment in the amount of about� the same or less than the initial dose, with subsequent doses separated by a time interval of at least 2 weeks.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment in the form of an initial dose of approximately 0.3 mg of the antibody or fragment per kg or less, and a plurality of subsequent doses of the antibody or fragment of about the same or less than the initial dose, with subsequent doses separated by a time interval, at least 2 weeks.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment in the form of an initial dose of approximately 0.1 mg of antibody or fragment per kg or less, and a plurality of subsequent doses of antibody or fragment in the number example�about the same or less than the initial dose, with subsequent doses separated by a time interval of at least 2 weeks.

Preferably, in the above embodiments of the invention in which the antibody or fragment is administered as an initial dose and a plurality of subsequent doses, the dose of the antibody or fragment is at least 0.01 mg of antibody or fragment per kg, at least 0.03 mg of antibody or fragment per kg, at least 0.05 mg of the antibody or fragment per kg or at least 0.09 mg of antibody or fragment per kg.

According to another aspect of the present invention, the antibody or fragment is administered as a fixed dose, independent of dose ratio and weight of the subject. According to one embodiment of the invention, the antibody or fragment is administered in the form of one or more fixed doses comprising 1000 mg or less of antibody or fragment, 750 mg or less of antibody or fragment, 500 mg or less of antibody or fragment, 250 mg or less of antibody or fragment, 100 mg or less of antibody or fragment, or about 25 mg or less of antibody or fragment. In another embodiment, the antibody or fragment is administered in the form of one or more fixed doses comprising at least about 1 mg of antibody or fragment, at least about 5 mg of antibody or fragment �Lee, at least about 10 mg of antibody or fragment.

In certain embodiments of the invention, the fixed dose is about 1 mg to about 10 mg, from about 1 mg to about 25 mg, from about 10 mg to about 25 mg, from about 10 mg to about 50 mg, from about 10 mg to about 100 mg, from about 25 mg to about 50 mg, from about 25 mg to about 100 mg, from about 50 mg to about 100 mg, from about 50 mg to about 150 mg, from about 100 mg to about 150 mg, from about 100 mg to about 200 mg, from about 150 mg to about 200 mg, from about 150 mg to about 250 mg, from about 200 mg to about 250 mg, from about 200 mg to about 300 mg, from about 250 mg to about 300 mg, from about 250 mg to about 500 mg, from about 300 mg to about 400 mg, from about 400 mg to about 500 mg, from about 400 mg to about 600 mg, from about 500 mg to about 750 mg, from about 600 mg to about 750 mg, from about 700 mg to about 800 mg, from about 750 mg to about 1000 mg. In a preferred embodiment of the invention, the fixed dose is selected from the group comprising from about 1 mg to about 10 mg, from about 1 mg to about 25 mg, from about 10 mg to about 25 mg, from about 10 mg to about 100 mg, from about 25 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150 mg to �rimero 200 mg, from about 200 mg to about 250 mg.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering to the people therapeutically effective amount of an antibody to IL-1β or its fragment, wherein after administration of the initial dose of the antibody or antibody fragment is administered one or more subsequent doses, wherein the allowable decrease in the concentration of the indicated antibody or fragment antibodies in the plasma of people below the level of about 0.1 μg/ml for a time period more about 1 week or less for about 6 months between administrations during a course of treatment specified initial dose and one or more subsequent doses. According to one embodiment of the invention, the permissible reduction in the concentration of the indicated antibody or fragment antibodies in the plasma of people below the level of about 0.07 μg/ml to about 0.05 μg/ml to about 0.03 μg/ml or about 0.01 μg/ml for a time period greater than about 1 week or less for about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, about 3 weeks or about 2 weeks between th�mi. According to one embodiment of the invention these levels in plasma are related to the levels measured for the individual that treating the antibody or antibody fragment in accordance with the invention. According to one embodiment of the invention, such an individual may be a patient suffering from the previously mentioned diseases, such as type 2 diabetes.

The invention provides that the antibody to IL-1β or the fragment used in accordance with the described methods, may be entered in any (listed here) dosages with any number (as listed) subsequent doses and in all (listed) intervals between doses, and any of these dosages, the number of subsequent doses and intervals between injections can be combined with each other in alternative modes to adjust therapeutic use. In certain embodiments of the invention, one or more subsequent doses are almost the same size or smaller than the entered dose. In another embodiment, one or more subsequent doses are almost the same size or larger than the initial dose. Preferably, the antibody to IL-1β or the fragment is administered by subcutaneous, intramuscular or intravenous injection. The invention should�trivet, each dose of the antibody or fragment may be administered in one or in several places.

According to another aspect of the invention includes a method of treatment or prophylaxis of a disease or condition in humans, using an antibody to IL-1β or the fragment of the antibody, wherein the disease or condition is type 2 diabetes, and the dose of the antibody or fragment is sufficient to achieve at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5 or at least 3.0 percentage improvement in the level of hemoglobin A1c. According to one embodiment of the invention, these parameter values relate to the values defined for the individual that treating the antibody or antibody fragment in accordance with the invention. According to one embodiment of the invention, such an individual may be a patient suffering from these diseases, such as type 2 diabetes.

In a preferred embodiment of the invention to improve the level of hemoglobin A1c is sufficient to satisfy regulatory principles for adoption (approval) of therapeutic agents in the treatment of type 2 diabetes. Methods for the determination of hemoglobin A1c are well known in the art. The invention provides that the dose of the antibody or fragment is sufficient to �of stijene improve hemoglobin A1c, may include any of the dosages described previously, any (described earlier), the number of subsequent doses and any (described earlier), the intervals between injections, as well as any combination of the dosages, the number of subsequent doses and intervals between doses described antibody or its fragment. In addition, improvement of hemoglobin A1c may occur in time, at least after about 1 month, about 2 months, about 3 months, about 4 months or about 5 months, and preferably after about 6 months or more, after about 7 months or more, after about 8 months or more, after about 9 months or more, after about 10 months or more, after about 11 months or more, or about 12 months or more after initial insertion of one or more doses of the antibody or fragment.

According to another aspect of the above-mentioned methods of treatment or prevention of type 2 diabetes, the method allows to achieve at least one of the following changes: reduce the level of sugar in blood glucose, reduce insulin resistance, reduce hyperinsulinemia, improve glucose tolerance, reduce C-reactive peptide (CRP), increase insulin secretion and reduce hyperglycemia, reducing the need for medical treatment �of abeta, reduction of body mass index (BMI), change in AUC glucose / C-peptide of insulin, lower glucose levels in the urine, reduction of acute-phase reactants, reducing the level of lipids in serum in improving the lipid profile in light of the risk of cardiovascular disorders. Ways to identify any such modifications are well known in the art. In addition, the invention provides that the achievement of one of the mentioned changes can occur in time, at least after about 1 month, about 2 months, about 3 months, about 4 months, or about 5 months, and preferably after about 6 months or more, about 7 months or more, about 8 or more months, about 9 months or more, about 10 or more months, about 11 months or more, or about 12 months or more after initial insertion of one or more doses of the antibody or fragment.

According to another aspect of the inventive method reduces or prevents associated with type 2 diabetes mellitus complication or condition selected from the group including retinopathy, renal failure, cardiovascular system and wound healing, wherein the method comprises administering the antibody to IL-1β or its fragment to the people. According to one embodiment of the invention, the complication or state�tion is a disease of the cardiovascular system, and disease of the cardiovascular system is atherosclerosis or peripheral vascular disease. In another embodiment of the invention, the complication or condition is the healing of wounds, and the specified wound healing is a diabetic ulcer. According to another aspect, the method prevents or suspends (lays) end-stage renal failure or diabetic neuropathy. According to one embodiment of the invention, the antibody to IL-1β or the fragment is administered in combination with at least one other generally accepted from a medical point of view, a way of treating a disease, condition or complication. In another embodiment of the invention, at least one other generally accepted from a medical point of view, the method of treating a disease, condition or complication is reduced or suspended, and treatment of the antibody to IL-1β or the fragment is carried out in a mode of regular dosages. In another embodiment of the invention, at least one other generally accepted from a medical point of view, the method of treating a disease, condition or complication reduce or suspend, and treatment of the antibody to IL-1β or the fragment cut. In another embodiment of the invention, at least one with another common �medical point of view, the method of treatment of the disease, condition or complication reduce or suspend, and treatment with antibody to IL-1β or the fragment to amplify (increase). In another embodiment of the invention, at least one other generally accepted from a medical point of view, the method of treating a disease, condition or complication remain at the same level, and treatment with antibody to IL-1β or the fragment reduce or suspend. In another embodiment, at least one other generally accepted from a medical point of view, the method of treating a disease, condition or complication and treatment with antibody to IL-1β or the fragment are reduced or suspended.

According to another aspect of the invention claimed a method of reducing the amount of C-reactive protein in a subject, comprising administering the antibody to IL-1β or its fragment to a subject. According to one embodiment of the invention, the antibody or antibody fragment is administered in the form of one or more doses comprising 1 mg of antibody or fragment per kg or less, 0.75 mg of antibody or fragment per kg or less, 0.5 mg of antibody or fragment per kg or less, 0.3 mg of antibody or fragment per kg or less 0.1 mg of antibody or fragment per kg or less, or 0.03 mg of antibody or fragment per kg or less. Preferably, the antibody or fragment is administered in the form of one or more doses, SOS�alausa, at least 0.01 mg of antibody or fragment per kg, at least 0.03 mg of antibody or fragment per kg, at least 0.05 mg of the antibody or fragment per kg, or at least 0.09 mg of antibody or fragment per kg. In another embodiment, the antibody or fragment is administered irrespective of the relationship between dose size and weight of the subject, in the form of one or more fixed doses comprising 500 mg or less of antibody or fragment, 250 mg or less of antibody or fragment, 100 mg or less of antibody or fragment, or about 25 mg or less of antibody or fragment. Preferably, the antibody or fragment is administered in the form of one or more fixed doses comprising at least about 1 mg of antibody or fragment, at least about 5 mg of antibody or fragment, or at least about 10 mg of antibody or fragment. In another embodiment, the antibody or antibody fragment binds to IL-1β with a dissociation constant of about 500 PM or less, 250 PM or less, about 100 PM or less, about 50 PM or less or about 25 PM or less, about 10 PM or less, about 5 PM or less, about 3 PM or less about 1 PM or less, about 0.75 PM or less, about 0.5 PM or less, about 0.3 PM or less, about 0.2 PM or less or about 0.1 PM or less. In dragomiresti of the invention after administration of the initial dose of the antibody is administered one or more subsequent doses separated from each other by a period at least about two weeks, at least about three weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, or at least about twelve months. In another embodiment, the invention is claimed the method of reducing the amount of C-reactive protein in a subject, wherein the subject is suffering from a renal disease (e.g., chronic kidney disease, renal failure). In another embodiment, the subject suffers from type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, insulin resistance and/or disease States characterized by insulin resistance. In another embodiment, the subject suffers from a disease or condition of pre-diabetes, dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome or painful SOS�of aania. The above dosages are given in mg (antibody or fragment per kg (of body weight of the individual, which is supposed to treat). The introduction of antibodies or fragments, characterized by the above dissociation constants, can be carried out by any of the listed doses and with any interval between doses dose (when administered two or more doses).

According to another aspect of the inventive methods are carried out jointly, at least one additional treatment method, and this additional method of treatment comprises administering at least one pharmaceutical composition containing the active agent, which is an antibody to IL-1β or the fragment. In another aspect of the inventive methods to prevent or reduce the need to use at least one additional treatment method, and this additional method of treatment comprises administering at least one pharmaceutical composition containing the active agent, which is an antibody to IL-1β or the fragment. According to another aspect, the inventive methods reduce the number, frequency or duration of at least one additional treatment method, and this additional method of treatment comprises administering at least one pharmaceutical composition, with�erasa active agent, not which the antibody to IL-1β or the fragment. According to one embodiment of the invention, at least one pharmaceutical composition comprising an active agent, which is an antibody to IL-1β or the fragment is selected from the group comprising sulfonylurea, meglitinide, biguanide, an inhibitor of alpha glucosidase, thiazolidinedione, a peptide similar to glucagon, and insulin. In another embodiment, the active agent is a sulfonylurea. In another embodiment, the active agent is meglitinide. In another embodiment, the active agent is biguanide. In another embodiment, the active agent is an inhibitor of alpha-glucosidase. In another embodiment, the active agent is thiazolidinedione. In another embodiment, the active agent is a peptide similar to glucagon. In another embodiment, the active agent is insulin. In another embodiment of the invention, at least one pharmaceutical composition comprising an active agent, includes two active agent. According to one embodiment of the invention, the two active agent represent sulfanilate�inu and biguanid. In another embodiment, two active agent represent thiazolidinedione and biguanide. In another embodiment, the treatment of at least one active agent retains at the same level. In another embodiment of the invention, the treatment of at least one active agent is reduced or suspended, while treatment with antibodies to IL-1β or its fragment remain at the same constant level of dosage regimen. In another embodiment of the invention, the treatment of at least one active agent is reduced or suspended and treatment with antibodies to IL-1β or the fragment cut. In another embodiment of the invention, the treatment of at least one active agent is reduced or suspended, while treatment with antibodies to IL-1β or the fragment is increased. In another embodiment, the treatment of at least one active agent remain at the same level, and treatment with antibodies to IL-1β or the fragment reduce or suspend. In another embodiment, the treatment of at least one active agent and treatment with antibodies to IL-1β or its fragment reduce or suspend.

According to another aspect of the invention includes a method of treatment for people �of zabolevaniya or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering a therapeutically effective amount of an antibody to IL-1β or its fragment to the people, wherein after administration of the initial dose of the antibody or antibody fragment is administered one or more subsequent doses, wherein the concentration of the antibody or fragment of antibody in the plasma of people support at the level of at least about 0.03 μg/ml, at least about 0.05 μg/ml, at least about 0.08 μg/ml, at least about 0.1 μg/ml, at least about 0.15 μg/ml, at least about 0.2 μg/ml, at least about 0.25 μg/ml, at least about 0.3 μg/ml, at least about 0.4 μg/ml, at least about 0.5 μg/ml, at least about 0.6 μg/ml, at least about 0.8 μg/ml, at least about 1 μg/ml, at least about 1.5 μg/ml, at least about 2 μg/ml, at least about 3 μg/ml, at least about 4 μg/ml or at least about 5 μg/ml during the course of treatment with the specified initial dose and one or more subsequent doses. According to one embodiment of the invention these levels in plasma are related to the levels specified for the individual undergoing treatment antibody or fragment�ntom in accordance with the invention. According to one embodiment of the invention, such an individual may be a patient suffering from one of the following diseases, such as type 2 diabetes.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering a therapeutically effective amount of an antibody to IL-1β or its fragment to the people, thus the introduction of antibodies to IL-1β or its fragment people leads to decreased production of one or more gene products selected from the group including leptin, resistin, visfatin, RANTES, IL-6, MCP-1, PAI-1, protein acylation stimulating (ASP), SAA3, pentraxin-3, factor inhibiting macrophage migration (MIF), IL-1RA, IL-12, IL-8 and TNF-α. According to one embodiment of the invention, the introduction of antibodies to IL-1β or its fragment people leads to decreased production of one or more gene products selected from the group including leptin, resistin and visfatin. In another embodiment of the invention, the introduction of antibodies to IL-1β or its fragment to the people leads to a decrease in expression of one or more gene products selected from the group comprising MCP-1, RANTES, IL-6, TNF-α and ne�tracksin-3. In another embodiment, the decrease of production of one or more gene products occurs from adipose tissue. In another embodiment, the decrease is recorded in the blood of people.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering a therapeutically effective amount of an antibody to IL-1β or its fragment to the people, thus the introduction of antibodies to IL-1β or its fragment men leads to increased secretion of adiponectin. According to one embodiment of the invention the increase in the secretion of adiponectin comes from adipose tissue. In another embodiment, the increase is detected in the blood of people.

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering a therapeutically effective amount of an antibody to IL-1β or its fragment people�m, according to the analysis of inhibition of IL-1β in whole blood of the people, allowing to determine induced IL-1β production of IL-8, an antibody to IL-1β or the fragment is characterized by a lower value IC50than the receptor antagonist IL-1β. According to one embodiment of the invention according to the analysis of inhibition of IL-1β in whole blood of the people, allowing to determine induced IL-1β production of IL-8, an antibody or fragment characterized IC50whose magnitude is less than about 90%, 80%, 70%, 60%, 50% the value of the IC50for the receptor antagonist IL-1β. According to another embodiment of the invention, the antibody or fragment characterized IC50whose magnitude is less than about 40%, 30%, 20%, 10% the value of the IC50for the receptor antagonist IL-1β, according to the analysis of inhibition of IL-1β in whole blood of the people, allowing to determine induced IL-1β production of IL-8). In a preferred embodiment of the invention, the antibody or fragment characterized IC50whose magnitude is less than about 8%, 5%, 4%, 3%, 2%, 1% the value of the IC50for the receptor antagonist IL-1β, according to the analysis of inhibition of IL-1β in whole blood of the people, allowing to determine induced IL-1β production of IL-8). According to one embodiment of the invention the IL1β receptor antagonist is anakinra (i.e. Kineret, Kineret®).

According to another aspect of the invention includes a method of treating disease in humans or condition selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering a therapeutically effective amount of an antibody to IL-1β or its fragment people, according to research described by Economides et, Nature Med., 9:47-52 (2003), included in the invention in the form of a link, an antibody or a fragment thereof in vivo inhibit stimulated IL-1β release of IL-6 in mice compared to the control antibody. According to one embodiment of the invention, the antibody or fragment in vivo inhibits stimulated IL-1β release of IL-6 in mice of at least about 10%, 20%, 30%, 40%, 50% compared with the control antibody. According to another embodiment of the invention, the antibody or fragment in vivo inhibits stimulated IL-1β release of IL-6 in mice of at least about 60%, 70%, 80%, 90%, 95% compared with the control antibody. According to one embodiment of the invention, the control antibody is an isotype antibody as a control.

According to another aspect, the invention includes a method of treating disease in humans or condition selected�about the group, including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance, wherein the method comprises administering a therapeutically effective amount of an antibody to IL-1R or its fragment to the people, in this case, the antibody or a fragment thereof inhibits induced Staphylococcus epidermidis cytokine production in whole blood compared with the control, in which the antibody was not used. According to one embodiment of the invention, the antibody or fragment exert stronger inhibitory effect on induced Staphylococcus epidermidis cytokine production in whole blood of people, at least approximately 10%, 20%, 30%, 40%, 50% compared with the control. According to another embodiment of the invention, the antibody or fragment has a stronger inhibitory effect on induced Staphylococcus epidermidis cytokine production in whole blood of people, at least approximately 60%, 70%, 80%, 90%, 95% compared with the control. According to one embodiment of the invention inhibiting cytokines are IL-1β, IL-1a, IL-6, IL-8, IL-1Ra, TNFa or IFNγ.

According to another aspect of the invention includes the use of antibodies to IL-1β or its fragment with a smaller value of IC50than the receptor antagonist IL-1β, according to the analysis of inhibition of IL-1β in whole blood of the people, makes it that�known to determine induced IL-1β production of IL-8) for the production of the composition, intended for use in the treatment of type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance. According to one embodiment of the invention, the receptor antagonist IL-1β is anakinra (i.e., Kineret, Kineret®).

According to another aspect of the invention provides for the use of antibodies to IL-1β or binding fragments for the production of drugs intended for the treatment or prevention of a disease or condition such as described. In all uses of the drug can be coordinated with treatment using a second active agent. In another embodiment, the invention provides for the use of a synergistic combination comprising an antibody according to the invention, for obtaining a drug intended for treatment of a patient having the symptoms or are at risk for the development of the mentioned diseases or conditions, the use of the medicinal product coordinated with treatment using a second active agent. In another close variant of the invention claimed composition, wherein the second active agent is another antibody, growth factor, cytokine or insulin. There are options you�of olnine of the invention, providing for any use from the mentioned above, according to which an antibody or fragment that bind IL-1β, contains a drug in an amount to effectively reduce the dosage of second active agent required to achieve a therapeutic effect.

According to another aspect of this invention provides a product comprising a container, a composition within the container containing the antibody to IL-1β or a fragment thereof, and a leaflet-liner in a package that contains instructions for administering the antibody or fragment to people in need in the treatment of the above-described methods of the present invention. According to one embodiment of the invention, the container further comprises a pharmaceutically acceptable carrier, excipient or diluent. In a close one embodiment, the composition inside the container further comprises a second active agent. In another close variant of the invention claimed composition, wherein the second active agent is another antibody, growth factor, cytokine or insulin.

Whales (sets) are also provided by the present invention. According to one embodiment of the invention, the kit comprises a therapeutically or prophylactically effective amount of antibodies to IL-1β or the fragment, packaging�x in the container, such as a vial or bottle, also contains a label affixed to the container or packaged with the container, and the label described the contents of the container and there are indications and/or instructions regarding use of the contents of the container for the treatment or prevention of a disease or condition in accordance with the previously described methods of the present invention. According to one embodiment of the invention, the container also contains a pharmaceutically acceptable carrier, excipient or diluent. In a close one embodiment, the composition inside the container further comprises a second active agent. In another close embodiment, the second active agent comprises another antibody, growth factor, cytokine or insulin.

According to one embodiment of the invention, the product kit (set) or a medication intended to treat or prevent a disease or condition in humans, and the disease or condition is selected from the group including type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulin secretion and insulin resistance. In one preferred embodiment of the invention, the disease or condition is selected from the group including type 2 diabetes, obesity and resistances�ü to insulin. In another embodiment, the instructions on the leaflet insert in the packaging or the label on the kit contain instructions for administering the antibody or fragment according to any of the above dosages, the number of subsequent doses and intervals between doses, as well as any combination of the described dosages, the number of subsequent doses and intervals between doses. In another embodiment, the container of the kit or product is a pre-filled syringe.

It should be understood that since this description includes methods of treatment using antibodies or fragments thereof that possess certain properties (such as Kd values or IC50), the invention includes the use of such antibodies or fragments thereof in the manufacture of drugs intended for use in accordance with these methods. Furthermore, the invention also includes antibodies or fragments thereof, having the described properties, as well as pharmaceutical compositions comprising these antibodies or fragments thereof, for use according to the described methods of treatment.

BRIEF description of the DRAWINGS

Fig.1 is a graphic representation of the results of experiments on the inhibition of IL-1β antibody, designated AV, and Kineret®, including inducive�ing IL-1 production of IL-8 in vitro.

Fig.2 is a graphic representation of the results of experiments on the inhibition of IL-1β antibody designated AB 5 and AB, including induced IL-1 release of IL-6 in vivo.

Fig.2B is a graphical representation of the results of experiments on the inhibition of IL-1β in vivo antibody labeled AV, including induced IL-1 release of IL-6, and comparative inhibition of human (box A) and murine (box B) IL-1β.

Fig.3 is a graphical representation of the concentrations of antibodies in the serum of rats after administration of 0.1, 1 or 10 mg/kg of antibody to IL-1β.

Fig.4 is a graphical representation of the concentrations of antibodies in the serum of Cynomolgus monkeys after administration of 0.3 or 3 mg/kg of antibody to IL-1β.

Fig.5 is a graphic simulation of the concentration profiles of antibodies to IL-1β in the plasma of Cynomolgus monkeys after five doses of 0.1, 0.3, 1 or 3 mg/kg, introduced on a monthly basis.

Fig.6 is a table showing the reduction of induced Staphyloccus epidermidis cytokine secretion in whole blood of people in the treatment of the antibody to IL-1β.

Fig.7 is a graphical representation of the pharmacokinetics AS in humans after administration of the antibody in the amount of 0.01 mg/kg.

Fig.8 is a graphical representation of the effects of the introduction AS in the amount of 0.01 mg/kg for SN�proposal CRP levels in humans.

Fig.9 is a graphical representation of the effects of the introduction AS in the amount of 0.03 mg/kg at reducing CRP levels in humans.

Fig.10 is a graphic representation of the levels of CRP in the control experiments, groups of men treated with placebo in the amount of 0.01 or 0.03 mg/kg.

Fig.11 is a graphic simulation of CRP levels in humans after administration of different doses of antibodies with properties similar to AV, in 28-day intervals.

Fig.12 is a graphical representation of the influence AV on type 2 diabetes in a mouse model of obesity induced by diet.

DETAILED disclosure of the INVENTION

IL-1β is a proinflammatory cytokine that is secreted by several cells of different types, including monocytes and macrophages. Being liberated as part of the inflammatory response, IL-1β causes a number of biological effects, mainly Mediaroom through the excitation of other inflammatory mediators such as corticotropin, platelet factor-4, prostaglandin E2 (PGE2), IL-6 and IL-8. IL-1β induces both local and systemic inflammatory effects through the activation of the receptor of IL-1, detected in almost all cell types.

The family of cytokines interleukin-1 (IL-1) is involved in a number of disease States such as rheumatoid arthritis (RA), osteoarthritis, Crohn's disease, not�specificeski ulcerative colitis (UC), septic shock, chronic obstructive pulmonary disease (COPD), asthma, homologous disease caused by graft versus host disease, atherosclerosis, T-cell leukemia adults, multiple myeloma, multiple sclerosis, stroke and Alzheimer's disease. The members of the family of IL-1 include IL-1α, IL-1β and IL-1Ra. Despite the fact that their ability combines the ability to bind with the receptors of IL-1 (IL-1R1 and IL-1R2), each of these cytokines are highly expressed particular genome and is particularly the primary amino acid sequence. Moreover, the physiological actions of these cytokines may be different from each other.

Compounds that disrupt the signaling receptor IL-1, have been explored as therapeutic agents for the treatment of IL-1-mediasound diseases, such as, for example, some of the earlier. These compounds include recombinant IL-1Ra (Amgen Inc., Thousand Oaks, CA), peptide, binding of IL-1 receptor (IL-1 receptor "trap" peptide, Regeneron Inc., Tarrytown, NY), and antibodies to IL-1β animal and recombinant antibodies to IL-1β, and fragments thereof.

As mentioned above, for use in the treatment of type 2 diabetes was suggested polypeptide receptor antagonist IL-1 (IL-1Ra) (WO 2004/002512). However, the need for effective means of treatment of type 2 diabetes persists, particularly for patients who do not want to do hedgehog�day, repeated injections. An additional problem of therapeutic treatments based on receptor antagonists, IL-1β, is the need for such therapy to take (to bind) a large number of receptors, which is a formidable task, because such receptors are widely represented in all cells except red blood cells (Dinarello, Curr. Opin. Pharmacol. 4:378-385, 2004). In most immunomediated diseases, such as described in the present invention, the amount of cytokine IL-1β, measured in body fluids or associated with activated cells is relatively small. Therefore, the method of treatment and/or prophylaxis, directly aimed at the ligand IL-1β, is an excellent strategy, especially with the introduction of antibodies to IL-1β with high affinity.

In the present invention, the claimed methods and associated products (products) intended for the treatment and/or prophylaxis in a mammal of type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, insulin resistance and/or disease States characterized by insulin resistance, with specific to IL-1β antibody or its fragment.

As shown hereinafter in Example 1, we surprisingly found that such an antibody (e.g., with high affinity) can be�ü considerably more efficient inhibitor of the metabolic pathway of IL-1, than IL-Ra (e.g., Kineret®), as shown below in Example 9, gives the possibility of achieving a therapeutic effect at lower doses and/or at a lower frequency than required for other drugs, such as recombinant IL-1Ra.

Methods of using antibodies to IL-1β or the fragment, such as those described herein may include the treatment of a subject suffering from type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, hypoinsulinemia, insulin resistance and/or disease States characterized by insulin resistance. The methods may also include the prevention of the manifestations of type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulin secretion, insulin resistance and disease States characterized by insulin resistance, or to prevent the manifestation of the described diseases in subjects at risk. The term "hyperinsulinemia" refers to the relative hyperinsulinemia resulting from insulin resistance.

Antibodies, humanized antibodies and antibody, is developed for people (Human Engineered Antibodies)

IL-1-binding antibodies (e.g., IL-1β) of the present invention can be obtained in the form of polyclonal antibodies, monoc�national antibodies (mAbs), recombinant antibodies, chimeric antibodies, CDR-grafted antibodies, fully human antibodies, single-chain antibodies and/or bespecifically antibodies as well as fragments, including variants and derivatives obtained by known methods, including, but not limited to these, enzymatic cleavage, peptide synthesis or recombination methods.

Antibodies typically comprise two heavy polypeptide chains and two light polypeptide chains, although the antibody with the single domain of one heavy and one light chain, antibody heavy chain, devoid of light chain, also provides. There are five types of heavy chains, called alpha, Delta, Epsilon, gamma and mu on the basis of the amino acid sequence of the constant domain of the heavy chain. These different types of heavy chains correspond to the five classes of antibodies, IgA (including IgA1and IgA2), IgD, IgE, IgG and IgM, respectively, including four IgG subclass, namely IgG1, IgG2, IgG3and IgG4. There are also two types of light chains, called Kappa (κ) or lambda (λ) based on the amino acid sequence of the constant domains. Panoramiczne antibody comprises a constant domain and variable domain. In the antigen-binding fragment of the antibody, the presence of a constant domain is not required. Disclosed in the Fig�attachment of antigen-binding fragments of antibodies may include Fab, Fab', F(ab')2and F(v) antibody fragments. As shown in more detail below, the IL-1β-binding fragments include fragments of the antibodies and antigen-binding polypeptide that binds to IL-1β.

Each of the sequences of the heavy chain and light chain of the antibody or antigen-binding fragment comprises the variable plot with three hypervariable sites (CDR) and framework (FR) non hypervariable sites (CDR). In this context, the terms "heavy chain" and "light chain" refers to the variable plot heavy chain and variable area light chain, respectively, unless otherwise noted. Hypervariable sites (CDRs) of the heavy chain is designated as CDR-H1, CDR-H2 and CDR-H3. Hypervariable sites (CDRs) of the light chain is designated as CDR-L1, CDR-L2 and CDR-L3. Variable and hypervariable sites of the sequence antibodies can be identified (i) using the normal rules known in the art, or (ii) alignment of sequences with known variable from the database sites. The identification of these sites are described in Antibody Engineering, edited by Kontermann and Dubel, Springer, New York, NY, 2001, and Dinarello, etc., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Database sequences of the antibodies described and available in the database "The Kabatman" on the site www.bioinf.org.uk/abs (supported by A. S. Martin, Department of Biochemstry & Molecular Biology University College London, London, England) and VBASE2 on the website www.vbase2.org as described Retter, etc., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Website database Kabatman" also includes General rules of identification of CDR. In this context, the term "CDR" refers to, as defined in Kabat et, Sequences of Immunological Interest, 5thed., U. S. Department of Health and Human Services, 1991, unless otherwise stated.

Polyclonal antibodies preferably are collected from animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the appropriate antigen and an adjuvant. Enhanced immune response (antibody formation) can be obtained by conjugation of a suitable antigen to a protein which is immunogenic subjected to immunization types, such as keyhole clam fissurellidae (keyhole limpet hemocyanin), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or transformative agent, for example, maleimidomethyl broadcast succinimide (conjugation through cysteine residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.

Immunize animals with antigen, immunogenic conjugate or derivative, mixing, e.g., 100 μg or 5 μg of the protein or conjugate (for rats or mice, respectively) with 3 volumes complete adju�of Anta freind and making injection solution intradermally (intracutaneously) at many places. A month later the animals are re-stimulated with the help of 1/5-1/10 of the original amount of peptide or conjugate in complete adjuvant of frand by subcutaneous injection in many places. 7-14 days after stimulating injections in animals take blood and determine the titer of antibodies in serum. Animals to stimulate until the titer comes out on the plateau. Preferably, animals stimulate the conjugate of the same antigen, but is associated with another protein and/or another cross-linking agent. Conjugates can be prepared as fusion proteins recombinant cell culture. For amplification of the immune response can also be used aggregating agents such as alum.

A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are typically highly specific and can be directed to a single immunodominant site in contrast to traditional drugs (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes). In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized by a homogeneous culture, do not contain impurities of other immunoglobulin with a different specificity and characteristics.

The monoclone�further antibodies, intended for use according to the present invention, can be obtained by the hybrid method, first described by Kohler and others (Nature, 256:495-7, 1975), or may be obtained by recombinant DNA methods (see, e.g., U.S. patent No. 4816567). Monoclonal antibodies can be isolated from phage libraries of antibodies using the methods described in, for example, Clackson et (Nature 352:624-628, 1991) and Marks and others (J. Mol. Biol. 222:581-597, 1991).

According to the method of hybridomas mice or other suitable animal hosts, such as hamsters or monkeys, immunize, as described in the invention, for the extraction of the lymphocytes that produce or are capable of producing antibodies, specifically binding to the protein used for immunization. Alternatively, lymphocytes can immunize in vitro. Then carry out the fusion of lymphocytes with myeloma cells using a suitable agent for the merger, such as polyethylene glycol, to obtain cells of hybridomas (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

Thus obtained cells of hybridomas are sown and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival nalivshihsya parental myeloma cells. For example, if the parental myeloma cells lack the enzyme �erase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, to produce remissions in childhood and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

Preferred are myeloma cells, which effectively gibridizatsiya (merge), support stable high-level production of antibody by the selected cells producing the antibodies, and are sensitive to the environment. Cell lines of myeloma people and hetero-myeloma mice and humans is also described for obtaining human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et, Monoclonal Antibody Production Techniques and Applications, pp.About 51 To 63 (Marcel Dekker, Inc., New York, 1987)). As an example, myeloma lines of mice, in particular, derived MOR-21 and M. C.-11 cancer mice that are available in the Distribution Center Cells of the Salk Institute, San Diego, California, USA (Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American type culture Collection, Rockville, Maryland, USA (American Type Culture Collection, Rockville, Md. USA).

Culture medium in which grown cells of hybridomas, check for the production of monoclonal antibodies to the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybrid cells, determined using the immunoprecipitation or analysis of binding in vitro, such as radioim�ideological analysis (RIA) or enzyme-linked immunoassay (ELISA, ELISA). The affinity of binding of the monoclonal antibody can be determined, for example, by analyzing Scatchard (Munson et, Anal. Biochem, 107:220 (1980)).

After determining that the cells of hybridomas produce antibodies of the desired specificity, affinity and/or activity, the clones can be subclavian using procedures of limited dilutions and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable for this purpose the culture medium include, for example, DMEM or RPMI-1640 medium. In addition, the cells of hybridomas can be grown in vivo in the form of ascitic tumors in animals. Monoclonal antibodies that are excreted by subklonov, suitably separated from the culture medium, ascites or serum conventional methods of purification of immunoglobulins, such as, for example, protein A-separatly, chromatography on hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

It is also envisaged that the antibody of the present invention can be used in the form of smaller antigen-binding fragments of antibodies, are well known in the art and described in the invention.

The present invention includes antibodies that bind IL-1 (e.g. IL-1β), which contain two panoramiczne heavy chains and two panoramiczne light chains. Alternatives�about, antibodies that bind IL-1β, can be constructs such as single chain antibodies or "mini" antibodies that retain the ability to bind to IL-1β. Such constructs can be generated are known in the art methods such as, for example, cloning by polymerase chain reaction (PCR also been other ideas where cloning) and the Assembly of single-chain antibodies for expression in E. coli (as described in Antibody Engineering, The practical approach series, J. McCafferty, H. R. Hoogenboom, and D. J. Chiswell, eds., Oxford University Press, 1996). In the constructs of this type variable portions of heavy and light chains of antibody molecules amplified by polymerase chain reaction (PCR) with cDNA. The resulting amplicons are then brought together, for example, at the next stage PCR using a DNA linker encoding a flexible linker protein consisting of amino acids Gly and Ser. This linker enables the variable portions of heavy and light chains to bend in such a way that the antigen-binding pocket was restored and the antigen is contacted with the affinity, often comparable to the affinity of the parent poliocephala dimeric immunoglobulin molecule.

Antibodies that bind IL-1 (e.g. IL-1β), and fragments thereof of the present invention include variants given here as an example antibodies, fragments and sequences. Options include� peptides and polypeptides, containing one or more amino acid substitution, deletion and/or insertion of possessing the same or almost the same affinity and specificity of binding epitope as one or more given here as an example antibodies, fragments and sequences. Thus, variants include peptides and polypeptides that contain one or more substitution, deletion and/or insertion in the amino acid sequence as compared with given by way of example antibodies, fragments and sequences, and such substitutions, deletions and/or insertions do not cause significant changes in affinity and specificity of binding epitope. For example, a variant antibody or fragment may be a result of one or more changes of antibody or fragment, wherein the modified antibody or fragment has the same or substantially the same affinity and specificity of binding epitope as the original sequence. Options can be natural, such as allelic variants or splicing variants, or may be artificially constructed. Options can be obtained from the relevant molecules of nucleic acids encoding these variants. Variants of these antibodies and IL-1β binding fragments may have variations in amino acid sequences of light and/or heavy� circuits, natural or entered using the engineering of native sequences in vitro using methods involving recombinant DNA. Natural options include somatic variants ("somatic" variants), which are generated in vivo from the corresponding nucleotide sequences of embryonic cell lines in the process of formation of antibodies in response to foreign antigen.

Variants of the antibodies and fragments that bind IL-1 (e.g. IL-1β), can also be obtained through mutagenesis. For example, changes in amino acids can be introduced randomly in the region encoding the antibody, and the resulting variants can be subjected to screening for their affinity binding with IL-1β or other property. Alternatively, amino acid substitutions can be introduced into selected areas of the antibodies to IL-1β, such as the hypervariable sites (CDR) of the light chain and/or heavy chain and/or in the area of the frame, and the resulting antibodies can be subjected to screening for their affinity binding with IL-1β or other activity. Amino acid substitutions include replacement of one or more amino acids in hypervariable segment (CDR), ranging from differences in single amino acid until the introduction of multiple permutations of amino acids within a given CDR, such as CDR3. According to another method, it is possible to determine the contribution of each OST�TKA inside CDR in the binding of IL-1β by replacing, at least one residue within the CDR with alanine. Lewis and others (1995), Mol. Immunol. 32: 1065-72. Residues that are not optimal from the point of view of binding to IL-1β, can then be replaced to determine a more optimum sequence. The invention also includes variants, obtained by the introduction of inserts (insertion) of amino acids to increase the size of the CDR, such as CDR3. For example, the majority of sequences of the light chain CDR3 contain nine amino acids. Sequences of light chains of antibodies that includes less than nine residues may be optimized from the point of view of binding to IL-1β by introducing insertion of appropriate amino acids to increase the length of the CDR.

Options can also be obtained by "shuffling" of light or heavy chains. Marks and others (1992), Biotechnology 10: 779-83. One (single) light (or heavy) chain can be combined with a library having a repertoire of heavy (or light) chains and the resulting set of variants is subjected to screening for the desired activity, such as binding to IL-1β. This enables screening of a larger number of samples with different heavy (or light) chains in combination with a single light (or heavy) chain compared with the possibilities offered by the libraries, including both sets (heavy and light) chains.

Antibodies and fragments by Dan�WMD to the invention, binding of IL-1 (e.g. IL-1β), include derivative is given here as an example antibodies, fragments and sequences. Derivatives include polypeptides or peptides, or variants, fragments or derivatives that have been chemically modified. Examples include covalently attached via the N atom one or more polymers such as water-soluble polymers, or attached via About carbohydrates, sugars, phosphates and/or other such molecules. Derivatives are modified in such a way that they differ from natural source or peptides or polypeptides or on the type or localization of attached molecules. Derivatives also include the deletion (removal) of one or more chemical groups, the original (from nature) present in the peptide or polypeptide.

IL-1β binding antibodies and fragments of the present invention can be bespecifically. Bespecifically antibodies or fragments may be of several configurations. For example, bespecifically antibodies may have similarities with the single antibodies (or fragments of antibodies), but to have two different antigen-binding site (variable region). Bespecifically antibodies can be obtained by chemical methods (Kranz and others (1981), Proc. Natl. Acad. Sci. USA 78: 5807), ways of "Polydome" ("polydoma techniques") (U.S. Patent No. 4474893) or ways to re�abinanti DNA. Bespecifically antibodies of the present invention can have binding specificity for at least two different epitopes, at least one of which is an epitope of IL-1β. IL-1β binding antibodies and fragments can also be getrootframe. Heteranthera are connected together two or more antibodies or binding fragments of antibodies (Fab), wherein each antibody or fragment has a different specificity.

Technologies of creation of recombinant DNA encoding the antigen-binding sites of antibody molecules, including the production of monoclonal antibodies included in the methods for producing antibodies and fragments that bind IL-1 (e.g. IL-1β), according to the present invention. DNA cloned in the bacterial expression system. One example of such method, suitable for the purposes of this invention, is used as a vector system of bacteriophage lambda with a leader sequence that causes the migration of the expressed Fab protein to the periplasmic space (between the bacterial cell membrane and cell wall) or its secretion. You can quickly get and be subjected to screening a large number of functional Fab fragments to find those that bind IL-1β. Such IL-1β-binding agents (Fab fragments having a real influence on� specificity to the polypeptide of IL-1β) are among the IL-1β binding antibodies and fragments of the present invention.

Antibodies and fragments that bind IL-1 (e.g. IL-1β), can be humanized antibodies or antibody, is developed for people (human engineered antibody). Temin a humanized antibody or antigen-binding fragment is a recombinant polypeptide comprising the antigen-binding part of the plot nonhuman antibody and part of the frame and/or constant regions of human antibodies. The term "antibody, is developed for the people", or a fragment of an antibody, refers to nonhuman (e.g., mouse) which has been modified, for example, deletions, insertions or substitutions of amino acids in certain positions, thus, to reduce or eliminate any detectivea immunogenicity of the modified antibodies in humans.

Humanized antibodies include chimeric antibodies and CDR-transplanted antibody. Chimeric antibodies are antibodies containing a variable fragment of a non-human antibody coupled to a constant area of the human antibody. Thus, chimeric antibodies the variable plot is basically superhuman, while the constant plot is human. Chimeric antibodies and methods for their preparation are described in Morrison et, Proc. Natl. Acad. Sci. USA, 81: 6841-6855 (1984), Boulianne, etc., Nature, 312: 643-646 (1984), and publicat�and PCT application WO 86/01533. Although they may be less immunogenic than murine monoclonal antibody, the introduction of chimeric antibodies have been associated with responses in the form of a human anti-mouse antibody (HAMA) against the inhuman parts of antibodies. Chimeric antibodies can also be obtained by gene splicing molecules of the murine antibody with the appropriate specificity of binding to the antigen, with the genes of a human antibody molecule of appropriate biological activity, such as ability to activate human complement and mediate antibody-mediated cellular cytotoxicity (ADCC). Morrison and others (1984), Proc. Natl. Acad. Sci., 81: 6851; Neuberger et (1984) Nature, 312: 604. One example is the replacement of the Fc section to another belonging to a different isotype.

The term "CDR-grafted antibody" means an antibody in which the hypervariable sites (CDRs) of anti-human "donor" antibody associated with skeleton plot "acceptor" antibody of human origin. Usually CDR-transplanted antibodies include more human sequences than chimeric antibodies, because they include sequences of constant sites, and the sequence variable (dummy) regions of human antibodies. So, for example, CDR-grafted humanized antibody of the present invention mo�et to include a heavy chain, contains a long amino acid sequence (e.g., about 5 or more, 10 or more, or even 15 or more contiguous amino acid residues) of the frame area of the human antibody (e.g., FR-1, FR-2 or FR-3 of human antibody) or, optionally, most or all of a frame region of a human antibody. CDR-transplanted antibodies and methods for their preparation are described in Jones et, Nature, 321: 522-525 (1986), Riechmann et, Nature, 332: 323 to 327 (1988) and Verhoeyen et, Science, 239: 1534-1536 (1988)). Ways that you can use for obtaining humanized antibodies are also described in U.S. patent 4816567, 5721367, 5837243 and 6180377. It is believed that CDR-transplanted antibody to a lesser extent, inducing immune response against sites of an antibody of nonhuman origin than the chimeric antibody. However, it was shown that the frame sequences of the donor antibody need to Express the binding affinity and/or specificity of the donor antibody, presumably due to the fact that these frame sequences affect the folding of the antigen-binding site of the donor antibody. Therefore, when nonhuman donor sequence hypervariable sites transplanted unchanged in frame sequences of human origin, obtained in Reza�adds the CDR-grafted antibody in some cases, you may lose the avidity of binding to antigen compared to the original nonhuman donor antibody. See, for example, Riechmann et, Nature, 332: 323 to 327 (1988) and Verhoeyen et, Science, 239: 1534-1536 (1988).

The antibodies are designed for people who include, for example "faced" ("veneered") antibodies and the antibodies obtained using the technology of human engineering™ (U.S. Patent 5869619). Technology human engineering™ is commercially available and includes modifying the non-human antibody or antibody fragment, such as a murine or chimeric antibody or antibody fragment, by specific changes in the amino acid sequence of the antibody for the production of modified antibodies with reduced immunogenicity in humans and which retains the desirable characteristics of the binding of the original non-human antibody. Usually the method involves the classification of amino acid residues nonhuman (e.g., murine) antibodies on the leftovers with "low risk", "moderate risk" and "high risk". The classification is carried out using the calculation of global risk/reward (global risk/reward calculation), allowing to estimate the predicted benefits of the implementation of specific substitutions (for example, relative immunogenicity in humans) and the risk that the replacement will affect the ability of the resulting antibody to fold and/or its antigen-binding properties. Thus, a situation with low CTE�the ood risk is that replacement in which the forecast is favorable, because it is predicted that it will lead to reduction of immunogenicity, significantly without affecting the antigen-binding properties. The situation with moderate risk position is, the replacement of which was expected to reduce immunogenicity, but are likely to affect the ability of the protein to fold and/or binding to antigen. It seems most likely that the provisions with a high degree of risk contain residues involved in the correct folding or binding to antigen. As a rule, the provisions of the low risk to non-human antibody is replaced by residues of human origin in locations with high risk of replacement is administered rarely, sometimes for humanization is carried out replacing the provisions with moderate risk, but not indiscriminately. The provisions containing Proline at variable sites of non-human antibodies, usually classified as at least moderate risk.

Specific human amino acid residue that can be used to replace this provision with a low or moderate risk in the sequence nonhuman (e.g., murine) antibodies, can be selected by aligning the amino acid sequence of the variable fragment nonhuman antibodies with suitable�appropriate site specific or consensus sequences of human antibodies. Amino acid residues in positions with a low or moderate level of risk can be appropriately substituted by residues of the sequence of the human antibody in accordance with the alignment. The means of producing protein for the people" in great detail described in Studnicka et, Protein Engineering, 7: 805-814 (1994), the U.S. patents 5766886, 5770196, 5821123 and 5869619, and in the publication of the PCT application WO 93/11794.

"Faced" (veneered) antibodies constitute inhuman or humanized (e.g., chimeric or CDR-transplanted antibody) antibody, designed to replace some facing the solvent (solvent-exposed) amino acid residues to further reduce their immunogenicity or enhance their function. I believe that the influence of residues located on the surface of the chimeric antibody, the correct folding of the antibody is unlikely. It is more likely that these residues are involved in immune response. Therefore, the "veneer" chimeric antibodies can include, for example, identification of exposed residues inhuman skeleton plot of the chimeric antibody and replacing at least one corresponding surface residues of the human skeleton plot. Veneer can be held by any suitable method of genetic engineering, including the use you described�e technology human engineering™.

According to another approach, the restoration of the avidity of binding can be achieved at the expense of "dehumanization" CDR-transplanted antibody. Dehumanization may include the return of residues from wireframe plots donor antibodies in the CDR-grafted antibody, restoring, thus, proper folding. Similar to the "dehumanization" can be achieved by (i) the introduction of fragments of the "donor" frame section in the "acceptor" antibody or (ii) transplantirovali fragments of the skeleton of the plot "donor" antibody "acceptor" antibody (along with transplanted donor CDRs).

Detail antibodies, humanized antibodies, antibodies, designed for people, and methods for their preparation are described in the book edited by Kontermann and Dubel, Antibody Engineering, Springer, New York, NY, 2001.

Examples of humanized antibodies or antibody for people include IgG, IgM, IgE, IgA and IgD. These antibodies can belong to any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can include the Kappa or lambda light chain. For example, the human antibody may include an IgG heavy chain or defined fragment, such as at least one of the isotypes IgG1, IgG2, IgG3 or IgG4. Such antibodies or fragments may include, for example, IgG1 heavy chain and a IgG1 light chain.

The antibodies and fragments can be human anti�partitions, such as antibodies, binding polypeptides of IL-1β and encoded by nucleic acid sequences that are the natural somatic variants of the nucleotide sequence of human immunoglobulin germ lines, and fragments, synthetic variants, derivatives and fusion derivatives. Such antibodies can be obtained by any known in the art method, such as the use of transgenic mammals (such as transgenic mice), in which the native repertoire of immunoglobulins substituted human V-genes in the chromosome of a mammal. It seems that these mammals in the normal operation is carried out VDJ-recombination and somatic hypermutation genes of human antibody germ lines, producing, thus, high-affinity antibodies with fully human sequences.

Human antibodies against the target protein can also be obtained using transgenic animals that have no endogenous secretion of immunoglobulin, but, in consequence of the use of genetic engineering, there are loci of human immunoglobulins. For example, WO 98/24893 discloses transgenic animals having a locus of human Ig, wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of lokusa� endogenous heavy and light chains. In WO 91/00906 also disclosed transgenic mammals are hosts that are not related to primates, capable of providing an immune response to the immunogen, wherein the antibodies have a constant and/or variable plots primates, and the loci encoding the endogenous immunoglobulin, substituted or been inactivated. WO 96/30498 and U.S. Patent No. 6091001 disclose the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as full or partial replacement of a constant or variable segment to obtain a molecule-modified antibody. In WO 94/02602 described mammals-owners, non people with inactivated endogenous loci Ig and functional loci of human Ig. U.S. patent No. 5939598 discloses methods of obtaining transgenic mice lacking endogenous heavy chain locus and expressed exogenous immunoglobulin containing one or more xenogeneic constant plots. Cm. also U.S. patent Nos. 6114598, 6657103 and 6833268.

When using transgenic animals described above can be obtained, the immune response to a selected antigen molecule, and antibody producing cells can be distinguished from the animal and used to obtain hybridomas secreting human monoclonal antibodies. The immunization protocols, adjuvants, etc. known in the tech� and are used in immunization, for example, transgenic mice, as described in WO 96/33735. This publication discloses monoclonal antibodies against a variety of antigenic molecules, including IL-6, IL-8, TNFα, human CD4, L-selectin, gp39 and tetanus toxin. Monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. In the publication WO 96/33735 shown that monoclonal antibodies against IL-8, immune cells isolated from transgenic mice immunized IL-8, block-induced IL-8 functions of neutrophils. Human monoclonal antibody with specificity against the antigen used to immunize transgenic animals are also described in WO 96/34096 and the application for U.S. patent No. 20030194404; and the application for U.S. patent No. 20030031667.

Transgenic animals useful for obtaining monoclonal antibodies also include mice Medarex HuMAb-MOUSE®, described in U.S. patent No. 5770429 and Fishwild, etc. (Nat. Biotechnol. 14:845-851, 1996) with gene sequences nontransgenic genes of human antibody encoding heavy and light chains of human antibodies. Immunization of mice, HuMAb-MOUSE® gives you the ability to produce fully human monoclonal antibodies to the protein target.

Ishida and others (Cloning Stem Cells. 4:91-102, 2002) also describe mice TransChromo Mouse (TCMOUSE™), �bearing measurment (megabase-sized segments) of human DNA, including the full locus of human immunoglobulin (hIg). Mouse TCMOUSE™ have a completely different repertoire hIgs, including all the subclasses of immunoglobulins (IgG1-G4). Immunization of mice TC MOUSE™ various human antigens causes a response in the form of formation of antibodies, including human antibodies.

Cm. also, Jakobovits et, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et, Nature, 362:255-258 (1993); Bruggermann et, Year in Immunol., 7:33 (1993); and U.S. patent No. 5591669, U.S. patent No. 5589369, U.S. patent No. 5545807; and the publication of the patent application U.S. No. 20020199213. The publication of the patent application U.S. No. 20030092125 described methods of influence on the immune response of the animal to the desired epitope. Human antibodies can also be generated activated in vitro In cells (see U.S. patents No. 5567610 and 5229275).

Human antibodies can also be obtained by using in vitro screening of libraries, presenting antibodies. Cm. Hoogenboom et (1991), J. Mol. Biol. 227: 381; Marks and others (1991), J. Mol. Biol. 222: 581. Have been described and can easily prepare different phage library presenting antibodies. The library can contain different sequences of human antibodies, such as human Fab, Fv and scFv fragments, which can be subjected to screening about the appropriate targets. Phage-presenting library can contain peptides or proteins that are not antibodies, which can be subjected to�rining to identify the agent, selectively binds IL-1β.

The development of methods for producing repertoires of genes of recombinant human antibodies and presentation of the encoded fragments of the antibodies on the surface of filamentous phage has provided direct ways of obtaining human antibodies. Antibodies obtained using the technology of phage display, are produced by bacteria in the form of antigen-binding fragments (typically, Fv or Fab fragments) and thus devoid of effector functions. Effector function can bring by using one of two strategic approaches: genetically engineered fragments can be completed either before the full antibodies for expression in mammalian cells, either before bespecifically fragments of antibodies with a second binding site that can trigger effector function.

The invention provides a method of obtaining specific to a target antibody or antigen-binding fragment comprising the stage of synthesis ragovoy library of human antibody library screening protein target or its fragment, separation of phage bound to the target, and obtaining the antibody from the phage. For example, one way of obtaining a library of antibodies for use according to the method of phage display comprises the steps of immunizing a non-human animal having a locus h�human immunoglobulin, the antigen target or its antigenic fragment to obtain an immune response, extracting immunized animal cells that produce antibodies; isolation of RNA from the selected cells, reverse transcription of RNA to obtain cDNA and cDNA amplification using a primer and introduction of cDNA into the vector phage display (phage display vector such that antibodies expressed by the phage. Recombinant specific to a target antibody of the present invention can be obtained by this method.

The process of phage display mimic immune selection through the presentation of a repertoire of antibodies on the surface of filamentous bacteriophage, with subsequent selection of phage by their binding to the selected antigen. One such method is described in WO 99/10494, where disclosed the allocation of high-affinity and functional agonistic antibodies for MPL-and msk receptors using such an approach. The antibodies of the present invention may be removed by screening a recombinant combinatorial libraries of antibodies, preferably phage libraries presenting scFv obtained using the human cDNA of the VLand VHderived from mRNA isolated from human lymphocytes. Methodological approaches for obtaining and screening such libraries are known in the art. See, for example, the patent�And No. 5969108. There are commercially available whales (sets) to create a phage presenting libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01; and the Stratagene SurfZAP.TM. phage display kit, catalog No. 240612). There are also other methods and reagents that can be used in the preparation and screening of libraries, presenting antibodies (see, e.g., Ladner et U.S. patent No. 5223409; Kang et al., PCT publication no WO 92/18619; Dower et al., PCT publication no WO 91/17271; Winter et al., PCT publication no WO 92/20791; Markland et al., PCT publication no WO 92/15679; Breitling et al., PCT publication no WO 93/01288; McCafferty et al., PCT publication no WO 92/01047; Garrard, etc., the PCT publication no WO 92/09690; Fuchs et (1991) Bio/Technology 9:1370-1372; Hay et (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et (1989) Science 246:1275-1281; McCafferty et, Nature (1990) 348:552-554; Griffiths and others (1993) EMBO J 12:725-734; Hawkins and others (1992) J. Mol. Biol. 226:889-896; Clackson et (1991) Nature 352:624-628; Gram et (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et (1991) Bio/Technology 9:1373-1377; Hoogenboom et (1991) Nuc Acid Res 19:4133-4137; and Barbas et (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

According to one embodiment of the invention for the isolation of human antibodies specific to the antigen target and having the desired characteristics, a human library VHand VLwas skanirovali selection of fragments of antibodies having the desired specificity. Libraries of antibodies used according to this method, are preferably�Xia scFv library, prepared and subjected to screening as described in the invention and well known in the art (McCafferty et al., PCT publication no WO 92/01047, McCafferty et al., (Nature 348:552-554, 1990; Griffiths and others, {EMBO J 12:725-734, 1993). Library of scFv antibodies preferably subjected to screening using the target protein as the antigen.

Alternatively, the Fd fragment (VH-CH1) and light chain (VL-CL) of antibodies are separately cloned by PCR and randomly recombine in combinatorial phage-presenting libraries, which are then selected for binding to a specific antigen. Fab fragments expressed on the surface of phage, i.e. physically associated with coding their genes. Thus, selection of the Fab binding to the antigen is accompanied by a selection of sequences encoding the Fab, which can then be amplified. After several cycles of binding to the antigen and repeated amplification is carried out "panning" (panning), Fab, specific against the antigen, enrich, and finally allocate.

In 1994, was described approach to the humanization of antibodies, called "selection under control" ("guided selection"). Breeding under control takes advantage (force) of the method of phage display for the humanization of murine monoclonal antibodies (see Jespers, L. S., etc., Bio/Technology 12, 899-903 (1994)). To do this, the Fd fragment of mising� monoclonal antibodies can be presenting in combination with a library of human light chains, and received hybrid library Fab subjected to selection with antigen. Thus, murine Fd fragment provides the matrix for breeding. Subsequently, the selected human light chains combine with the human library Fd fragments. Selection received in the library provides a fully human Fab.

It was described many methods for obtaining human antibodies from phage-presenting libraries (see, e.g., Hoogenboom, etc., J. Mol. Biol., 227:381 (1991); Marks et, J. Mol. Biol, 222:581-597 (1991); U.S. patents Nos. 5565332 and 5573905; Clackson, T. and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, a powerful tool was the in vitro selection and evolution of antibodies derived from phage-presenting libraries (see Burton, D. R., and Barbas III, C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., and others, to Appy. Rev. Immunol. 12, 433-455 (1994); published patent application U.S. No. 20020004215 and WO 92/01047; the publication of the patent application U.S. No. 20030190317; and U.S. patents Nos. 6054287 and 5877293.

In the work of Watkins, "Screening of Phage-Expressed Antibody Libraries by Capture Lift," Methods in Molecular Biology, Antibody Phage Display: Methods and Protocols 178: 187 to 193 (2002), and in the publication of the patent application U.S. No. 20030044772, published March 6, 2003, describes methods of screening phage libraries expressing antibodies or other binding molecules by capture lift" (capture lift, a method involving immobilization on a solid substrate molecules that are candidates for linking.

As the only source of human VLand VHsegments selected experiments "mix and matching" ("mix and match", in which different pairs of the source selected VLand VHsegments subjected to screening for binding to the target, with the purpose of selection of preferred combinations of VL/VHpairs. In addition, to further improve the quality of the antibody, VLand VHthe preferred segments VL/VHvapor(s) can be subjected to random mutations, preferably within any one of CDR1, CDR2 or CDR3 of the plots of VHand/or VLin a manner similar to the process of somatic mutation in vivo, responsible for the increased affinity of antibodies during a natural immune response. This mutation resulting in a change in affinity in vitro, can be completed (perfected) by amplification of the VLand VHsites using PCR primers complementary CDR1, CDR2 and CDR3 of the VHor CDR1, CDR2 and CDR3 of the VLaccordingly, preach�m in these primers introduced a random mixture of the four nucleotide bases at certain positions, so that the resultant PCR products encode VLand VHsegments, which were implemented random mutations in CDR3 sections VHand/or VL. These randomly mutated VLand VHthe segments can be re-screened for binding to the antigen target.

After screening and selection of antibodies that are specific in relation to a target, from a recombinant library, presenting immunoglobulins, from the set of display package (e.g., from the phage genome) can distinguish a nucleic acid encoding the selected antibody, and subclinical into other expression vectors by standard methods using recombinant DNA. If desired, the nucleic acid can act on to create other forms of antibodies according to the present invention, as described below. For expression of recombinant human antibodies selected by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and injected into a host mammal as described in the invention.

Suppose that the technology of phage display can be performed in the mutator strain of the bacterium or the host cell. Mutator strain is a host with a genetic defect causing mutation Rapley�gazirovannoi within it the DNA of the parent DNA. Examples of mutator strains are NR9046mutD5 and NR9046 mut T1.

Suppose also that the technology of phage display can be performed using phage-assistant, which is a phage used to infect cells containing the genome of the defective phage, the function of which is to complement the defect. The genome of the defective phage can be fahmida or phage with remote sequences of genes encoding some features. Examples of phage-helpers include M13K07, M13K07 gene III No. 3; and phage displaying or encoding a binding molecule fused with the capsid protein.

Antibodies are also prepared by methods of screening phage display using hierarchical dual combinatorial approach as described in WO 92/01047, according to which an individual colony containing clone or heavy (H) or light (L) chains, are used to infect a complete library of clones encoding the other chain (L or H), and the resulting double-stranded specific binding element is chosen with the aid of technology, phage display, such as described in the invention. This method is described in Marks et (Bio/Technology, 10:779-783, 1992).

How to display peptides on the surface of yeast and bacterial cells have also been used to identify antigen-specific antibodies. See, for example, �atent USA No. 6699658. Library of antibodies can be attached to the yeast proteins, such as agglutinin, effectively mimicking the presentation of antibody cell surface of b-cells of the immune system.

In addition to technology phage display, antibodies can be isolated using renderers ribosomal mRNA and renderers bacterial cells. Breeding polypeptide using ribosome display as described in Hanes et (Proc. Natl Acad Sci USA, 94:4937-4942, 1997) and U.S. patents Nos. 5643768 and 5658754 issued by Kawasaki. Ribosome display is also useful for rapid, large-scale mutational analysis of antibodies. Using the approach of selective mutagenesis can be obtained antibodies with improved activity, which can be selected with the use of technology ribosomal display.

Antibodies and fragments that bind IL-1 (e.g. IL-1β), can include one or more areas that do not bind IL-1β, but instead are responsible for other functions, such as the half-life of circulation, direct cytotoxic effect, detected tagging or activation cascade of the endogenous complement of the recipient or endogenous cellular cytotoxicity. The antibodies or fragments may include full or part of constant section and may be any isotype, including IgA (e.g., IgA1 or IA2), IgD, IgE, IgG (e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In addition or instead, including a constant phase, antigen-binding compounds of the present invention may contain an epitope tag (epitope tag), the epitope of the receptor recycling (salvage receptor epitope), a label for diagnostic or purification, or a cytotoxic fragment, such as radionuclide or toxin.

Const plot (if any) of the antibodies and fragments can be γ1, γ2, γ3, γ4, μ, β2, δ or ε type, preferably of the γ type, more preferably of the γ type, and a constant area of the human light chain may be of the κ or λ type (including λ1, λ2and λ3subtypes) but is preferably belongs to the κ type.

Options also include antibodies or fragments comprising a modified Fc plot, and a modified Fc unit includes at least one amino acid modification related to Fc site neatening type. Variant Fc plot can be constructed (relative to a comparable molecule comprising the Fc plot neatening type) so as to bind Fc receptors with a greater or lesser affinity.

For example, IL-1β binding antibodies and fragments of the present invention may include a modified Fc plot. Fc plot refers to natural or synthetic polypeptides homologous to the C-terminal house�at IgG, which is produced by cleavage of IgG with papain. IgG Fc has a molecular weight of about 50 kDa. The antibodies and fragments of the present invention can be used Fc full area or only a fragment, which increases the half life. In addition, many modifications acceptable amino acid sequence, since the natural activity is not in all cases is necessary or desirable.

If desired, the Fc portion may be subjected to mutation to inhibit its ability to fix complement and bind an Fc receptor with high affinity. For mouse Fc IgG substitution of Ala residues for Glu 318, Lys 320, and Lys 322 makes the protein unable to direct ADCC. Substitution of Glu for Leu 235 inhibits the ability of the protein to contact the Fc receptor with high affinity. Also known different mutations of human IgG (see, e.g., Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke et al., 1994, The Immunologist 2: 125).

In some embodiments the invention provides antibodies or fragments with a modified Fc region, where the natural Fc plot modified to increase the half-life of the antibody or fragment in a biological environment, for example, time half-life from serum or half-time measured in an in vitro study. How to change the original form Fc plot of IgG are also described in U.S. patent �6998253.

In certain embodiments of the invention may be desirable to modify the antibody or fragment to increase its half-life from serum, such as adding to a fragment of an antibody molecule, such as PEG or other water soluble polymers, including polysaccharide polymers, to increase the half-life. This can also be achieved, for example, by introducing the epitope binding receptor recycling in a fragment of an antibody (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag, so then drain it with a fragment of the antibody, either at the end or in the middle, e.g., by DNA or peptide synthesis) (see international publication number WO96/32478). The epitope binding receptor recycling refers to an epitope of an Fc section of an IgG molecule (e.g., IgG1, IgG2, IgG3or IgG4) responsible for the increase of in vivo half-life of serum IgG molecule.

The epitope binding receptor recycling may include the area in which one or more amino acid residues from one or two loops of the Fc domain moved to a similar position of a fragment of the antibody. Even more preferably, have been moved three or more residues from one or two loops of the Fc domain. Even more prefer�till then, the epitope was taken from the CH2 domain of the Fc portion (e.g., IgG) and moved to CH1, CH3, or VHregion, or more than one such region of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc section and moved to CLor VLor in both regions of the antibody fragment. Cm. also international application WO 97/34631 and WO 96/32478 that describe the Fc variants and their interaction with the receptor recycling.

Mutation of residues within the plots of binding to the Fc receptor may result in a change in effector function, such as a modified ADCC or CDC activity or an altered half-life. Potential mutations include insertion, deletion or replacement of one or more residues, including substitution by alanine, a conservative substitution, a non-conservative substitution, or replacement with an amino acid residue other IgG subclass in the same position (for example replacement of IgG1 residue with a corresponding IgG2 residue at the same position). For example, it was shown that mutation of serine at position 241 amino acid sequence of IgG4 to Proline (found at that position in IgG1 and IgG2) leads to the production of a homogeneous antibody, as well as increase the half-life of serum and improvement of distribution in the tissues compared to the original chimeric IgG4 (Angal et, Mollmmunol. 30:105-8, 1993).

Fragments of antibodies are areas of intact�about poliocephala antibodies, such as antigen-binding or variable fragments of intact antibodies. Examples of fragments of antibodies include Fab, Fab', F(ab')2and Fv fragments; dyatel; linear antibodies; single-stranded molecules of antibodies (e.g., scFv); fragments multispecificity antibodies, such as bespecifically, thespecification and multispecific antibodies (e.g., diately, Triatel, tetrathele; Manantial; chelating recombinant antibodies; of tritel or Bitel; intracel; nanotesla; small modular immunopharmaceuticals agents (SMIP), anectine, fusion proteins based on the binding domain of immunoglobulins; carmelitane (camelized) antibody; VHH-containing antibodies; and any other polypeptides obtained from fragments of antibodies.

The present invention encompasses IL-1β binding fragments of antibodies, including any of the above-mentioned sequences of the heavy or light chains that bind IL-1β. The term "fragments" in this text refers to the sequence of three or more amino acids (e.g., 4 or more, 5 or more, 6 or more, 8 or more, or even 10 or more contiguous amino acids) of the antibodies and include Fab, Fab', F(ab')2and F(v) fragments, or individual variable fragments of light or heavy chains or their parts. IL-1β-binding fragments include, for example. Fab, Fab', F(ab')2, Fv and scFv. These fragmentise have the Fc fragment of intact antibody, quickly removed from the circulation and may have reduced nonspecific binding to tissues than intact antibody. Cm. Wahl and others (1983), J. Nucl. Med., 24: 316-25. These fragments can be obtained from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to get a Fab fragment) or pepsin (to obtain F(ab')2fragments).

In vitro studies and studies on cells to determine the binding of IL-1β to its receptor IL-1 type I (IL-1R1), including research, allowing to determine the presence of molecules (such as antibodies, antagonists, or other inhibitors) that bind to IL-1β or IL-1R1, well known in the art (see, e.g., Evans and others (1995), J. Biol. Chem. 270:11477-11483; Vigers and others (2000), J. Biol. Chem. 275:36927-36933; Yanofsky and others (1996), Proc. Natl. Acad. Sci. USA 93:7381-7386; Fredericks and others (2004), Protein Eng. Des. Sel. 17:95-106; Slack and others (1993), J. Biol. Chem. 268:2513-2524; Smith and others (2003), Immunity 18:87-96; Vigers et (1997) Nature 386:190-194; Ruggiero and others (1997), J. Immunol. 158:3881-3887; Guo and others (1995), J. Biol. Chem. 270:27562-27568; Svenson and others (1995), Eur. J. Immunol. 25:2842-2850; Arend and others (1994), J. Immunol. 153:4766-4774). Recombinant receptors of IL-1 type I, including the human receptor of IL-1 type I, such studies are commercially available in various sources (see, for example, R&D Systems, SIGMA). The receptor of IL-1 type I can be expressed by the construct or the expression vector, introduced into a suitable�existing host using standard methods of molecular biology and transfection, known in the art. Expressed the receptor of IL-1 type I can then identify and purify for use in studies on the binding or, alternatively, be used directly in the associated with cells of the form.

For example, the binding of IL-1β to its receptor IL-1 type I may be determined by immobilizing an IL-1β binding antibodies, the interaction (contact) of IL-1β with immobilized antibody and determining contacted IL-1R with the antibody, and the interaction of a soluble form of IL-1RI with the bound IL-1β/antibody and determining contacted soluble IL-1RI complex. The Protocol may also involve the interaction of soluble IL-1RI with immobilized antibody before exposure to IL-1β to confirm that soluble IL-1RI not contacted with the immobilized antibody. This Protocol can be carried out using the instrument Biacore® analysis of binding kinetics. Such a Protocol can also be used to elucidate, allows or blocks the antibody or other molecule binding to IL-1β with its receptor IL-1 type I.

For other studies of the binding of IL-1β to IL-1RI is possible to determine the possibility or blocking binding of IL-1β to its receptor IL-1 type I by comparing the binding of IL-1β to IL-1RI in the presence or in the absence of antibodies to IL-1β or IL-1β binding fragments. The block will be identified by visible �lowering the binding of IL-1β to its receptor IL-1 type I in the presence of antibodies to IL-1β or IL-1β binding fragments compared to a control sample, contains the corresponding buffer or diluent but not containing antibodies to IL-1β their IL-1β-binding fragments. The analysis results can be evaluated qualitatively as an indication of the presence or absence of blocking or can be quantified as a percentage or rate of attenuation of binding due to the presence of antibody or fragment.

Alternative or in addition, when IL-1β binding antibody or IL-1β binding fragment substantially blocks the binding of IL-1β to IL-1RI, the binding of IL-1β to IL-1RI is reduced at least 10-fold, alternatively, at least about 20-fold, alternatively, at least about 50-fold, alternatively, at least about 100 times alternatively at least about 1000 times, alternative, at least about 10,000 times or more compared to the binding of IL-1β and IL-1RI in the same concentrations in the absence of antibody or fragment. Another example: when IL-1β binding antibody or IL-1β binding fragment substantially permits the binding of IL-1β to IL-1RI, the binding of IL-1β to IL-1RI is at least about 90%, alternatively, at least about 95%, alternatively, at least about 99%, alternatively, at least about 99.9%, alternatively, at least about 99.99%, alternatively, at least approximately 9.999%, alternatively, at least about 99.9999%, alternatively, virtually identical to the binding of IL-1β to IL-1RI with the same concentrations in the absence of antibody or fragment.

Separate embodiments of this invention can imply that antibodies that bind IL-1β, or fragments that bind IL-1β bind to the same or substantially the same epitope as that of one or more antibodies, shown here as an example. Alternative or in addition, IL-1β binding antibody or IL-1β binding fragments compete with an antibody having the sequence of the variable segment AS described in the application U.S. No. 11/472813 (sequence given below) for binding. Alternative or in addition, the present invention encompasses IL-1β binding antibodies and fragments that bind to an epitope contained in the amino acid sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1) epitope, bound to the antibody designated AB 5 and AB (application U.S. No. 11/472813). It is implied that with the help of several known in the art methods can easily determine whether associated IL-1β binding antibody or fragment with the same epitope or substantially the same epitope as that of one or more antibodies, shown here as an example, such as, for example, the antibody designated AB.

For example, the key�new amino acid residues (epitope), associated with IL-1β binding antibody or fragment may be determined using a set of peptides, such as, for example, a peptide set PepSpot™ (JPT Peptide Technologies, Berlin, Germany), in which the scan of twelve amino-acid peptides spanning the complete amino acid sequence of IL-1β, and each peptide overlap of 11 amino acids previous peptide synthesized directly on a membrane. The membrane carrying the peptides, then probe antibody information about the epitope of which want to get, for example at a concentration of 2 μg/ml for 2 hours at room temperature. The binding of an antibody with the peptide attached to the membrane, it is possible to register using a secondary HRP-konjugierten goat anti-human (or mouse, if applicable) antibody, followed by detection of enhanced chemiluminescence (ECL). Peptide spot(a) corresponding to specific amino acid residues or sequences of the Mature protein of IL-1β, which is a positive indication of the binding with the antibody, indicate the epitope associated with a specific antibody.

Alternative or in addition it is possible to conduct experiments in competition with the antibody, and such studies are well known in the art. For example, to determine whether the associated antibody or fragment with an epitope contained with peptide seq�lovatelli, comprising amino acids ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1), which corresponds to residues 83-105 of the Mature protein of IL-1β, you can compare the antibody with unknown specificity, with any given as an example of an antibody (for example, AV) of the present invention, about which it is known that it binds to an epitope contained in the sequence. Study the competition binding can be done, for example, using a Biacore instrument® to conduct kinetic binding assays or ELISA. In this study, the antibody with an unknown epitope specificity detected by its ability to compete with the known comparator antibody (e.g., AV) for binding. Competition for binding to a specific epitope defined by a reduction of binding with the epitope of IL-1β is known comparator antibody (e.g., AB) on at least about 50%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% or about 100%, specifies to bind substantially the same epitope.

Taking into account the identification of IL-1β-binding sites are shown as an example, antibodies and/or epitopes recognized by antibodies described, it is understood that can� to be received by the secondary antibodies with similar binding characteristics and therapeutic or diagnostic benefit, which corresponds to the embodiment of the invention.

Antigen-binding antibody fragments include fragments that retain the ability to specifically contact the antigen, usually by maintaining the antigen-binding fragment of the antibody. It is generally accepted that the function of binding of the antibody to the antigen can be performed by fragments poliocephala antibodies. Examples of antigen-binding sites comprise: (i) the Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2the fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridge at the hinge region; (iii) Fd fragment, representing the VH and CH1 domains; (iv) Fv fragment, representing the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward and others (1989) Nature 341:544-546), which is a VH domain; and (vi) isolated the hypervariable segment (CDR). Single-chain antibodies are also provided for the term antigen-binding portion (fragment) of the antibody. IL-1β binding antibodies and fragments of the present invention include monovalent or multivalent, or Monomeric or multimeric (e.g. tetrameric), linking domains based CDR with a frame or without a frame (scaffold) (e.g., protein or carbohydrate skeleton).

IL-1β binding antibody or frag�patients under stood according to the present invention can be part immunoadhesin molecules of larger size, formed by covalent or noncovalent Association of the antibody or part of an antibody with one or more other protein or peptide. Examples of such immunoadhesin molecules include the use of kernel area of streptavidin to obtain a tetrameric scFv molecule (Kipriyanov, S. M. and others (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal segment of polyhistidine to obtain bivalent and biotinylated scFv molecules (Kipriyanov, S. M. and others (1994) Mol. Immunol. 31:1047-1058). Antibodies and fragments comprising immunoadhesin molecules, can be obtained by standard methods of recombinant DNA, as described in the invention. Preferred antigen-binding sites are full domains or pairs of full domains.

IL-1β binding antibodies and fragments of the present invention also include fragments, domain antibodies (dAb) (Ward et, Nature 341:544-546, 1989), including the VHdomain. IL-1β binding antibodies and fragments of the present invention also include diately, which are bivalent antibodies in which VHand VLdomains expressed as a single polypeptide chain, but using a linker that is too short for mating the two domains of the same chain that contributes to the pairing of complementary domains, belonging to different chains, and the establishment of a second antigens�anywaysi plots (see, for example, in EP 404097; WO 93/11161; Holliger and others, Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak, etc., Structure 2:1121-1123, 1994). Diatel can be bespecifically or monospecifičeskoj.

IL-1β binding antibodies and fragments of the present invention also include single-chain fragment antibodies (scFv) that binds to IL-1β. scFv comprises a variable fragment of a heavy chain antibody (VH), functionally associated with the variable fragment of a light chain antibody (VL), and a variable fragment of a heavy chain and a variable fragment of a light chain together or individually, form a binding site that binds IL-1β. scFv may include VHa fragment on the N-end and VLa fragment From the end. Alternatively, scFv can include VLa fragment on the N-end and VHa fragment From the end. Moreover, since the two domains of Fv fragment, VLand VH, are encoded by separate genes, they can be combined using the methods of recombination, using a synthetic linker that enables to receive them as a single protein chain in which the VLand VHplots coupled with the formation of monovalent molecules (known as single chain Fv (scFv); see, for example. Bird et (1988) Science 242:423-426; and Huston et (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

scFv can (not necessarily) further comprise a polypeptide linker between the variable fragment�m heavy chain and a variable fragment of a light chain. Such polypeptide linkers typically contains from 1 to 50 amino acids, alternatively, from 3 to 12 amino acids, alternative, 2 amino acids. An example of a polypeptide linker to link the heavy and light chains of scFv comprises a five-membered amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2). Other examples include one or more tandem duplication of this sequence (e.g., a polypeptide comprising from two to four repeats Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2) to create linkers).

IL-1β binding antibodies and fragments of the present invention also include antibodies that contain only the heavy chain (heavy chain antibody, HCAb). Exceptions to H2L2the structure of conventional antibodies are some isotypes of the immunoglobulins found in camelids (camelids) (camel, one-humped camels and llamas; Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J. Mol. Biol. 275: 413), carpet sharks (Nuttall and others, Mol Immunol. 38:313-26, 2001), sand sharks (Greenberg, etc., Nature 374:168-73, 1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and the spotted Chimera (spotted ratfish) (Nguyen, et al., "Heavy-chain antibodies in camelidae family; a case of evolutionary innovation," 2002 Immunogenetics 54(1): 39-47). Apparently, these antibodies can form antigen-binding sites, using only the variable fragments of the heavy chain, as these functional antibodies are dimers only of heavy chains (called antibodies, aderrasi�and only the heavy chain of heavy-chain antibodies" or "HCAb"). Accordingly, according to some embodiments of the invention IL-1β binding antibodies and fragments may be an antibody containing a heavy chain specifically binding to IL-1β. For example, antibodies containing only heavy chains belonging to the IgG class and not with light chains are produced by animals of the camelidae family, including camels, one hump camels and llamas (Hamers-Casterman et, Nature 363:446-448 (1993)). HCAb have a molecular mass of about 95 kDa is the molecular mass of approximately 160 kDa conventional IgG antibodies. Their binding domains consist only of the variable domains of the heavy chains, often referred to as VHHto distinguish them from the traditional VH(Muyldermans et, J. Mol. Recognit. 12:131-140 (1999). Variable domain of antibodies containing only the heavy chain, sometimes called nanotesla (nanobody) (Cortez-Retamozo et, Cancer Research 64:2853-57, 2004). Library of nanutel can be obtained from the immunized one-humped camels, as described in Conrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombination methods.

Due to the fact that the first constant domain (CH1) was removed in the process of splicing during mRNA processing due to loss of consensus splicing signal), variable domain (VHH) immediately below the hinge region, CH2and CH3domains (Nguyen and others, Mol. Immunol. 36:515-524 1999); Woolven, etc., Immunogenetics 50:98-101 (1999)). Suppose that VHHcamelids (Camelid) recombines with a constant sections of IgG2 and IgG3, which includes a hinge section, CH2 and CH3 domains and lacking the CH1 domain (Hamers-Casterman, etc., see above). For example, Lam IgG1 antibody is normal isotype (H2L2), where VHrecombines with a constant phase containing a hinge region, CH1, CH2 and CH3 domains, whereas IgG2 and IgG3 Lama are isotypes containing only the heavy chain having no CH1 domain and do not contain light chains.

Although HCAb devoid of light chains, they have antigen-binding repertoire. The genetic mechanism of reproduction HCAb reviewed Nguyen, etc., Adv. Immunol 79:261-296 (2001) and Nguyen and etc., Immunogenetics 54:39-47 (2002). The sharks, including baleen (carpet) shark (nurse shark) found similar Monomeric V-domains containing the antigen receptor. Irving, etc., J. Immunol. Methods 248:31-45 (2001); Roux, etc., Proc. Natl. Acad. Sci. USA 95:11804 (1998).

VHHinclude a small intact antigen-binding fragments (e.g., fragments of approximately 15 kDa, 118-136 residues). It was found that VHHdomains camels binds to the antigen with high affinity (Desmyter et, J. Biol. Chem. 276:26285-90, 2001), while the affinity of the VHHusually located in the mu range and is comparable with the affinity of the Fab and scFv fragments. VHHhighly soluble and more stable�ini, than the corresponding derivatives of scFv and Fab fragments. Until recently it was quite difficult to get the VHfragments in soluble form, but improvements in solubility and specific binding can be achieved if the frame remains, replace the residues that are more similar to VHH(see, e.g., Reichman et, J Immunol Methods 1999, 231:25-38.). VHHcontain amino acid substitutions that make them more hydrophilic and prevent prolonged interaction with BiP (protein linking the heavy chain of immunoglobulin), which is usually associated with the H-chain in the endoplasmic reticulum (ER) in the process of folding and assembling until then, until it replaces the L-chain. Due to the increased hydrophilicity VHHsecretion from ER is improved.

Functional VHHcan be obtained by proteolytic cleavage of HCAb immunized camelids, direct cloning of genes VHHfrom b-cells of immunized camelids that leads to the production of recombinant VHHor from naive (naive) or synthetic libraries. VHHwith the desired specificity towards the antigen may also be obtained by the technology of phage display. The use of VHHin phage display much easier and more efficient than Fab or scFv fragments, as in cloning and expression to obtain the functionality of�professional antigen-binding fragment needs only one domain. Muyldermans, Biotechnol. 74:277-302 (2001); Ghahroudi et, FEBS Lett. 414:521-526 (1997); and Van der Linden, etc., J. Biotechnol. 80:261-270 (2000). Methods for the production of antibody containing heavy chain camelids, also described in patent publications U.S. No. 20050136049 and 20050037421.

Technology ribosomal display can be used for identification and selection of scFv molecules and/or VHHwith the right activity and affinity of binding. Irving, etc., J. Immunol. Methods 248:31-45 (2001). Ribosome display and selection have the potential of obtaining and presentation (display) of large libraries (1014).

Another embodiment of the invention allows to obtain similar to VHHmolecules using the "kanalizacji" ("verbliebene", camelisation) through modification of the VHneverblue origin, such as human VHHto improve their solubility and prevent non-specific binding. This is achieved by replacing residues VHby VLresidues similar to VHHsimulating, thus, more soluble VHHfragments. It is expected that carmelitane VHfragments, especially based on the human frame, will have a significantly reduced immune response when administered to a patient in vivo and, accordingly, will have significant advantages for therapeuti�Eskom use. Davies, etc., FEBS Lett. 339:285-290 (1994); Davies, etc., Protein Eng. 9:531-537 (1996); Tanha, etc., J. Biol. Chem. 276:24774-24780 (2001); and Riechmann, etc., Immunol. Methods 231:25-38 (1999).

Available in a huge variety of different expression systems for the production of IL-1β fragments, including Fab fragments, scFv, and VHH. For example, systems for the expression of both prokaryotic and eukaryotic origin may be used for large-scale production of antibody fragments and fusion proteins antibodies. Particularly useful are the expression systems that allow the secretion of a large number of fragments of the antibody into the culture medium.

Develop especificacao Fab-scFv ("Bitel", bibody) and thespecifics Fab-(scFv)(2) ("tritel", tribody) described Schoonjans, etc. (J Immunol. 165:7050-57, 2000) and Willems and others (J Chromatogr In Analyt Technol Biomed Life Sci. 786:161-76, 2003). To obtain Bitel or tritel scFv molecule fused with one or both of the chains VL-CL (L) and VH-CH1(Fd), for example, to get the tritel two scFv connect with C-end Fab, while upon receipt of Bitel one scFv connect with C-end Fab. "Minitel", consisting of scFv fused to CH3 by using a peptide linker (hingeless, hingeless) or via an articulated plot IgG described Olafsen, etc., Protein Eng Des Sel. 2004 Apr; 17(4):315-23.

Intratel (intrabodies) are single-chain antibodies expressed intracellularly, which can affect the function of vnutri�mocnych proteins (Biocca, etc., EMBO J. 9:101-108, 1990; Colby et, Proc Natl Acad Sci USA. 101:17616-21, 2004). Intratel containing cell signal sequences which retain the construct of antitela in the intracellular region, can be obtained as described Mhashilkar et (EMBO J 14:1542-51, 1995) and Wheeler et al., (FASEB J. 17:1733-5. 2003). Transtel (transbodies) are antibodies that can penetrate into the cells and in which domains of the protein transduction (PTD) fused with single chain variable fragment (scFv) antibodies (Heng., Med Hypotheses. 64:1105-8, 2005).

IL-1β binding antibodies and fragments of the present invention also include antibodies, representing small modular immunopharmaceutical agents (SMIP) or fusion proteins based on the binding domain of immunoglobulins that are specific with respect to the protein target. These constructs are single-chain polypeptides containing the antigen-binding domains fused to immunoglobulin domains, necessary for the implementation of effector functions of antibodies. See, for example, WO 03/041600, patent publication U.S. 20030133939 and patent publication U.S. 20030118592.

IL-1β binding antibodies and fragments of the present invention also include immunoadhesin. One or more CDRs may be incorporated into a molecule either covalently or ecovalence to obtain immunoadhesin. Immunoadhesin may incorporate the CDR(s) as part of a larger p�dipeptide chain can covalente to connect the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) ecovalence. CDR-areas described in the invention, allow immunoadhesins specifically to contact with IL-1β.

IL-1β binding antibodies and fragments of the present invention also includes mimetics of antibodies, including one or more sections, binding of IL-1β, built on an organic or molecular skeleton (scaffold) (such as a protein or carbohydrate skeleton). Proteins that have a relatively pronounced three-dimensional structure, usually called protein cages (protein scaffolds) can be used as reagents for the design of mimetics of antibodies. These frames typically contain one or more parcels subject to a specific or random change in the sequence, wherein the randomization sequence is often performed to obtain a library of proteins, which can be selected the desired products. For example, a mimetic of an antibody may contain a chimeric polypeptide binding not-immunoglobulins, has a domain similar to immunoglobulin containing an armature having two or more loops, exposed to a solution containing another CDR of the parent antibody, is introduced into each of the loops, and having selective binding activity toward a ligand associated with the parent�Kim antibody. Protein non-immunoglobulin frameworks have been proposed to obtain proteins with new properties (properties) binding (Tramontane, etc., J. Mol. Recognit. 7:9, 1994; McConnell and Hoess, J. Mol. Biol. 250:460, 1995). Other proteins have been tested as frames and were used for the presentation (display) of randomized residues in alpha-helical surfaces (Nord and others, Nat. Biotechnol. 15:772, 1997; Nord, etc., Protein Eng. 8:601, 1995), the loop between alpha-helices in alpha-helical bands (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops created by disulfide bridges, such as in young of protease inhibitors (Markland, etc., Biochemistry 35:8045, 1996; Markland, etc., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164:243, 1995; Wang and others, J. Biol. Chem. 270:12250, 1995). How to use frames to obtain mimetics of antibodies described in U.S. patent 5770380 and patent publications U.S. 2004/0171116, 2004/0266993 and 2005/0038229.

Preferred antibodies to IL-1β or fragments of such antibodies for use according to the invention typically bind to human IL-1β with high affinity (e.g., as determined with BIACORE), such as, for example, the equilibrium dissociation constant (KDfor IL-1β of about 10 nm or less, about 5 nm or less, about 1 nm or less, about 500 PM or less, or more preferably about 250 PM or less, about 100 PM or less, about 50 PM or less, an example�about 25 PM or less, about 10 PM or less, about 5 PM or less, about 3 PM or less about 1 PM or less, about 0.75 PM or less, about 0.5 PM or less, or about 0.3 PM or less.

The antibodies or fragments according to the invention can, for example, be contacted with IL-1β with IC50about 10 nm or less, about 5 nm or less, about 2 nm or less, about 1 nm or less, about 0.75 nm or less, about 0.5 nm or less, about 0.4 nm or less, about 0.3 nm or less, or even about 0.2 nm or less, as determined using enzyme-linked immunosorbent assay (ELISA). Preferably, the antibody or antibody fragment of the present invention shall not enter into cross-react with targets than IL-1. For example, antibodies and fragments of the present invention can be contacted with IL-1β, but not associated with IL-1α to the extent discoverable, or have at least about 100 times (e.g., at least about 150 times, at least about 200 times, or even at least about 250 times) greater selectivity of binding of IL-1β compared to the binding of IL-1α. Antibodies or fragments used according to the invention, in some embodiments of the invention inhibit induced IL-1β expression of serum IL-6 in animals, at least 50% (e.g., at least 0%, at least 70%, or even at least 80%) in comparison with the level of IL-6 in serum of animals stimulated with IL-1β, which has not been entered, the antibody or fragment according to the present invention. Antibodies can bind IL-1β, but to prevent or substantially prevent binding of the bound ligand IL-1β with its receptor IL-1 type I (IL-1RI). Unlike many of the known IL-1β binding antibodies that block or substantially prevent the binding of IL-1β to IL-1RI, antibody designated AB 5 and AB (application U.S. No. 11/472813), selectively associate the ligand IL-1β, but leave open the possibility of binding of the bound ligand IL-1β to IL-1RI. For example, the antibody designated AB, binds to the epitope of IL-1β, but leaves the possibility of binding of the bound IL-1β to IL-1RI. In some embodiments of the invention, the antibody may reduce the affinity of interaction of the bound IL-1β to IL-1RI. Accordingly, the invention includes, in an appropriate aspect, the IL-1β binding antibody or IL-1β binding antibody fragment having at least one of the above characteristics. Any of the above-mentioned antibodies, fragments of antibodies or polypeptides of the present invention may be humanized or designed for people, as described in the invention.

Many antibodies to IL-1 (e.g. IL-1β) and fragments, Izv�STN from the prior art, can be used according to the claimed methods, including, for example, antibodies described or designed using the methods disclosed in the following patents and patent applications: U.S. patent 4935343; U.S. patent 2003/0026806; U.S. patent 2003/0124617; WO 2006/081139; WO 03/034984; WO 95/01997; WO 02/16436; WO 03/010282; WO 03/073982. WO 2004/072116, WO 2004/067568, EP 0267611 B1, EP 0364778 B1, and the patent application U.S. 11/472813. As limitiruesh examples of antibody AB 5 and AB (application for U.S. patent No. 11/472813, WO 2007/002261), which can be used according to the invention. Sequences of the variable regions AB 5 and AB below:

AB 5

LIGHT CHAIN

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSKLHSGV

PSRFSGSGSGTDYSLTISNLEOEDIATYFCLQGKMLPWTFGGGTKLEIK (SEQ ID NO: 3)

The underlined sequences are (left to right) CDR1, 2 and 3.

HEAVY CHAIN

OVTLKESGPGILKPSOTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDG

DESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCARNRYDPPWFVDWGOGT

LVTVSS (SEQ ID NO: 4)

The underlined sequences are (left to right) CDR1, 2 and 3.

AV

LIGHT CHAIN

DIOMTOSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYYTSKLHSGV

PSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPWTFGQGTKLEIK (SEQ ID NO: 5)

The underlined sequences are (left to right) CDR1, 2 and 3.

HEAVY CHAIN

OVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDG

DESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCARNRYDPPWFVDWGOGT LVTVSS (SEQ ID NO: 6)

The underlined sequences are (left to right) CDR1, 2 and 3.

Described in the invention�and antibodies and fragments of antibodies can be obtained by any suitable method. Suitable methods for obtaining such antibodies and fragments of antibodies are known in the art. Other methods for producing such antibodies and fragments of antibodies described in the invention as an integral part of the invention. The antibody, antibody fragment, or polypeptide of the present invention, as described in the invention can be isolated or purified to any degree. In this context, a "dedicated connection" refers to a compound isolated from its natural environment. The purified compound is a compound, the purity of which is increased so that the compound exists in a form more pure than it exists (i) in its natural environment, or (ii) in the conditions of a synthesized source and/or amplification in laboratory conditions, wherein "purity" is a relative term and does not necessarily mean "absolute purity."

The pharmaceutical composition

Antibodies and fragments of antibodies that bind IL-1 (e.g. IL-1β) for use according to the present invention can be included in compositions, especially pharmaceutical compositions, for use according to the methods of the present invention. Such compositions comprise a therapeutically or prophylactically effective amount of IL-1β binding antibody or antibody fragment according to �the turbine zobretenie in admixture with a suitable carrier, for example, a pharmaceutically acceptable agent. Usually, antibodies and fragments of antibodies that bind IL-1, according to the present invention before the preparation of the pharmaceutical compositions are substantially purified for administration to an animal.

Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavoring and diluting agents, emulsifying agents, suspendresume tools, solvents, excipients, buffers, carriers for delivery, drugs affecting the correct tonicity, co-solvents, wetting (wetting) funds komplektuyuschie funds, buffering agents, antimicrobials, and surfactants.

Neutral buffered saline or saline (physiological) solution, mixed with albumin are examples of suitable carriers. Pharmaceutical compositions may include antioxidants such as ascorbic acid, low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA (ethylenediaminetetraacetic acid); �acharnae alcohols, such as mannitol or sorbitol; counterions of the salts, ions such as sodium; and/or nonionic surfactants such as tween, pluronic or polyethylene glycol (PEG, PEG). Also as an example of suitable means for increasing toychest include the halides of alkali metals (preferably sodium chloride or potassium), mannitol, sorbitol and similar. Suitable preservatives include benzalkonium chloride, thimerosal, finitely alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and similar. As preservative can also be used hydrogen peroxide. Suitable co-solvents include glycerin, propylene glycol and PEG. Suitable komplektuyuschie agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropylcellulose. Suitable surfactants or wetting agents include esters of sorbitol, Polysorbate, such as Polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol and similar. As buffers, you can use normal buffers, such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Tae buffer can have a pH of about 4-5.5, and Tris-buffer can have a pH of about 7-8.5. Additional pharmaceutical agents are described in Remington's Pharmaceutical Sciences, 18th Edition, edited by A. R. Gennaro, Mack Publishing Company, 1990.

<> The composition may be in liquid form or dried by freeze-drying or freeze-dried and may include one or more bioprotector, filler, surfactant, high molecular weight structural additive and/or filler (see, for example, U.S. patents 6685940, 6566329 and 6372716). According to one embodiment of the invention the composition includes iprotector representing nevosstanovlenie sugar, such as sucrose, lactose or trehalose. Bioprotector typically include in such quantity that when the reconstruction of the resulting formulation was isotonic, although hypertonic hypotonic or weakly formulations may also be suitable. In addition, the number of bioprotector should be sufficient to prevent an unacceptable degree of degradation and/or aggregation of the protein during lyophilization. For example, the concentration of bioprotector for sugars (e.g., sucrose, lactose, trehalose) in preparations before freeze drying is from about 10 mm to about 400 mm. In another embodiment, the composition includes a surfactant, such as, for example, nonionic surfactants and ionic surfactants, such as Polysorbate (for example, Polysorbate 20, Polysorbate 80); poloxamer (e.g. gender�xamar 188); phenyl ethers of polyethylene glycol (e.g., Triton); sodium dodecyl sulfate (SDS); laurilsulfate sodium; octylglucoside sodium; lauryl-, myristyl-, linoleyl - or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl - or stearyl-sarcosine; linoleyl, myristyl - or cetyl-betaine; lauramidopropyl, cocamidopropyl, linoleamide, myristamide, alminoprofen - or isostearamide-betaine (for example, lauramidopropyl); ministernotomy, alminoprofen - or isostearamide-dimethylamine; metalcolor sodium, Cocoyl - or centripetality; and the MONAQUAT series™. (Mona Industries, Inc., Paterson, N. J.), polyethylene glycol, polypropylene glycol and copolymers of ethylene glycol and propylene glycol (e.g., pluronic PF68 etc). The number of surfactants that may be contained in the preparations prior to freeze drying, may be, for example, about 0.001-0.5%. High molecular weight structural additives (e.g., fillers, binders) may include, for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, sodium salt of carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, dextran, dextrin, dextrine, sucrose, Tilos, pregelatinization starch, sulfate ka�ice, the amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, acidic disodium phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragakant, microcrystalline cellulose, starch and Zein. The approximate concentration of high molecular weight structural additives comprise from 0.1% to 10% by weight. In other embodiments of the invention in the composition may include a filler (e.g., mannitol, glycine).

The compositions can be suitable for parenteral administration. Given as an example of a composition suitable for injection or infusion (infusions) animal by any available qualified employee in a manner such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, inside the lesion (intralesional), vnutriuretralnami, transdermal, oral administration and the introduction by inhalation. The formulation for parenteral administration is typically a sterile, non-pyrogenic, isotonic aqueous solution, optionally containing pharmaceutically acceptable�contain preservatives.

Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and suitable for injectable organic esters, such as ethyloleate. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions., including saline and buffered environment. Carriers for parenteral administration include sodium chloride solution, ringer's solution with dextrose, the dextrose with sodium chloride, ringer's solution with lactose, or fixed oils. Carriers for intravenous administration include liquid and nutritious fillers, electrolyte fillers, such as those based on ringer's solution with dextrose, etc. May also be present preservatives and other additives, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, etc. in General, Remington s Pharmaceutical Science, 16th Ed., Mack Eds., 1980, included in the invention in the form of a link.

The pharmaceutical composition described in the invention, can be formulated for controlled or sustained (continuous) delivery, providing a local concentration of the product (e.g., a bolus, the effect of delayed suction), long lasting (continuous) release and/or increased stability or half-time in a particular local environment. Invent�their provides, in certain embodiments of the invention, such compositions may include a substantially greater amount of antibody or fragment in the original drug, while effective amount of antibody or fragment, is really released and available at any time, it appears, in accordance with the invention, significantly less than the original drug. The compositions can include combinations (formulations) IL-1β binding antibodies, fragments of antibodies, nucleic acids or vectors of the present invention with particulate preparations of polymeric compounds such as polylactic acid, polyglycol acid, etc., and means, such as biodegrability matrix (matrix), suitable for injection of microspheres, microencapsulated particles, microcapsules, biodegradable granules, liposomes, and implantable devices for delivery, providing controlled or prolonged release of the active agent, which can then be delivered in the form of an injection agent slow suction. The technology of preparation of such funds for the controlled or sustained delivery is known, and we developed and used a variety of polymers for controlled release and drug delivery. Such polymers are usually of biodegra�interface and biocompatible. Polymer hydrogels, including gels, formed by complexation of enantiomeric polymer or polypeptide segments, and the temperature - or pH-sensitive hydrogels may be desirable to allow the effect of delayed absorption due to the soft and water conditions of encapsulation (traps, trapping) bioactive proteinaceous agents (e.g., antibodies). See, for example, the description of a controlled release polymeric microparticles for delivery of pharmaceutical compositions in the publication of the PCT application WO 93/15722.

Suitable materials for this purpose include polylactic acid called PLA (see, e.g., U.S. patent 3773919), polymers of poly-α-hydroxycarbonic acids such as poly-D-(-)-3-hydroxybutyric acid (EP 133988 A), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, etc., Biopolymers, 22: 547-556 (1983)), poly-2-hydroxyethyl-methacrylate (Langer et, J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. Other biodegradable polymers include polylactide, Polyacetal, polyarteritis and polycholorinated. Composition for prolonged release can also include liposomes, which can be obtained by any known in the art (see, e.g., Eppstein and others, Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The device itself or products of its degradation�tion must be non-toxic to the target tissue and should not worsen the condition. This can be achieved in routine screening models target disorders in animals or, if such models are not available in normal animals.

Microencapsulation recombinant proteins for prolonged-release has been successfully performed with human growth hormone (human growth hormone, rhGH), interferon- (rhIFN--), interleukin-2, and MN rgp120. Johnson and others, Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et, Bio/Technologv. 8:755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds (Plenum Press: New York, 1995), pp.439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. patent No. 5654010. Were developed formulations for the prolonged release of these proteins with the use of a copolymer of lactic and glycolic acid (poly-lactic-coglycolic acid, PLGA) due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids, can be quickly removed from the human body. Moreover, degradiruemosti this polymer may depend on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp.1-41. Additional examples of compositions for extended release is described for example in EP 58481 A, U.S. patent No. 3887699, EP 158277 AND, in the patent Canada No. 1176565, U. Sidman et, Biopolymers 22, 547 [1983], . Langer and others, Chem. Tech. 12, 98 [1982], Sinha and others, J. Control. Release 90, 261 [2003], Zhu and others, Nat. Biotechnol. 18, 24 [2000] and Dai and others, Colloids Surf In Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also intended for use as part of or together with the compositions of the present invention. Bioadhesive are synthetic and natural materials, is capable of adhering to biological substrates for long time intervals. For example, carbopol and polycarbophil are both synthetic crosslinked polyacrylic acid derivatives. Bioadhesive delivery systems based on natural materials include, for example, hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a natural mucopolysaccharide consisting of D-glucuronosyl residues and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular matrix of vertebrate tissues, including connective tissue and in synovial fluid and in the vitreous body and aqueous humor. Esterified derivatives of hyaluronic acid have been applied to obtain microspheres for use in delivery as biocompatible and biodegradable (see, for example, Cortivo, etc., Biomaterials (1991) 12:727-730; EP 517565; international publication WO 96/29998; Illum, etc., J. Controlled Rel. (1994) 29:133-141). Examples of compositions according to the invention containing hyaluronic keys�otoo, include polymeric ester of hyaluronic acid in an amount of from about 0.1% to about 40% (by weight) of the IL-1β binding antibody or fragment in relation to the polymer based on hyaluronic acid.

In the composition of compositions for delivery according to the present invention can be used as biodegradable and non biodegradable polymer matrix. Such a polymer matrix can include natural or synthetic polymers. Biodegradable matrices are preferred. The time period during which the release occurs, depends on the choice of polymer. Usually the release during the period from several hours to three to twelve months is most desirable. Examples of synthetic polymers that can be used to obtain the biodegradable delivery system include:

polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkenes, polyalkylphenol, polyalkoxy, polyalkenoate, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolide, polysiloxane, polyanhydride, polyurethanes and their copolymers, politicsnow acid, bolivarianos acid, alkylaryl, hydroxyethylcellulose, ethers of cellulose, esters of cellulo�s, nitrocellulose, polymers based on esters of acrylic and methacrylic acids, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethylmethylcellulose, cellulose acetate, cellulose propionate, acetabular cellulose, acetated cellulose, carboximetilzellulozu, cellulose triacetate, sodium salt of cellulose sulfate, polymethyl methacrylate, polimetilmetakrilat, polybutylmethacrylate, polyisobutylene, polyacrylnitrile, polyisobutylcyanoacrylate, polyarylethers, polivinilatsetat, polymethylacrylate, polyisopropylene, polyisobutylene, paleoclimatologist, polyethylene, polypropylene, polyethylene glycol, polyethylene oxide, polyethylene terephthalate, polyvinyl alcohols, polyvinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone. Examples of natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives (substitution, additions of chemical groups, such as, for example, alkyl, alkylene, hydroxylation, oxidation and other modifications routinely carried out by the specialists), albumin and other hydrophilic proteins, Zein and other prolamins and hydrophobic proteins, copolymers and mixtures thereof. Generally speaking, these materials are broken down (degraded) or in the process of the enzyme�active hydrolysis or exposure to water by surface or bulk erosion in vivo. The polymer (not necessarily) is in the form of a hydrogel (see, e.g., WO 04/009664, WO 05/087201, Sawhney, et, Macromolecules, 1993, 26, 581-587), capable of absorbing up to about 90% of its weight in water and further, optionally, is (transversely) crosslinked multivalent ions or other polymers.

Delivery systems also include polimernye systems that are lipids, including sterols, such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono -, di - and triglycerides; system release based hydrogels, elastique systems; systems based on peptides; wax coatings; tablets, extruded using conventional binders and excipients, partially fused implants, etc. are Typical examples include, but are not limited to only these: (a) erosional systems in which the product is contained inside the matrix, such as described in U.S. patents №№4452775, 4675189 and 5736152, and (b) diffusional systems in which the product at a controlled rate out of the polymer, such as described in U.S. patents №№3854480, 5133974 and 5407686. Liposomes containing the product can be obtained by known methods, such as, for example (DE 3218121; Epstein et, Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang, etc., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; the patent application of Japan 83-118008; U.S. patents №№4485045 and 4544545; and EP 102324).

Pharmaceutical compositions containing an antibody or fragment that bind IL-1β, can be formulated for inhalation in the form, such as, for example, dry powder. Solutions for inhalation can be prepared in a condensed liquid in an aerosol spray for delivery by aerosol. According to another formulation, the solutions can be sprayed. Additional pharmaceutical compositions for administration to the lungs include described, for example, in the publication of the PCT application WO 94/20069, which describes the delivery into the lungs of chemically modified proteins. For delivery to the lungs, the particle size should be suitable for delivery to peripheral portions of the lungs. For example, the particle size may be from 1 μm to 5 μm; at the same time may be used and particles of larger size, for example, if the particle is fairly porous.

Some formulations containing IL-1β binding antibodies or fragments of antibodies, can be administered orally. Recipes entered in this way can be with or without carriers customarily used in the preparation of solid dosage forms such as tablets or capsules. For example, a capsule may be designed to release the active portion of the formulation in the place of the gastrointestinal tract, where the bioavailability reaches a maximum and desestima degrades�I minimum. Can additional funds be included to enhance absorption of selectively binding agent. Diluents, flavorings, low melting waxes, vegetable oils, lubricants, suspendresume funds, agents that promote decomposition of the tablets, and binders may also be used.

Another recipe (preparation) may include an effective amount of IL-1β binding antibody or fragment in a mixture with non-toxic excipients that are suitable for manufacture of tablets. Solutions in the form of standard doses can be prepared by dissolving the tablets in sterile water or other suitable media. Suitable fillers include, but are not limited to these, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders, such as starch, gelatin or acacia, or lubricating agents such as magnesium stearate, stearic acid or talc.

Suitable and/or preferred pharmaceutical formulations can be adjusted, taking into account the present invention and common knowledge of the technology of preparation of medicines, depending on the intended route of administration, delivery format and desired dosage. Regardless of the method of administration, the effective dose can be calculated in accordance with the mass �ate patient the body surface area or body size. Further refinement of the calculations to determine the appropriate dosage for treatment involving each of the formulations described herein, known in the art and routinely carried out by specialists. Suitable dosages can be determined using relevant data dose - response.

Taking into account this statement of invention, additional formulations are obvious, including IL-1β binding antibodies and fragments in combination with one or more other therapeutic agent. For example, in some formulations of IL-1β binding antibody, antibody fragment, nucleic acid or vector of the present invention is combined with a second inhibitor of the signaling pathway of IL-1. Representatives of such second inhibitors are (but not limited to, antibodies, fragments of antibodies, peptides, polypeptides, corresponding mixtures, nucleic acids, vectors and pharmaceutical compositions, such as, for example, described in documents US№№6899878, 2003022869, 20060094663, 20050186615, 20030166069, WO/04022718, WO/05084696, WO/05019259. For example, the composition may include IL-1β binding antibody, antibody fragment, nucleic acid or vector according to the invention in combination with IL-1β binding antibody, fragment, or nucleic acid, or vecto�Ohm, encoding such antibody or fragment.

The pharmaceutical composition may include antibodies or fragments that bind IL-1β, in combination with other active agents. Such combinations are combinations to achieve a goal. Combinations that form part of the invention are combinations of antibodies to IL-1β and fragments, such as, for example, described in the invention, at least one additional agent, selected from the list below. Listed below active agents are provided to illustrate and not limit the list of possible. The combination can also include more than one additional agent, e.g., two or three additional funds, if thus prepared composition can perform its intended function.

The invention also refers to pharmaceutical compositions containing one or more active agents may be administered separately from the IL-1β binding antibodies or fragments, and the introduction of these methods separately can be delivered in the same or in different moments of time, for example, on the same day or on different days. The introduction of other active agents can be carried out by standard methods of medical practice, known in the art, or the method of administration can be modified (for example�EP, large intervals, lower doses, the introduction of late) when used in conjunction with the introduction of the IL-1β binding antibodies or fragments, such as described in the invention.

Active agents or in combination with antibodies or fragments of the present invention include non-steroidal anti-inflammatory drugs (NSAID) such as aspirin, ibuprofen, and other propionic acid derivatives (alminoprofen, benoxaprofen, bulochnikova acid, carprofen, fenbufen, fenoprofen, Florien, flurbiprofen, indoprofen, Ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and toxaphen, acetic acid derivatives (indomethacin, acemetacin, alclofenac clidanac, diclofenac, fenclofenac, fenclova acid, fentiazac, furofenac, ibufenac, isoxepac, exping, sulindac, tiopinac, tolmetin, zidometacin and zomepirac), the derivative Funambol acid (flufenamic acid, meclofenamic acid, mefenamic acid, niflumova acid and telenova acid), derivatives biphenylcarboxylic acid (diflunisal and flufenisal), oxicam (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (Amazon, baseperiod, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). Other combinations of flucythrinate cyclooxygenase-2 (COX-2). Other active agents for combinations include steroids, such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. This combination may be particularly useful because one or more of the side effects of the steroid can be reduced or even eliminated by gradually reducing the dose of steroid needed to treat patients in combination with the inventive antibodies and fragments.

Alternative or in addition can be used therapeutic method of treating at least one or more additional active agent active on a different mechanism (other way): 1) sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and/or meglitinide (for example, Repaglinide, nateglinide), which markedly stimulates insulin secretion; 2) biguanides (e.g., Metformin) promote the utilization of glucose by reducing glucose production by the liver and reducing the supply of glucose from the intestines; 3) inhibitors of alpha-glucosidase (e.g., acarbose, miglitol) slow the breakdown of carbs and, as a result, absorption from the intestinal tract and reduce afternoon hyperglycemia; 4) thiazolidinediones (e.g., troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, tropic�areas englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP 221, MK-0767, ciglitazone, aleglitazar, CLX 0921, darglitazone, MS 92768, VM 152054) enhance the action of insulin, thereby contributing to the utilization of glucose in peripheral tissues; 5) a peptide similar to glucagon, including DPP4 inhibitors (e.g., sitagliptin); and 6) insulin stimulating glucose utilization by tissues and inhibits the flow of glucose from the intestines. Glucagon-like peptide-1 (GLP-1), DPP-IV-resistant analogues (mimetics incretin), inhibitors of DPP-IV, insulin, insulin analogues, agonists, PPAR-gamma, PPAR agonists double-acting agonists or analogues of GLP-1 inhibitors RTRV, SGLT inhibitors, agents that increase insulin secretion, RXR agonists, inhibitors of glycogen-synthase-kinase-3, insulin sensitizers, immune modulators, agonists of adrenergic receptors beta-3, agonists, Pan-PPAR inhibitors llbeta-HSDl, analogues amilina, biguanides, inhibitors of alpha-glucosidase, meglitinides, thiazolidinediones, sulfonylureas, etc. can also be used as the other active agent(s) (see, e.g., Nathan, 2006, N. Engl. J. Med. 355:2477-2480; Kahn et al., 2006, N. Engl. J. Med. 355:2427-2443). In another embodiment, the active agent may be an inhibitor of HMG Co-A reductase inhibitors (e.g. statins).

It is also envisaged that the antibody to IL-1β or the fragment, introduced the subject in accordance with the invention�, can be introduced in combination with treatment with at least one additional active agent such as, for example, any of the previously active agents. According to one embodiment of the invention, the treatment of at least one active agent to continue at the same level. In another embodiment of the invention, the treatment of at least one active agent reduce or suspend (for example, when the subject is stable), while treatment with antibody to IL-1β or the fragment continues at a constant dosing regimen. In another embodiment of the invention, the treatment of at least one active agent reduce or suspend (for example, when the subject is stable), and treatment with antibody to IL-1β or the fragment reduce (e.g., reduce the dose, reduce the frequency of dosing, shorten treatment program). In another embodiment of the invention, the treatment of at least one active agent reduce or suspend (for example, when the subject is stable), and treatment with antibody to IL-1β or the fragment of increase (e.g., increase the dose, increase the frequency of dosing, increase the duration of treatment). In another embodiment, the treatment of at least one active agent retains at the same level, while treatment�e antibody to IL-1β or the fragment reduce or suspend (for example, reduce dosage reduce the frequency of dosing, shorten treatment program). In another embodiment, the treatment of at least one active agent and treatment with antibody to IL-1β or the fragment reduce or suspend (for example, reduce the dose, reduce the frequency of dosing, shorten treatment program).

The pharmaceutical composition used according to the invention may include a therapeutically effective amount or prophylactically effective amount of IL-1β binding antibodies or fragments. The term "therapeutically effective amount" refers to the amount effective at dosages and compliance time periods required to achieve the desired therapeutic effect. A therapeutically effective amount of the antibody or antibody fragment may vary depending on factors such as stage of disease, age, sex and weight of the individual, and the ability of the antibody or part thereof to cause the desired response in the individual. A therapeutically effective also is the amount for which any toxic or detrimental effects of the antibody or fragment of antibody are outweighed by therapeutically beneficial effects. The term "prophylactically effective amount" refers to the amount, effective with respect �of ozirovo and time periods, necessary to achieve the desired prophylactic result.

A therapeutically or prophylactically effective amount of a pharmaceutical composition containing IL-1β-binding.the antibody or fragment will depend, for example, for therapeutic purposes, such as indications (symptoms), treatment which uses an arrangement, the mode of introduction, the status of the subject. The pharmaceutical composition is administered in a therapeutically or prophylactically effective amount for the treatment of IL-1-dependent state. According to the present invention a "therapeutically or prophylactically effective amount of IL-1β binding antibody or fragment according to the present invention is an amount that can treat or prevent one or more symptoms of IL-1-dependent disease in a subject, as described in the invention.

Ways to use

According to the present invention antibodies against IL-1β in the effective amount can be used for the treatment and/or prevention of type 1 diabetes, type 2 diabetes, obesity, hyperglycemia, hyperinsulinaemia, insulin resistance and disease States characterized by insulin resistance. Such methods can be used to treat mammals (e.g., humans) suffering from type 2 diabetes, type 1 diabetes, obesity, hyperg�ikeji, hyperinsulinaemia, insulin resistance and disease States characterized by insulin resistance, or for preventing such conditions in subjects at risk.

The terms "preventing", "prevent", "prevention", "preventing", "suppression", "suppress", "inhibit" and "inhibition" refer to the mode of action (such as the introduction of a compound or pharmaceutical composition) initiated (e.g., before the manifestation of clinical symptoms of the disease state) to prevent, suppress and reduce, temporarily or permanently, the appearance (manifestation) of clinical signs of the disease state. Such prevention, inhibition or reduction need not be absolute to be useful.

The terms "treatment" and "cure" in this context, refers to method steps (such as the introduction of a compound or pharmaceutical composition) initiated after the onset of clinical symptom of the disease condition, to eliminate, reduce, suppress or enhance temporarily or permanently clinical manifestation or development of the disease condition. Such treatment need not be absolute to be useful.

In this context, the term "in need of treatment" refers to the proposition expressed by people�m, caring for the individual, that the patient needs treatment or will benefit from treatment. This judgment may be based on a variety of factors that are in the area of competence (experience, knowledge) caring person, but includes the knowledge that the person is sick or will be sick because of the conditions that can be treated using the method or substance according to the present invention.

In this context, the term "in need of prevention" refers to the judgment made by the person who cares for the individual, that the patient in need of prevention or will benefit from prevention. This judgment may be based on a variety of factors that are in the area of competence (experience, knowledge) caring person, but includes the knowledge that people will be sick or may become sick because of the conditions that can be prevented by the use of a method or substance according to the invention.

The term "therapeutically effective amount" in this context refers to the amount of a substance (e.g., antibodies), by itself or in pharmaceutical compositions, which can cause any detected positive effect on any symptom, aspect or symptom of the disease condition when administered to a patient (e.g., in the form of one or more doses). �which the effect does not have to be absolute, to be useful.

In this context, the term "insulin resistance" refers to the state when the normal amount of insulin is not able to induce normal physiological or molecular response.In some cases, increased compared with physiological amounts of insulin, or secreted endogenous or introduced exogenously, able to overcome the insulin resistance, fully or partially, and to cause a biological response.

Antibodies against IL-1β or fragments can be administered to the people in an effective amount for the treatment and/or prophylaxis of type 2 diabetes, type 1 diabetes, obesity, hyperglycemia, hyperinsulinaemia, insulin resistance and/or disease States characterized by insulin resistance. Other disease or condition that can be treated with antibodies to IL-1R or the corresponding fragments of the present invention include pre-diabetes, dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome and painful condition. The invention also includes methods of using such antibodies or fragments, to reduce the incidence or severity of diseases, or stabilize complications or conditions associated with type 2 diabetes, such as retinopathy, renal failure, cardiovascular with�the system (for example, atherosclerosis, peripheral vascular disease), and healing of wounds (e.g. diabetic ulcers).

The invention also includes methods of using antibodies to IL-1β and fragments to reduce the level of C-reactive protein (CRP) in a subject as described in the invention. CRP is an acute phase protein that is produced predominantly by hepatocytes under the influence of cytokines such as IL-1, IL-6 and tumor necrosis factor (TNF). According to the electronic version of the 2007 guidelines on treatment of internal disease UpToDate®, despite the lack of specificity of the causes of inflammation (e.g., infection, chronic kidney disease, autoinflammatory disease, cancer), the data is more than 30 epidemiological studies have shown a significant Association between increased concentrations of CRP in serum or plasma and the widespread underlying atherosclerosis, the risk of recurrent cardiovascular attacks among patients with established disease, and the occurrence of heart attacks among individuals who are at risk for atherosclerosis. The interaction with the underlying disease of the kidneys, leading to renal failure with oxidative stress and post-synthetic modification of proteins, dialysis, associated pollutants, and the impact of dialysis membranen serum proteins, and infection associated with re-introduction of the access site, and subsequent systemic infection lead these patients to exorbitant freight incentives inflammation. As the levels of purification of serum creatinine from falling as the weakening of the kidney, there is a proportional increase of inflammatory mediators in serum (e.g., TNF, IL-6, IL-1), and the obvious attempts of the body to deal with this situation by increasing (but not sufficient) production of anti-inflammatory mediators IL-1 RA and IL-10. This is an inflammatory condition in patients with chronic renal failure leads to instability of atherosclerotic plaques due to direct apoptosis of smooth muscle cells of blood vessels. Increased levels of cytokines leads to one of the two major causes of death in these patients - a startling increase in deaths from cardiovascular disease due to myocardial infarction and strokes. A direct illustration of this increased risk is seen in clarifying the levels of CRP in patients: when dividing patients into four groups depending on the level of CRP in the group with higher CRP levels mortality is about 35% within 12 months. Thus, the present invention discloses the use of antibodies to IL-1β or fragments according to the invention to reduce the levels of CRP in tacapariyanto (for example, the subjects suffering from kidney disease). Reduction of CRP levels in a subject, as described in the invention is a suitable way to achieve a corresponding proportional reduction in morbidity and mortality from cardiovascular diseases.

According to one embodiment of the invention, the antibody to IL-1β or the fragment is administered to a subject suffering from at least one of the above diseases, conditions or complications, and the subject also receives at least one otherwise acceptable from a medical point of view, the treatment of the disease, condition or complication (eg, medication, drug therapy, active agent). In another embodiment of the invention, at least one otherwise acceptable from a medical point of view, the treatment of the disease, condition or complication is reduced or suspended (e.g., when the subject is stable), while treatment with antibody to IL-1β or the fragment is maintained at a constant dosing regimen. In another embodiment of the invention, at least one otherwise acceptable from a medical point of view, the treatment of the disease, condition or complication is reduced or suspended (e.g., when the subject is stable), and treatment with antibody to IL-1β or the fragment reduce (e.g., reduce d�of Siroki, reduce the frequency of dosing, shorten treatment program). In another embodiment of the invention, at least one otherwise acceptable from a medical point of view, the treatment of the disease, condition or complication is reduced or suspended (e.g., when the subject is stable), and treatment with antibody to IL-1β or the fragment of increase (e.g., increase the dose, increase the frequency of dosing, increase the duration of treatment). In another embodiment of the invention, at least one otherwise acceptable from a medical point of view, the treatment of the disease, condition or complication leave at the same level and treatment with antibody to IL-1β or the fragment reduce or suspend (for example, reduce the dose, reduce the frequency of dosing, shorten treatment program). In another embodiment of the invention, at least one otherwise acceptable from a medical point of view, the treatment of the disease, condition or complication and treatment with antibody to IL-1β or the fragment reduce or suspend (for example, reduce the dose, reduce the frequency of dosing, shorten treatment program).

In the preferred methods of treatment or prophylaxis of the above diseases or conditions (e.g., type 1 diabetes, type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity, ones�spine to insulin) antibody to IL-1β or a fragment thereof is administered to people according to the above-mentioned number of doses, the size of the dose and/or the intervals between injections. Alternatively, an antibody to IL-1β or the fragment can be administered in the form of one or more initial doses in the above amounts that are smaller than the values of one or more subsequent doses. The decrease in the value of the initial dose (dose) may increase the efficacy and/or tolerability. For example, in one limitiruesh embodiment of the invention, you can enter one or more initial doses (e.g., 1, 2, 3, 4, 5) the antibody or fragment in the amount of ≤1 mg/kg (e.g., ≤0.9 mg/kg ≤0.8 mg/kg ≤0.7 mg/kg ≤0.6 mg/kg, ≤0.5 mg/kg, ≤0.4 mg/kg ≤0.3 mg/kg ≤0.2 mg/kg ≤0.1 mg/kg, ≤0.05 mg/kg ≤0.03 mg/kg ≤0.01 mg/kg), followed by introduction of one or more subsequent doses, the magnitude of which is greater than the value of the initial dose(s) (e.g., ≥0.01 mg/kg, ≥0.03 mg/kg, ≥0.01 mg/kg, ≥0.3 mg/kg≥0.5 mg/kg ≥0.6 mg/kg, ≥0.7 mg/kg, ≥0.8 mg/kg, ≥0.9 mg/kg, ≥1.0 mg/kg, ≥1.5 mg/kg, ≥2 mg/kg ≥2.5 mg/kg, ≥3 mg/kg, ≥3.5 mg/kg, ≥4 mg/kg, ≥4.5 mg/kg, ≥5 mg/kg). The invention provides that each dose of the antibody or fragment may be administered in one or more places.

Methods for the treatment or prophylaxis of a disease or condition in accordance with the present invention may involve a pre-defined or "routine" schedule for the introduction of the antibody or fragment. In this context, a routine schedule for the introduction refers zaranee defined and assigned to a time interval between doses. Routine schedule may involve the same or different duration time intervals, pre-determined schedule. Any individual combination is a routine schedule up until the pre-determined that appropriate graph includes the introduction of a specific day.

The invention also provides for antibodies to IL-1β or the fragment used according to the claimed methods can be introduced in combination with more traditional methods of treatment and pharmaceutical compositions (e.g., active agents). Such compositions may include, for example, inhibitors of DPP-IV, insulin, insulin analogues, agonists, PPAR-gamma, PPAR agonists double-acting agonists or analogues of GLP-1 inhibitors RTR IN, SGLT inhibitors, agents that increase insulin secretion, RXR agonists, inhibitors of glycogen-synthase-kinase-3, insulin sensitizers, immune modulators, agonists of adrenergic receptors beta-3, agonists, Pan-PPAR, inhibitors of 11beta-HSD1, analogues amilina, biguanides, inhibitors of alpha-glucosidase, meglitinides, thiazolidinediones, sulfonylureas, etc. (see, for example, Nathan, 2006, N. Engl. J. Med. 355:2477-2480; Kahn et al., 2006, N. Engl. J. Med. 355:2427-2443). In some embodiment, the antibodies and fragments used according to the invention, can prevent or delay the need for additional method�x treatment or pharmaceutical compositions. In other embodiments of the invention, the antibodies or fragments can reduce the amount, frequency or duration of additional methods of treatment or pharmaceutical compositions.

Alternative, methods of treatment or prevention of a disease or condition according to the present invention can involve a timetable for the introduction of the antibody or fragment based on the presence of symptoms and/or changes any of the mentioned assessments (e.g., HbA1c, blood sugar levels on an empty stomach, results oral test glucose tolerance (OGTT), AUC glucose/C-peptide of insulin, use of drug treatment of diabetes, insulin sensitivity, levels of cytokines in serum, CRP levels, indicators of quality of life, improve BMI) as a means to determine, when you should enter one or more subsequent doses. Similarly, this approach can be used as a means for determining whether to increase or decrease subsequent dose based on the effect caused by the introduction of the previous dose.

The diagnosis of such diseases and conditions in patients or, alternatively, the risk of developing these diseases and conditions may be carried out in accordance with standard medical practices known in the art. Clinical studies (esti�) therapeutic or prophylactic effect on the above-mentioned diseases and conditions, performed after administration of antibodies against IL-1β or fragments, are well known in the art and can be used as ways of monitoring (control) of the effectiveness of the methods of the present invention.

For example, the response to treatment of type 2 diabetes can be assessed on the basis of efficiency improvement of hemoglobin A1c (HbA1c) in the primary endpoint, see, e.g., Reynolds, etc., BMJ, 333(7568):586-589, 2006). The improvement in HbA1c, an indicator of therapeutic efficacy may vary depending on the measurement source baseline values in the patient with greater reduction often corresponds to a higher initial starting value, but less often decrease is consistent with lower initial base value. According to one aspect of the present invention, the process must result in a decrease in HbA1c of at least about 0.5% (e.g., at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4% or more) compared with levels prior to the introduction of the doses.

In order to evaluate the effectiveness of the treatment, you can also define one or more subsequent secondary endpoints of the study), such as, for example, ravensara fasting blood (for example, glucose) (for example, reduction to≤130, ≤125, ≤120, ≤115, ≤110, ≤105, ≤100; alternative, a decrease of >20%, >30%, > 40%, > 50%, > 60%, > 70%, > 80%, > 90%, > 95% compared with levels before the introduction of dose), 120 minute oral test glucose tolerance (OGTT) (for example, ≤200, ≤190, ≤180, ≤170, ≤160, ≤150, ≤140), AUC glucose/ C-peptide of insulin (e.g., >25%, > 50%, > 60%, > 70%, > 80%, > 90%, >100% increase from baseline, prior to dose), reducing medical treatment of diabetes (e.g., insulin, oral hypoglycemic agents), increase insulin sensitivity, levels of cytokines in serum (e.g., normalization), CRP levels (for example, a reduction≥0.2, ≥0.4, ≥0.6, ≥0.8, ≥1.0, ≥1.4, ≥1.8, ≥2.2, ≥2.6, ≥3.0 mg/l; alternatively, a decline of >20%, >30% and >40%, >50% and >60%, > 70%, > 80%, > 90%, > 95% compared with baseline, prior to dose), quality of life, improved BMI (down 1%, 3%, 5%), pharmacokinetics, etc. (Saudek, etc., JAMA, 295:1688-97, 2006; Pfutzner, etc., Diabetes Technol Ther. 8:28-36, 2006; Norberg, etc., J Intern Med. 260:263-71, 2006).

Similarly, you can evaluate the effectiveness in relation to other diseases or conditions using one or more of the previously mentioned end points and/or others known in the art. For example, the effect on hyperglycemia can be assessed by measuring levels of sugar (i.e., glucose) in the blood on an empty stomach, effect� to hyperinsulinemia can be assessed by measuring the levels of insulin and/or levels of C-peptide, impact on obesity can be assessed by measuring the weight and/or BMI, and the effect on insulin resistance can be assessed using OGTT.

Alternative or additionally, the subjects treated in accordance with the invention, there may be a decrease in the level of triglycerides in the blood, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more compared with baseline (before treatment) level. Alternative or additionally, the subjects treated in accordance with the invention, there may be a decrease in the level of free fatty acids, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more compared with baseline (before treatment) level.

EXAMPLES

The following examples are intended only to illustrate the implementation in practice of this invention and do not limit the invention. All the above-mentioned patents and literature references in their entirety are included in the invention is in the form of links.

EXAMPLE 1

Inhibition of IL-1β high-affinity antibody against IL-1β in vitro studies on cells using induced IL-1 release of IL-8 as a means for information

The inhibitory effect of IL-1β-specific antibodies compared with the effect of the inhibitor metabolic pathways of IL-1 Kineret® (anakinra, anakinra), which is not an antibody and represents Soboh� recombinant receptor antagonist IL-1. Fresh heparinized peripheral blood was taken from healthy donors. 180 μl of whole blood was placed in a 96-well plate and incubated with various concentrations of antibodies AV (application for U.S. patent No. 11/472813, WO 2007/002261) and 100 PM rhIL-1β. For samples treated with Kineret®, Kineret® and rhIL-1β were combined in a ratio of 1:1 prior to mixing with the blood. The samples were incubated for 6 hours at 37°C with 5% CO2. Cells whole blood was literally 50 µl of 2.5% Triton X-100. The concentration of interleukin-8 (IL-8) in cleared lysates was determined using ELISA (Quantikine human IL-8 ELISA kit, R&D Systems) according to manufacturer's instructions. The concentration of IL-8 in samples treated AV and Kineret®, compared to a control sample treated with anti-KLH control. The results are shown in Fig.1 and summarized in Table 6. IC50the concentration of antibody required for 50% inhibition of release of IL-8 induced by IL-1β.

Table 1
IC50(PM)
AV1.9
Kineret®53.4

These results show the effectiveness AS in vitro by measurement of inhibition induced IL-1 β vysvobozhdeny� IL-8. These results, showing greater efficacy of the antibody in comparison with Kineret®, indicate that the antibody will possess inhibitory activity against IL-1β in vivo.

EXAMPLE 2

Inhibition of the biological activity of human IL-1β in vivo IL-1β-specific antibodies according to the effect on induced IL-1 release of IL-6

To confirm the effectiveness AS in vivo, its ability to block the biological activity of human IL-1β was tested on mice. Details of the study described Economides, etc., Nature Med., 9: 47-52 (2003). Briefly, male mice S/B16 mice (Jackson Laboratory, Bar Harbor, Maine) using intraperitoneal injection introduced titrated doses AW, other antibodies to IL-1β, AB5, or control antibodies. 24 hours after injection of antibodies to mice did subcutaneous injection of recombinant human IL-1β (rhIL-1β) (manufactured by PeproTech Inc., Rocky Hill, NJ) in an amount of 1 μg/kg two hours after injection of rhIL-1β (time to peak response IL-6), mice are killed, collected the blood and processed to obtain serum. The levels of IL-6 in serum was determined using ELISA (BD Pharmingen, Franklin Lakes, NJ), following the instructions of the manufacturer's Protocol. The percentage of inhibition was calculated based on the ratio of IL-6 measured in the serum of experimental animals, to the amount of IL-6, a specific serum to�control animals multiplied by 100).

The results are shown in Fig.2. The ability to inhibit the activity of IL-1β in vivo was evaluated as a function of stimulated IL-1β levels of IL-6 in serum. As can be seen from Fig.2, antibodies AB and AB 5 antibodies are effective inhibitors of the activity of human IL-1β in vivo. These results also show that a single injection AV or AB 5 can block the systemic effects of stimulation of IL-1β and that such antibodies are useful for inhibiting the activity of IL-1β in vivo.

A similar experiment was conducted to further demonstrate the capability AS to neutralize murine IL-1β in vivo, to justify the use of this antibody in mouse models of disease. It was found that AB has affinity to human IL-1β greater than the affinity to murine IL-1β is about 10,000 times, and the efficiency of in vitro (no research data D10.G4.1) about 1000 times greater than that for mouse IL-1β. In the model in mice C57BL/6 with IL-6 for information, the mice did intraperitoneal injection IV (3 or 300 μg) or PBS as control for 24 hours prior to subcutaneous injection of human (Fig.2, inset A) or mouse (Fig.2B, inset In a) IL-1β (20 ng). After 2 hours, blood was collected and analyzed samples of serum levels of IL-6 using ELISA. These data show that the maximum suppression of the levels of IL-6 (~ 75%), the Indus�yovanny human IL-1β, observed at 3 mg (box A), while sub-maximal suppression levels of IL-6 (~ 50%) induced murine IL-1β was observed at 300 ág (insert In). These results are consistent with observations, showing a much greater affinity and efficacy in vitro antibody AS in relation to human IL-1β than to murine IL-1β. In addition, data indicate that this antibody may be used in vivo in suitable mouse models of disease using the most appropriate dosage than would be required for the treatment of people for whom the antibody has a much greater affinity and efficiency. In the case of other antibodies to IL-1β, such as, for example, described or cited herein, antibodies that do not have this property significantly higher affinity and efficacy in relation to human IL-1β in comparison with murine IL-1β, similar to higher doses in mouse models may not be necessary.

EXAMPLE 3

Pharmacokinetics of antibodies to IL-1β after administration to rats of a single dose intravenously or subcutaneously

To study the pharmacokinetic profile of the antibody to IL-1β, marked IV, was administered to adult male rats as an intravenous (IV) bolus in the tail vein in an amount of 0.1, 1.0 or 10 mg/kg (groups 1, 2 and 3, respectively) or subcutaneous int� blades in the amount of 1.0 mg/kg (group 4). Blood samples were collected through sampling from the jugular vein or from the orbital sinus at certain time intervals up to 91 days after administration of the dosage. The blood samples were centrifuged to obtain serum. The samples were analyzed to determine the concentration of antibodies against IL-1β using ELISA based on the use of alkaline phosphatase as follows.

IL-1β (Preprotech) was diluted to a concentration of 0.5 μg/ml in PBS, 50 µl of this solution was added to the cells titration microplates Nunc-Immuno Maxisorp (VWR) and incubated overnight at 2-8°C. the antigen Solution was removed, after which all the cells were added with 200 µl of blocking buffer [1% bovine serum albumin (BSA) in 1X PBS containing 0.05% tween 20] and incubated for 1 hour at room temperature. After blocking cells were washed three times with buffer for washing (1X PBS containing 0.05% tween 20). Standards, samples and controls were diluted with a solution for dilution of the sample (25% rat serum in 1X PBS containing 1% BSA and 0.05% tween 20). Standard solutions of antibodies to IL-1β was prepared by using serial two-fold dilutions from 2000 to 0.24 ng/ml. Each repeat and dilution of standard solutions, sample and reference solutions (50 ál) was transferred into a blocked titration microplates and incubated for 1 hour at 37°C. After incubation the cells three�dy was washed with buffer for washing. Alkaline phosphatase, conjugated with goat antibody (H+L) human IgG (Southern Biotech Associates Inc, Birmingham, AL) was diluted 1/1000 solution for dilution of conjugate (1% BSA in 1X PBS containing 0.05% tween 20). 50 ál of diluted conjugate was added to all cells except the control (BLANK) cells, which added 50 ml of pure solution for dilution of conjugate. The plates were incubated for 1 hour at 37°C, after which all cells were washed three times with a solution for washing and three times with deionised water. The substrate p-nitrophenylphosphate (1 mg/ml in 10% diethanolamine buffer, pH 9.8) was added to each cell, the appearance of color occurred within 1 hour at room temperature, after which the reaction was terminated by addition of 50 μl of IN NaOH. The absorption (optical density) at 405 nm was measured with a spectrophotometer to read the tablets SPECTRAmax M2 Plate Reader (Molecular Devices, Menlo Park, CA) and built a standard dependence A concentration (ng/ml) of the antibody-standard. Conducted regression analysis and determined the concentration of the sample and reference solutions from the standard curve by interpolation. The quantification limit was 40 ng/ml.

As shown in Fig.3, the concentration in serum fall bi-exponentially among groups IV doses. Compartmental analysis conducted on the data for individual animals, � the resulting pharmacokinetic parameters were averaged for each group of dispensing with the exception of those animals, which was generated RAHA response. Levels of antibodies to IL-1β in serum were falling with an average half-life alpha phase from 0.189±0.094 to 0.429±0.043 days (from 4.54 to 10.3 hours) and with an average half-life in beta phase from 9.68±0.70 to 14.5±1.7 days. Among rats that received subcutaneous dose AS 1 mg/kg, the levels in serum reached a peak of 4.26±0.065 µg/ml 2-3 days and fell over time, elimination half-life of 2.59±0.25 days.

EXAMPLE 4

Pharmacokinetics of antibodies to IL-1β after administration of a single intravenous dose to cynomolgus monkeys

Antibody against IL-1β, marked AS, once injected adult male and female cynomolgus monkeys at an intravenous bolus in the amount (dose) of 0.3, 3.0 or 30 mg/kg). blood Samples of animals were collected before the introduction of dose, 5 minutes, 4 hours, 8 hours, and through 1, 2, 4, 8, 11, 15, 22, 29, 43 56 days. The samples were analyzed to determine the concentration of antibodies against IL-1β using ELISA based on the use of alkaline phosphatase as follows.

A solution of IL-1β was diluted to a concentration of 0.5 μg/ml in PBS, 50 µl of this solution was added to the cells titration microplates Nunc-Immuno Maxisorp (VWR) and incubated overnight at 2-8°C. the antigen Solution was removed, after which all the cells were added with 200 µl of blocking buffer [1% bovine serum albumin (BSA) in 1X PBS containing 0.05% tween 20] and incubated for 1-4 hours at room temperature�ture. After blocking the cells of each plate were washed three times with buffer for washing (1X PBS containing 0.05% tween 20). Standards, samples and controls were diluted with a solution for diluting the sample (2% normal Cynomolgus serum (NCS) in 1X PBS containing 1% BSA and 0.05% tween 20). Standard solutions of antibodies to IL-1β was prepared by using serial two-fold dilutions from 8000 ng/ml. Each repeat and dilution of standard solutions, sample and reference solutions (50 ál) was transferred into a blocked titration microplates and incubated for 1 hour at 37°C. After primary incubation, cells were washed three times with buffer for washing, has added all the cells with 50 ál biotinylated rhIL-1β. Then the plates were incubated for 1 hour at 37°C. the Cells were washed three times with buffer for washing and spent a third incubation with 50 ál of diluted alkaline phosphatase, conjugated with streptavidin, which was added to all cells except the control (BLANK) cells, which added 50 ml of pure solution for dilution. The plates were incubated for 30 minutes at 37°C, after which all cells were washed three times with buffer for washing and three times with deionised water. The substrate p-nitrophenylphosphate (1 mg/ml in 10% diethanolamine buffer, pH 9.8) was added to each cell. The appearance of colour was in the dark for 1 hour p�and room temperature, then the reaction was terminated by addition of 50 μl of IN NaOH. The absorption (optical density) at 405 nm was measured in all cells with a spectrophotometer to read the tablets SPECTRAmax M2 Plate Reader (Molecular Devices, Menlo Park, CA). Built standard dependency A405concentration (ng/ml) used as standard antibodies to IL-1β. Conducted 4-parametric regression analysis and determined the concentration of the sample and reference solutions from the standard curve by interpolation. The quantification limit was 40 ng/ml.

For groups that received single doses of 0.3 and 3 mg/kg, the levels of antibodies to IL-1β in serum were falling with an average half-life alpha phase 9.40±2.00 hours, followed by half-life in beta phase 13.3±1.0 days (Fig.5). In cynomolgus monkeys that received a single intravenous injection of 30 mg/kg, the levels of antibodies to IL-1β in serum were falling faster, with an average half-life alpha phase 10.9±3.2 hours, with a subsequent half-life in beta phase 7.54±1.79 days. Modeling of profiles in the coordinates of the plasma concentration - time for doses of 0.1, 0.3, 1 and 10 mg/kg, injected with a five month intervals, was also carried out and shown in Fig.5.

Example 5

The influence of antibodies to IL-1β in research on the human system from

Identified as in vitro models h�island human cells (from) treated with high glucose to mimic the microenvironment in diabetes type 2. Antibodies to IL-1β can be used in the system from human to test the impact on the function of beta cells (insulin release in response to glucose), the proliferation of beta cells and apoptosis.

Islets isolated from the pancreas of many donors of human organs with no history of diabetes or metabolic disease as described (Linetsky, etc., Diabetes 46:1120-1123, 1997; Oberholzer, etc., Transplantation 69:1115-1123, 2000; Ricordi, etc., Diabetes 37:413-420, 1988, Maedler, etc., Proc. Natl. Acad. Sci. USA 101:8138-8143, 2004; WO 2004/0002512). Islets were culturebully on tablets covered with extracellular matrix isolated from bovine corneal endothelial cells (Novamed Ltd., Jerusalem), allowing the cells to attach to the dies, and saving their functional integrity. Islets were culturebully in the medium CMRL 1066 containing 100 units/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (GIBCO, Gaithersburg, MD). For stimulation of insulin secretion culture medium was replaced by culture medium, additionally containing 5, 11 or 33 mm glucose, with or without the addition of fatty acids.

To measure the release of insulin in response to glucose, from washed and preincubated for 30 minutes in the bicarbonate-buffered Krebs-ringer (KRB) containing 3.3 mm glucose and 0.5% BSA. Then KRB replaced in KRB containing 3.3 mm glucose for 1 hour, after which withstood� an additional 1 h in KRB, containing 16.7 mm glucose. The from was extracted with 0.18 M HCl in 70% ethanol for determination of insulin using the kit for determination of human insulin by the method of radioimmunoassay (CIS Biointemational, Gif-sur-Yvette, France). Apoptosis of beta-cells can be measured in various ways. For example, the cells are subjected to double staining in a standard way end tagging breaks dUTP, MediaLounge deoxynucleotidyl-transferase (terminal deoxynucleotidyl transferase-also been other ideas where dUTP nick-end labeling (TUNEL), and also for insulin. In parallel, apoptosis was confirmed by detection of activated caspase-3 or the expression of Fas, as described (see, e.g., WO 2004/002512; Maedler et al., 2004, ibid.).

Example 6

The influence of antibodies to IL-1β in the research system of the rat pseudoinsulata

Alternative or in addition to models from human, can be used system rat pseudoinsulata as in vitro models to investigate the effect of antibodies against IL-1β. For example, pseudostate can be obtained and tested as described in U.S. patent 20060094714. Pancreas (pancreata) four rats Sprague Dawley were divided into small pieces, washed three times with buffer salts Hanks and HEPES (buffer Hanks-Hepes) and processed (digested) by collagenase (Liberase, 0.25 mg/ml, Roche Diagnostic Corp., Indianapolis, Ind., USA) at 37°C in a shaker water bath for 10 minutes. Then �assalamou the pancreatic tissue is washed three times with 50 ml of buffer Hanks-Hepes to remove collagenase, and the residue remnant tissue was filtered through a filter with a pore size of 250 microns. The filtrate was mixed with 16 ml of 27% (weight/volume) Ficoll (Sigma, St. Louis, Mo., USA) in buffer Hanks-Hepes and centrifuged in a Ficoll gradient (23%, 20.5% and 11% respectively; 8 ml of each concentration) at 1600 rpm for 10 minutes at room temperature. The pancreatic islets were concentrated in interfase between 11% and 20.5%, and between 20.5% and 23% depending on the size of the islets. Islets were collected from two interfaz, washed twice with Hanks buffer-Hepes containing no calcium, and suspended in 5 ml of Hanks buffer-Hepes without calcium, containing 1 mm EDTA, and incubated for 8 minutes at room temperature. Trypsin and Tnkase I have added to the suspension of the islets to a final concentration of 25 μg/ml and 2 μg/ml, respectively, and suspension were incubated with shaking at 30°C for 10 minutes. Cleavage of the trypsin was stopped by adding 40 ml of RPMI medium 1640 (GIBCO Life Technologies, Invitrogen, Carlsbad, Calif.) with 10% FBS. Then, digested with trypsin, the cells of the islets was filtered through a nylon filter with a pore size of 63 micron (PGC Scientific, Frederick, Md.) to remove large cellular clusters. Then dispersed cells of the islets were washed, counted and seeded into 96-well plates with V-shaped bottom (2500 cells per well). Then dispergirovannoyj suspension of cells of islets were centrifuged at 1000 Rev/�in within 5 minutes. Buffer Hanks-Hepes was removed and replaced by 200 μl of RPMI medium 1640 containing 10% FBS, 1% penicillin-streptomycin and 2 mm L-glutamine. The next step 96-well plates were centrifuged at 1000 rpm for 5 minutes to collect the dispersed cells of the islets that concentrated on the V-shaped bottom, forming pseudostate. These pseudostate then culturebully over night in an incubator for cell culture at 37°C with 5% CO2, and then used for research.

Example 7

The effect of antibodies to IL-1β on insulin sensitivity in models in animals.

The effectiveness of antibodies to IL-1β as an insulin-sensitizing agent in vivo can be measured by the action of antibodies to insulin and effects on glucose consumption using a power model of insulin resistance. Male rats Sprague-Dawley 6 weeks of age were kept on a diet high in fat and carbohydrates, containing 60% fructose, 10% pork fat (lard) and 0.06% magnesium. Two days after the beginning of such nutrition rats randomly divided into groups depending on the dose of antibody (varying from 0.1 to 5 mg/kg body weight), the method of administration (subcutaneously, intravenously or intraperitoneally) and the frequency of administration (daily or twice weekly). Animals of the control groups received either only the buffer (media), libon relevant antibody. The intake of fluids and food was measured every day and dual Protocol power used to ensure that the three groups consumed the same diet. After 5 weeks were used to determine the levels of glucose, insulin and triglycerides in serum in a half-starved condition (the night before the blood sampling, the animals were given a limited amount of food, and in the morning took away the blood). The Protocol was continued an additional 9 weeks, during which conducted tests for glucose tolerance (OGTT) in conscious animals in a half-starved condition by sampling of blood for measurement of glucose and insulin after oral administration of glucose load (100 mg/100 g body weight). Levels of glucose and triglycerides in serum were measured spectrophotometrically, insulin levels were measured by radioimmunoassay (Linco, St. Louis, MO).

Example 8

Antibodies to IL-1β for the treatment model of type 2 diabetes (T2D) in animals Psammomys obesus

therapeutic efficacy of antibodies to IL-1β in preventing the reduction of beta cell mass observed in patients suffering from type 2 diabetes, assessed in rodents (gerbils) Psammomys obesus with insulin resistance and are prone to diabetes, associated with obesity, diet induced, and diabetes initially is associated with hyperinsulinemia and hyperglycemia, and gradually progressives�th to the degree of severe hyperglycemia, accompanied by a temporary increase in the proliferative activity of beta cells and prolonged increase in the rate of loss of beta cells with destruction of the architecture of the islets (Donath, etc., Diabetes 48:738-744, 1999). To determine the effect of antibodies to IL-1β on induced hyperglycemia apoptosis of beta-cells and impaired proliferation in pancreatic islets Psammomys obesus during development of diabetes, the antibody was injected predisposed to diabetes animals (switched on high calorie diet) at different doses in the range of from 0.1 to 5 mg/kg of body weight subcutaneously, intravenously or intraperitoneally, and the introduction of antibodies was repeated at intervals in the range from daily to weekly. Control groups of animals were either kept on a low calorie diet, or switched to a high calorie diet and "treated" by one buffer (carrier) or an antibody that is irrelevant to the case. Subgroups of animals killed at 4, 7, 14, 21 and 28 day, immediately thereafter, blood was collected and used to determine levels of glucose, insulin and triglycerides in plasma. The pancreas is also removed, some were frozen at -70°C for later determination of insulin, and the remainder was fixed in 10% formalin solution in phosphate buffer, sealed with paraffin and did slicers to analyze the expression of Fas, IL-1β and ince�Lina, as well as the proliferation and apoptosis of beta-cells. This analysis allows to define the action to prevent or delay the symptoms of diabetes, protection from induced hyperglycemia apoptosis of beta-cells, reduced proliferation and reduced beta cell mass and normalization of the content of insulin in the pancreas.

Example 9

The use of antibodies to IL-1β for the treatment of type 2 diabetes in humans

Described in the invention the antibody to IL-1β or fragments can be administered to the patients (people) according to the invention for therapeutic treatment and/or prophylaxis of type 2 diabetes. Specifically, in one example, the antibody to IL-1β with the above-mentioned properties (described earlier AB) used for therapeutic treatment of patients having signs and symptoms of type 2 diabetes. More specifically, the safety and efficacy of antibodies to IL-1β for the treatment of type 2 diabetes was demonstrated on one or more clinical studies in humans, including, for example, the following tests.

Were conducted clinical studies of treatment in patients with diabetes type 2, double-blinded with placebo as a control. Patients meeting the following criteria for such studies in accordance with the diagnostic criteria of diabetes 2 timeamerican Diabetes Association (American Diabetes Association, ADA):

- the concentration of fasting blood glucose ≥126 mg/DL (≥7.0 mmol/l) (must be measured within 28 days prior to the day zero);

OR

- symptoms of hyperglycemia (eg, thirst, polyuria, weight loss, blurred vision) And irregular/random level plasma glucose ≥200 mg/DL (≥11.1 mmol/l) (must be measured within 28 days prior to the day zero), and HbA1c>7.5% and ≤12% (DCCT standard) were included in the study consecutively in groups, and within each group were randomly established patients treated with antibody to IL-1β or placebo. To minimize risk to patients, safety and tolerability checked at each dosing level before moving to the next higher dose level. Group of patients and the number of subjects who participated in the study are given in the table below:

AntibodyPlacebo
GroupMethod of administrationThe number of subjectsDoseThe number of subjects
1iv* or sc** 50.01 mg/kg1
2iv or sc50.03 mg/kg1
3iv or sc50.1 mg/kg1
4iv or sc50.3 mg/kg1
5iv or sc51.0 mg/kg1
6iv or sc53.0 mg/kg1
* iv - intravenously, intravenous
** sc - subcutaneous, subcutaneously

On the first day of the study, the antibody or placebo administered subcutaneously or by intravenous infusion with a constant speed for 30 minutes. Safety assessment, including the entry of undesirable effects, medical examinations, basic signs, clinical laboratory research� (for example, blood chemistry, Hematology, urinalysis), the levels of cytokines in plasma and electrocardiogram (ECG) was performed using standard methods of medical practice, known in the art. Blood samples were collected before the introduction of dose and after various time periods (e.g., days) after injection for determination of HbA1c, lipid profile, including free fatty acids, cholesterol of high density and low density cholesterol, the levels of antibodies to IL-1β (pharmacokinetics), the responses to the antibody to IL-1β, levels of cytokines (e.g., IL-1β, IL-6, TNFα), CRP, sodium, potassium, creatinine, AST, ALT, blood pressure. Can be also carried out studies of other cytokines and lymphokines such as, for example, described in the invention. You can take additional blood samples later than the original, in cases where the levels of the introduced antibodies to IL-1β did not fall below the limit of detection. The measurements are performed at certain points after treatment.

Clinical observation of treatment of type 2 diabetes was made on the basis of efficiency improvement of hemoglobin A1c (HbA1c) in the primary endpoint (see, e.g., Reynolds, etc., BMJ, 333(7568):586-589, 2006). Improvement of HbA1c is an indicator of therapeutic efficacy of treatment with antibody to IL-1β, and should normally lead to a reduction of at least about 0.5% or more. Also �predelut one or more secondary endpoints to evaluate the effectiveness of the treatment of type 2 diabetes, such as, for example, the levels of sugar (e.g., glucose) in the blood on an empty stomach (for example, ≤130, ≤120), 120-minute oral test glucose tolerance (OGTT), AUC glucose/ C-peptide of insulin (for example, increase >50%, >60%), reducing medical treatment of diabetes (e.g., insulin, oral hypoglycemic agents), increase insulin sensitivity, levels of cytokines in serum (e.g., normalization), the CRP levels, indicators of quality of life, improved BMI (down 1%, 3%, 5%), pharmacokinetics, etc. (Saudek, etc., JAMA, 295:1688-97, 2006; Pfutzner, etc., Diabetes Technol Ther. 8:28-36, 2006; Norberg, etc., J Intern Med. 260:263-71, 2006). Further analysis of the lipid profile samples includes the following tests are carried out in accordance with generally accepted standard methods known in the art.

ResearchMethod
Electrophoresis of lipoproteinsGel electrophoresis
Apolipoprotein A-I (arc A-I) in serumNephelometry
Apolipoprotein A-II (arc A-II) in serumNephelometry
Polip�protein b-48 (arc b-48) in serum ELISA
Apolipoprotein b-100 (Aro b-100) in serumNephelometry
Apolipoprotein Cs (Aro Cs) in serumImmunonephelometry Aro CII and Apo CIII
Apolipoprotein E (Aro E) in serumNephelometry
Apolipoprotein J (Aro J) in serumELISA
Amyloid A in serumNephelometry
Free fatty acids in plasma (FFA)Colorimetry
Glycerol in plasmaColorimetry
LCAT in serumELISA
Protein-a carrier of aethers of cholesterol in serum (BPAH) Serum cholesteryl ester transfer protein (CETP)ELISA
Hepatic lipase in serum Serum hepatic lipase (HL)Fluorometry
Paraoxonase 1 in serum paraoxonase 1 (PON1)UV/colorimetry

Pharmacokinetic analysis

Samples for pharmacokinetic analysis were obtained at 0, 1, 2, , 4, 7, 9±1, 11±1, 14±1, 21±2, 28±2, 42±3 and 56±3 day. Preliminary analysis of the pharmacokinetics AS in subjects with type 2 diabetes who received a single dose of 0.01 mg/kg intravenously, showed that the profiles based on the concentration in serum from time to time have a terminal half-life of 22 days, clearance of 2.9 ml/day/kg and volume of distribution Central compartment 50 ml/kg, which is very close to the volume of serum (Fig.7).

Analysis of glucose and HbA1c in the blood

Samples were prepared and measured the levels of glucose in the blood 0, 7, 14±1, 21±2, 28±2, 42±3 and 56±3 day and on the day of screening (mass screening). Research data on these samples to reduce levels of blood glucose are shown below for samples obtained from the first two groups, divided by dosages (the top line for each subject). Samples were prepared and measured HbA1c at 0, 28±2, 42±3 and 56±3 day and on the day of the screening. Research data on these samples to reduce HbA1c levels shown below for samples obtained from the first two groups, divided by dosages (bottom row for each subject).

The group with the dose of 0.01 mg/kg

Day 21 (lab)td align="left">
SubjectScreeningDay 0 (laboratory)Day 7 (laboratory)Day 14 (laboratory)Day 28 (laboratory)Day 42 (laboratory)Day 56 (laboratory)
5200.00247.00212.00179.00213.00Is at 242.00235.00200.00
8.408.608.507.109.30
1211.00232.00235.00236.00199.00221.00252.00204.00
9.309.609.509.109.80
2229.0 131.00160.00191.00193.00224.00204.00207.00
9.008.808.408.609.00
11290.00283.00300.00177.00308.00278.00292.00302.00
11.8011.6011.4011.2011.80
3175.00158.00175.00Is at 154.00Is at 154.00162.00183.00170.00
8.208.207.707.808.10
4238.00255.00270.00275.00289.00278.00255.00245.00
8.509.4010.5010.6010.40

The group with the dose of 0.03 mg/kg

SubjectScreeningDay 0 (laboratory)Day 7 (laboratory)Day 14 (laboratory)Day 21 (lab)Day 28 (laboratory)Day 42 (laboratory)Day 56 (laboratory)
6 222.00164.00148.00166.00162.00145.00207.00
8.408.408.208.40
7208.00109.00101.00120.0081.00108.00113.00
8.007.506.706.40
8364.00287.00289.00260.00237.00
12.4012.00
9204.00128.00124.00117.00112.00113.00
7.907.507.10
12275.00235.00250.00126.00168.00
9.9010.30
10332.00398.00243.00187.00220.00
11.5011.50

These data show that the claimed invention lower limits of dosages of antibodies to IL-1β be useful for achieving a therapeutic effect (e.g., reduced levels of glucose and/or HbA1c) in a subject after a single injection of the antibody.

Analysis of C-reactive protein

C-reactive protein (CRP), which is secreted by the liver in response to various stress triggers, including IL-6, produced in response to IL-1 was also measured in serum at the same time points as the samples for pharmacokinetic studies. Was developed PK/PD model (modeling of pharmacokinetics/pharmacodynamics), including dvukhkomponentnoi model for the level of antibody in serum and the concentration-dependent indirect response antibodies on the rate of production of CRP with a linear ratio, low gas consumpt�entom elimination of CRP. On the basis of model calculations after a single dose of the antibody in the amount of 0.01 mg/kg intravenously in subjects with type 2 diabetes mellitus CRP fell within 7-10 days to 66±22% compared to 100% before the introduction of dose (Fig.8). After a single dose of the antibody in the amount of 0.03 mg/kg intravenously in subjects with type 2 diabetes mellitus CRP fell within 7-10 days to 40±12% compared with 100% before the introduction of dose (Fig.9). Control data for placebo are shown in Fig.10. Based on these data and model predictions support the expected levels of CRP after monthly injections of the antibody comprise about 40% at 0.03 mg/kg, 16.5% at 0.1 mg/kg, 6.2% at 0.3 mg/kg, 1.9% at 1 mg/kg and 0.66% at 3 mg/kg per month (Fig.11). These data indicate that to obtain a therapeutic effect in a subject after a single injection of the antibody (e.g., reduction of CRP levels) provided by the invention the antibody to IL-1β, you can enter a frequency of once a month or less.

Based on the results of the first clinical studies conducted additional clinical trials. Such tests may include one or more of the above dosages, as well as, or alternatively, one or more other dosages of the antibody to IL-1β, longer duration of treatment and/or observation period and a larger number of patients in the group (at least approximately 10, 50, 100, 200, 300, 400, 500, 750, 1000) according� the invention. In addition, these and other studies can be used to determine the time required to obtain the desired therapeutic effect, on the basis of a change of a specific parameter (for example, reducing blood sugar, reducing HbA1c, reducing CRP), a well as to determine the duration of the favorable therapeutic effects on the basis of a change of a specific parameter (for example, reducing blood sugar, reducing HbA1c, reducing CRP) prior to the introduction of additional doses.

Example 10

The effect of antibodies to IL-1β on the function of adipocytes and insulin resistance

In vitro studies using culturebound adipocytes can be used to demonstrate the reduction (e.g., blocking) induced IL-1β insulin resistance using antibodies to IL-1β. Cells preadipocyte line 3T3-L1 derived from ATS (No. CL-173), was raised in 7% CO2at 37°C in DMEM with 25 mm glucose and 10% calf serum and induced their differentiation into adipocytes. Briefly, 2 days after confluence the medium was replaced by DMEM containing 25 mm glucose and 10% fetal calf serum (FCS), supplemented by isobutylmethylxanthine (0.25 mm), dexamethasone (0.25 μm), insulin (5 µg/ml) and pioglitazone (10 μm). The medium was removed after 2 days and replaced by DMEM containing 25 mm glucose and 10% FCS, supplemented with insulin (5 μg/ml) and pig�Autonom (10 μm) for 2 days. Then every 2 days spent updating DMEM with 25 mm glucose and 10% FCS. Adipocytes 3T3-L1 used in 8-15 days after the start of the differentiation Protocol.

Human preadipocyte (Biopredic International, Rennes, France) were reared in 5% CO2at 37°C in DMEM Ham''s F12 containing 15 mm HEPES, 2 mm L-glutamine, 5% FCS, 1% antifungal solution, ECGS/H-2, hEGF-5 and HC-500 from an additional set to medium for the cultivation of preadipocytes (Promocell, Heidelberg, Germany). Differentiation into adipocytes was induced after confluence by replacing DMEM medium on Ham''s F12 with 15 mm HEPES, 2 mm L-glutamine and 3% FCS, supplemented with Biotin (33 µm), insulin (100 nm), Pantothenate (17 µm), isobutylmethylxanthine (0.2 mm), dexamethasone (1 μm) and rosiglitazone (10 μm). The medium was removed after 3 days and replaced on Ham''s F12 containing 15 mm HEPES, 2 mm L-glutamine and 10% FCS, supplemented with Biotin (33 µm), insulin (100 nm), Pantothenate (17 μm) and dexamethasone (1 μm). Then every 2 days spent updating the same environment for the cells. Human adipocytes were used 15 days after the start of the differentiation Protocol. Human preadipocyte can also be obtained from alternative sources, such as, for example, cell lines HAA and HMV (Lonza, Allendale, NJ).

The role of IL-1β in the induction of insulin resistance (reduced insulin sensitivity) culturebound adipocytes shown by incubation ... �of cítov with IL-1β (e.g., 20 ng/ml, 48 hours), followed by incubation with different concentrations of insulin (e.g., 0.5 nm, 100 nm; 20 min) and subsequent measurement of glucose transport after addition of 2-[3H]deoxyglucose. Insulin resistance was defined as reduced consumption of glucose, and the effect of antibodies to IL-1β on the decline (e.g., blocking) of insulin resistance is easily measured on this system cell culture of adipocytes.

The role of IL-1β in direct stimulation of the production of adipokines and cytokines by adipocytes (e.g., leptin, resistin, visfatin, IL-6, MCP-1 (CCL2), RANTES, PAI-1, protein acylation stimulating, SAA3, pentraxin-3, factor inhibiting macrophage migration, IL-1RA, IL-12, IL-8, IL-6, TNF-α) was determined using kulturarbeit adipocytes in the absence or in the presence of different concentrations of IL-1β for different time periods as described above and measuring the levels of adipocytes and cytokines in the conditioned culture medium using ELISA or other commonly used methods. In addition, determined the effect of treatment of cultures of adipocytes stimulated with IL-1β neutralizing antibody against IL-1β in the induction of secretion of adipokines and/or cytokines associated with insulin resistance. Similarly, measured the effect of treatment with antibody to IL-1β on the inhibition of insulin-sensitizing ... �Keane, of adiponectin. To find out whether neutralizes treatment with antibody against IL-1β effects of endogenously produced IL-1 released from immune/inflammatory cells (e.g., macrophages) during inflammation of fat tissue, various amounts of human macrophages (derived from monocytes or different cell lines of monocytes) were culturebully with the above-described cultures of adipocytes in the absence or in the presence of antibody to IL-1β and measured the modulation of adipokines and cytokines. In addition, its modulatory effect on the secretion of adipokines and cytokines after treatment of the subject in vivo an antibody to IL-1β was measured in the circulation (e.g., serum, plasma) as a means of demonstrating effectiveness.

Example 11

Inhibition of cytokine production in whole human blood by antibody to IL-1β

Measurement of cytokines in the blood when the disease or during treatment of diseases may be useful for determining the severity of the disease or response to therapy. Usually determine the levels of cytokines in serum, but this method is not necessarily measured total (aggregate) cytokines. Many cytokines may be located inside the cells (intracellular). In addition, the ability of cells to produce the cytokine may be more useful information than the level of circulating cytokines.

A method of stimulating Zelin�th blood was used to determine cytokine production and the effect of treatment with antibody to IL-1β. Blood samples were collected from patients in sterile heparinization tubes, then 250 μl of whole blood was added to each sterile cryoprobes Coming in 4 ml orange top, prepared as follows:

Control series

All tubes are pre-filled with 550 μl RPMI. In test tube 1 (control) was added 200 μl RPMI and tubes 2-10 was added 100 µl RPMI. In each of the test tubes 2-10 was added 100 µl of a solution of antibody to IL-1β (AB7).

Series for research

A similar series of solutions of antibodies were prepared as described in detail below.

All tubes were well mixed using a vortex for 10 seconds. In the control tube series A1-10, then added 100 ál of RPMI, again stirred on a vortex for 10 seconds, cover tightly spun and recorded, and placed the tubes in a thermostat. In test tubes series for studies B1-10 added 100 µl killed by heating Staphylococcus epidermidis (final concentration 1:1000 stock solution that resulted in a ratio of bacteria:leukocytes 10:1), the tubes were mixed on a vortex for 10 seconds, closed the lids and placed in a thermostat at 37°C. After 24 hours of incubation the cultures were literally the Triton (final concentration 0.5%) to highlight the content of the cells, the lysates were frozen. After lysis of the cultures of whole blood tubes three times powerpill of freezing and thawing and measured the levels of cytokines by standard methods of the study of cytokines using ELISA for human TNFα, IL-6, IFNγ, IL-8, IL-1α, IL-1Ra and IL-1β (R&D Systems, Minneapolis, MN).

The cytokines measured in test tubes control series containing only sterile culture medium and the antibody (where specified), reflect the spontaneous level of stimulation. It was found that in healthy people, the levels of various cytokines measured after incubation for 24 hours, is very low. In patients whose diseases are not treated, the levels can be higher. Tube series for studies also contain a dead heat Staphylococcus epidermidis, stimulating the production of several cytokines. If treatment with antibody to IL-1β is effective, it should show up in reducing the production of cytokines.

As shown in Fig.6, high-affinity antibody to IL-1β AB7 was a very effective inhibitor of the production of IL-1β in human blood. On average, three human samples, the antibody inhibited the production of IL-1β induced Staphylococcus epidermidis, 50% at 0.1 PM and 75% at 3 PM. At 100 PM the inhibition was 100%. Interferon gamma (IFNγ) is induced by Staphylococcus epidermidis, a AB7 reduced IFNγ induced Staphylococcus epidermidis, 75% at 100 PM.

Example 12

The effect of antibodies against IL-1β on diabetes netacnih diabetic mice

In order to demonstrate the effectiveness of the antibodies to IL-1β on the model of diabetes in mice, females netacnih mice with diabetes (Non-Obese Diabetic (NOD) mous, NOD mouse) (Jackson Laboratories, Bar Harbor, ME) aged 3-4 weeks were placed in a vivarium in the conditions of absence of pathogenic microorganisms. Different doses of the antibody against IL-1β (for example, from 3 to 600 μg) was diluted in a suitable carrier (e.g., PBS) and injected prediabetic female NOD mice, beginning not later than 6 weeks of age, using different methods of injection (e.g., intraperitoneally, subcutaneously, intravenously) through certain time intervals (e.g., weekly, biweekly, once a month). The level of blood glucose was monitored using glucometer (Glucometer Encore; Bayer, Elkhart, IN) at intervals of a week, starting at 10 weeks of age. Mice with glucose levels 200 mg/DL in two consecutive measurements recognized diabetic (manifestation of diabetes usually occurred at the age of around 15 to 20 weeks and the incidence rate reached a maximum of about 90% by 30 weeks of age). Data is counted as the percentage of animals that are not cases of diabetes mellitus in the course of the experiment. The difference between curves was tested using the log rank test that compares the distribution throughout the observation period.

In another model in NOD mice the efficacy of antibodies against IL-1b was demonstrated on the model of the disease accelerated by cyclophosphamide (CY) (Reddy and d�., Histochem J. 33:317-327, 2001; Cailleau, etc., Diabetes 46:937-940, 1997; Reddy and others, Histochem J., 34:1-12, 2002; Harada, etc., Diabetologia 27:604-606, 1984; Nicoletti, etc., Eur J Immunol 24:1843-7, 1994). Males (or females) neadiabaticheskikh NOD mice (Jackson Laboratories, Bar Harbor, ME) aged 4-8 weeks were placed in a vivarium in the conditions of absence of pathogenic microorganisms. Mice injection was administered a single dose of CY (Sigma) in an amount of 200 mg/kg and progressively reduced through various time periods from "accelerating" the nature of the model (e.g., once a week, twice a week) for 2 to 3 weeks were injected with either different doses of antibodies to IL-1β (e.g., 3 μg, 30 μg, 150 μg, 600 μg) or isotype antibody as a control, diluted in a suitable carrier (e.g., PBS), using different methods of injection (e.g., intraperitoneally, subcutaneously, intravenously). The levels of glucose in the urine (glucosuria) was monitored three times a week, and the levels of glucose in the blood once a week with a glucose meter, starting from the day prior to injection of CY. Mice with glucose levels in the urine >20 mm/l in two consecutive measurements recognized diabetic, and low levels of glucose in the urine under the influence of antibodies to IL-1β was considered a measure of efficiency.

In another model, the effectiveness of antibodies to IL-1β was evaluated on the model of recurrent diabetes in the transplantation of pancreatic islets (if neutorgena TRANS�of Lantana) (Mellgren, etc., Diabetologia 29:670-2, 1986; Sandberg, etc., Clin Exp Immunol 108:314-7, 1997). Females neadiabaticheskikh NOD mice (Jackson Laboratories, Bar Harbor, ME) aged 4-8 weeks were placed in a vivarium in the conditions of absence of pathogenic microorganisms. The pancreatic islets were obtained from males or females neadiabaticheskikh NOD mice 5-6 weeks of age until visible (marked) leukocyte infiltration and transplanted under the kidney capsule of the female spontaneously diabetic NOD mice 15-20 weeks of age (from 400 to 450 islets/mouse). The temporary stabilization of blood sugar levels (transient normoglycemia) observed shortly after transplantation, but approximately 6 days after transplantation hyperglycemia usually appears again. The mice were injected with either different doses of antibodies to IL-1β (e.g., 3 μg, 30 μg, 150 μg, 600 μg) or isotype antibody as a control, diluted in a suitable carrier (e.g., PBS), using different methods of injection (e.g., intraperitoneally, subcutaneously, intravenously). The levels of blood glucose was monitored before and after transplantation once or twice a week with a glucose meter. Mice with glucose levels > 200 mg/DL in two consecutive measurements recognized diabetic, and low levels of glucose in the blood under the action of antibodies to IL-1β was considered a measure of efficiency.

Example 13

Treatment models of diabetes, induce�created by the introduction of small doses of streptozotocin, in mice C57BL/K.

In order to demonstrate the effectiveness of the antibodies to IL-1β on the model of hyperglycemia caused by repeated injection of streptozotocin (STZ) in small doses, and on the model of insulin mellitus (Sandberg, etc., Biochem Biophys Res ComMun 202:543-548, 1994; Reddy, etc., Ann N Y Acad Sci 1079:109-113, 2006), mice of the C57BL/K 4-8 weeks of age (Jackson Laboratories, Bar Harbor, ME) were placed in a vivarium in the conditions of absence of pathogenic microorganisms. According to this "accelerated" model, mice received five injections of STZ (40 mg/kg) daily and were subjected to accelerated treatment (starting from the day before STZ injection) across different time intervals (e.g., one, two or three times a week) for one to three weeks with or without different doses of antibodies to IL-1β (e.g., 3 μg, 30 μg, 150 μg, 600 μg) or isotyping antibody as a control, diluted in a suitable carrier (e.g., PBS), using different methods of injection (e.g., intraperitoneally, subcutaneously, intravenously). The levels of glucose in the blood was monitored once a week using a glucometer, starting with the day prior to STZ injection. Mice with glucose levels > 200 mg/DL in two consecutive measurements recognized diabetic, and low levels of glucose in the blood under the action of antibodies to IL-1β was considered a measure of efficiency.

Example 14

Treatment of type 2 diabetes on the model �of Irene, caused by diet

The effectiveness of antibodies to IL-1β was tested on a model of type 2 diabetes is obesity caused by diet (DIO). According to this model, mice which were kept on a diet high in fats, after a few weeks became fat, and had a reduced glucose tolerance and impaired insulin secretion in response to injection of a loading dose of glucose. Male mice C57BL/6 6-weeks of age were kept on normal diet (ND, Teklad, 5% kcal from fat) or a diet with high fat and sucrose (Surwit''s high fat, high sucrose diet, HFD, Research Diets #D12331, 58% kcal from fat). Dosage antibodies began a day earlier. The tested antibody to IL-1β (AB7) and the isotype of the human IgG2 antibody as a control were injected intraperitoneal injections. Antibodies were injected twice a week for 4 weeks. Body weight was also monitored twice a week. After 4 weeks, the mice were tested for glucose tolerance (GTT). In the GTT test, mice were fasted from the night, and then intraperitoneal injection received glucose (1 g/kg Glucose in the blood taken from tail cuts, measured at 0, 15, 30, 60, 90 and 120 minutes after injection using a glucometer FreeStyle. Fig.12 shows that mice kept on a diet with a high content of fat in 4 weeks, had weakened tol�runtest to glucose compared with mice, kept on normal diet (Fig.12). The introduction of the tested antibodies to IL-1β defended HFD mice from reduced glucose tolerance. Within 60 minutes of holding GTT test, the mice that were dosed injected antibody to IL-1β in an amount of 1 mg/kg, showed himself to be significantly better than mice treated with control antibody IgG2 (*, p<0.05). It should be emphasized that positive results in this mouse model were obtained despite the fact that the antibody AB has a significantly lower affinity (~10000 times) and effective in vitro against murine IL-1β compared with human IL-1β as described in Example 2 above.

All the cited invention references, including publications, patent applications and patents in their entirety are included in the invention is in the form of links.

The terms "including", "having", "containing" is used to denote open terms, i.e., meaning "including, but not limited to(I) except as otherwise specified. In all cases, the use of open terms to describe the properties or element according to the invention provides that instead of an open term can be used indoor without violating the spirit and scope of the invention. An enumeration of the intervals of values, except as otherwise specified, serves as the single�public goal of reducing the volume of the enumerated individually reduce each value within the interval, and each separate value is included in the description as if it were listed separately. All the described methods can be performed in any suitable order unless otherwise stated and is not contrary to the context. Examples or examples of the terms (e.g., "such as") provided for better coverage of the invention and does not limit the nature and scope of the invention, unless otherwise specified. No words in the description can't be treated as undeclared elements, if they are essential for practical use according to the present invention.

In the description of the preferred embodiments of the invention, including the best-known authors ways. Variations of these preferred embodiments of the invention may become obvious to those skilled upon reading the above description. The authors believe that to exercise such options as appropriate will be experts, moreover, the authors believe that the invention will be practically used not only as it is specifically described. Accordingly, the invention includes all modifications and equivalent replacements listed in the claims of the subjects (objects) that are part of the invention in accordance with applicable law. Balayage, any combination of the above-described elements in all possible combinations included in the invention, unless specified otherwise and not inconsistent with the context.

1. A method for the treatment in humans of a disease or condition selected from the group including type 2 diabetes, insulin resistance, decreased insulin production, hyperglycemia, hyperinsulinemia and obesity, characterized in that the method includes the introduction of human antibody to IL-1β or its fragment.

2. A method according to claim 1, characterized in that the disease or condition is a type 2 diabetes.

3. A method according to claim 1, characterized in that the disease or condition is an insulin resistance.

4. A method according to claim 1, characterized in that the disease or condition is a hyperglycemia.

5. A method according to claim 1, characterized in that the disease or condition is a hyperinsulinemia.

6. A method according to claim 1, characterized in that the disease or condition is a obesity.

7. A method according to claim 1, characterized in that after the introduction of the initial dose of the antibody or antibody fragment by adding one or more subsequent doses.

8. A method according to claim 7, characterized in that after the introduction of the initial dose of the antibody or antibody fragment by adding two or more subsequent doses.

9. A method according to claim 1,�lichudis, that after administration of the initial dose of the antibody or antibody fragment by adding one or more subsequent doses, and the value of the specified one or more subsequent doses are about the same or less than the value of the initial dose.

10. A method according to claim 1, characterized in that after the introduction of the initial dose of the antibody or antibody fragment by adding one or more subsequent doses, wherein at least one of the subsequent doses at the value exceeds the initial dose.

11. A method according to claim 1, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 250 PM or less.

12. A method according to claim 11, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 10 PM or less.

13. A method according to claim 12, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 1 PM or less.

14. A method according to claim 13, characterized in that the antibody or antibody fragment binds to human IL-1β with a dissociation constant of about 0.5 PM or less.

15. Method according to any one of claims.7-14, wherein the initial dose and each one or more subsequent dose are separated from each other by an interval of at least about 2 weeks.

16. �] according to claim 15, wherein the initial dose and each one or more subsequent dose are separated from each other by an interval of at least about 1 month.

17. A method according to claim 16, wherein the initial dose and each one or more subsequent dose are separated from each other by an interval of at least about 3 months.

18. A method according to claim 17, wherein the initial dose and each one or more subsequent dose are separated from each other by an interval of at least about 6 months.

19. A method according to claim 18, wherein the initial dose and each one or more subsequent dose are separated from each other by an interval of at least about 12 months.

20. A method according to claim 1, characterized in that the antibody or antibody fragment is administered in the form of one or more doses of the antibody or fragment of 3 mg/kg or less.

21. A method according to claim 20, characterized in that the antibody or antibody fragment is administered in the form of one or more doses of the antibody or fragment of 1 mg/kg or less.

22. A method according to claim 21, characterized in that the antibody or antibody fragment is administered in the form of one or more doses of the antibody or fragment of 0.5 mg/kg or less.

23. A method according to claim 22, characterized in that the antibody or antibody fragment is administered in the form of one or more doses of the antibody or fragment at 0.1 mg/kg or less.

24. A method according to claim 23, characterized in that antic�lo or antibody fragment is administered in the form of one or more doses of the antibody or fragment 0.03 mg/kg or less.

25. A method according to claim 24, characterized in that the antibody or antibody fragment is administered in the form of one or more doses of the antibody or fragment of 0.01 mg/kg or less.

26. Method according to any one of claims.20-25, characterized in that the value of one or more doses is at least 0.01 mg of antibody or fragment per kg.

27. A method according to claim 1, characterized in that the antibody to IL-1β or the fragment of the antibody is a neutralizing antibody.

28. A method according to claim 1, characterized in that the antibody to IL-1β or the fragment of the antibody binds to an epitope of IL-1β in such a way that the bound antibody or fragment substantially permits the binding of IL-1β to its receptor IIL-1 (IL-1Ra).

29. A method according to claim 1, characterized in that the antibody or antibody fragment does not bind to IL-1α, IL-1R or IL-IRa to the extent available for detection.

30. A method according to claim 1, characterized in that the antibody or antibody fragment binds with an epitope contained in the sequence ESVDPKNYPKKKMEKRFVFNKIE.

31. A method according to claim 1, characterized in that the antibody or antibody fragment binds to an epitope incorporating Glu64 of Il-1β.

32. A method according to claim 1, characterized in that the antibody or antibody fragment binds to amino acids 1-34 of the N-end of IL-1β.

33. A method according to claim 1, characterized in that the antibody or antibody fragment are designed for the person or humanitero�prisoners who had been.

34. A method according to claim 1, characterized in that the antibody or fragment of the antibody are human.

35. A method according to claim 1, characterized in that the antibody to IL-1β or the fragment is administered by subcutaneous, intravenous or intramuscular injection.

36. A method according to claim 1, characterized in that the antibody or fragment is administered as a fixed dose, irrespective of the relationship between dose and body weight of the subject.

37. A method according to claim 36, characterized in that the antibody or fragment is administered in the form of one or more doses of 500 mg or less of antibody or fragment.

38. A method according to claim 37, characterized in that the antibody or fragment is administered in the form of one or more doses of 250 mg or less of antibody or fragment.

39. A method according to claim 38, characterized in that the antibody or fragment is administered in the form of one or more doses of 100 mg or less of antibody or fragment.

40. A method according to claim 39, characterized in that the antibody or fragment is administered in the form of one or more doses of 25 mg or less of antibody or fragment.

41. A method according to claim 36, characterized in that the antibody or fragment is administered in the form of one or more doses, at least 1 mg of antibody or fragment.

42. A method according to claim 36, characterized in that the antibody or fragment is administered in the form of one or more doses in a size from about 10 mg to about 100 mg of antibody or fragment.

43. A method according to claim 11, characterized in that the dose of the antibody of refragment is sufficient to improve hemoglobin A1C, at least 0.5 percent.

44. A method according to claim 43, characterized in that the dose of the antibody or fragment is sufficient to improve hemoglobin A1C of at least 1 percent.

45. A method according to claim 44, characterized in that the dose of the antibody or fragment is sufficient to improve hemoglobin A1C of at least 2 percent.

46. A method according to claim 45, characterized in that the dose of the antibody or fragment is sufficient to improve hemoglobin A1C of at least 3 percent.

47. A method according to claim 11, characterized in that the method is sufficient to achieve at least one of the following changes: reduce the level of sugar in blood glucose, reduce insulin resistance, reduction of hyperinsulinemia, increase in glucose tolerance, reduction in C-reactive protein (CRP), reduction of hyperglycemia, reduction in the need for medical treatment of diabetes, lower BMI, change in AUC glucose / C-peptide of insulin, reduction of acute phase reactants, decrease lipids in the serum in improving the lipid profile.

48. A method according to claim 11, characterized in that the method reduces or prevents a complication or condition associated with type 2 diabetes selected from the group including retinopathy, renal failure, cardiovascular system and wound healing, when it� the method comprises administering the antibody to IL-1β or its fragment to the people.

49. A method according to claim 48, characterized in that the complication or condition is a disease of the cardiovascular system, and is a disease of the cardiovascular system is atherosclerosis or peripheral vascular disease.

50. A method according to claim 48, characterized in that the complication or condition is wound healing, and this healing wound is a diabetic ulcer.

51. A method according to claim 1, characterized in that the method is carried out jointly, at least one additional treatment method, and this additional method of treatment comprises administering at least one pharmaceutical composition containing the active agent, which is an antibody to IL-1β or the fragment.

52. A method according to claim 1, characterized in that the method prevents or causes a delay is necessary to use at least one additional treatment method, and this additional method of treatment comprises administering at least one pharmaceutical composition containing the active agent, which is an antibody to IL-1β or the fragment.

53. A method according to claim 1, characterized in that the method reduces the number, frequency or duration of at least one additional treatment method, and this additional method of treatment includes the introduction, at the very� least a pharmaceutical composition containing the active agent, which is an antibody to IL-1β or the fragment.

54. Method according to any one of claims.51 to 53, characterized in that said at least one pharmaceutical composition comprising an active agent, which is an antibody to IL-1β or the fragment is selected from the group comprising sulfonylurea, meglitinide, biguanide, an inhibitor of alpha glucosidase, thiazolidinedione, a peptide similar to glucagon, and insulin.

55. A method according to claim 54, characterized in that the active agent is a sulfonylurea.

56. A method according to claim 54, characterized in that the active agent is meglitinide.

57. A method according to claim 54, characterized in that the active agent is biguanides.

58. A method according to claim 54, characterized in that the active agent is an inhibitor of alpha-glucosidase.

59. A method according to claim 54, characterized in that the active agent is thiazolidinediones.

60. A method according to claim 54, characterized in that the active agent is a peptide similar to glucagon.

61. A method according to claim 54, characterized in that the active agent is insulin.

62. A method according to claim 1, characterized in that after the introduction of the initial dose of the antibody or antibody fragment by adding one or more subsequent doses, wherein the concentration of the indicated antibody or antibody fragment in plasma l�dei is maintained at a constant level, at least about 0.03 μg/ml during the course of treatment the initial dose and one or more subsequent doses.

63. A method according to claim 62, characterized in that the concentration of the indicated antibody or antibody fragment in the plasma is maintained at a constant level, at least about 0.1 μg/ml during the course of treatment.

64. A method according to claim 63, characterized in that the concentration of the indicated antibody or antibody fragment in the plasma is maintained at a constant level, at least about 0.3 μg/ml during the course of treatment.

65. A method according to claim 1, characterized in that according to studies of inhibition of IL-1β in whole blood of the people, which measure induced IL-1β production of IL-8, an antibody or a fragment thereof has a lower value of IC50than the receptor antagonist IL-1.

66. A method according to claim 65, characterized in that the receptor antagonist IL-1 anakinra is used.

67. The use of antibodies to IL-1β or its fragment having a lower value of IC50than the receptor antagonist IL-1 according to studies of inhibition of IL-1β in whole blood of the people, which measure induced IL-1β production of IL-8, in the manufacture of a composition intended for use in the treatment of type 2 diabetes, insulin resistance, decreased production of insulin, hyperglycemia, hyperinsulinaemia and obesity, with antagonist-the-counter�Directors of IL-1 anakinra is.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to a compound of formula (I), or its tautomer, or a pharmaceutically acceptable salt, where each of Z1 and Z2: N and CR, where at least, one of Z1 and Z2 represents CR, and each R: H, C1-C4 alkyl and -N(R3)(R3); W: -O-, -N(C1-C4) alkyl and -C(R6)(R6) -, and each R6: H and C1-C4 alkyl, or two R6, bound with the same carbon atom, are taken together with the formation of =O, R1: a phenyl and heterocycle, which represents a saturated or unsaturated 5-6-member monocyclic ring, containing 1-3 heteroatoms, selected from atoms N, S and O, or a 8-12-member bicyclic ring, each cycle of which is selected from a saturated, unsaturated and aromatic cycle, containing 1-2 nitrogen atoms, where R1 is optionally substituted with one or more substituents, independently selected from halogen, C1-C4 alkyl, =O, fluorosubstituted C1-C2 alkyl, -O-R3, -(C1-C4 alkyl)-N(R3)(R3), -N(R3)(R3) and -C(O)-N(R3)(R3), R2: a phenyl and heterocycle, which represents an unsaturated 5-6-member monocyclic ring, containing 1-2 heteroatoms, selected from atoms N and O, or represents dihydrobenzofuranyl, where R2 is optionally substituted with 1-2 substituents, independently selected from a halogen, -C≡N, C1-C4 alkyl, C1-C2 fluorosubstituted alkyl, -O-R3, -(C1-C4 alkyl)-N(R3)(R3) and -N(R3)(R3); each R3: -C1-C4 alkyl; or two R3 are taken together with a nitrogen atom, which they are bound with, with the formation of a 4-8-member unsaturated heterocycle, optionally containing one additional heteroatom, selected from N and O, where in case when R3 represents an alkyl, the said alkyl is optionally substituted with two -OH groups, and when two R3 are taken together with a nitrogen atom, which they are bound with, with the formation of a 4-8-member saturated heterocycle, the said saturated heterocycle is optionally substituted with fluorine by any carbon atom; and is substituted with hydrogen by any capable of substitution nitrogen atom; p equals 1, 2 or 3; X2 is selected from -C(=O)-♣, -C(=O)-O-♣, -C(=O)-NH-♣, -S(=O)2-NH-♣ and -C(=O)-NH-CR4R5-♣, where: ♣ represents a site, by which X2 is bound with R1; and each R4 and R5 represents hydrogen. The invention also relates to compounds of formulas (IV), (V), (VI), particular the compounds, a pharmaceutical composition based on the compound of formulas (I), (IV)-(VI) and to a method of treatment, based on the application of the said compounds.

EFFECT: novel heterocyclic compounds, possessing sirtuin-modelling activity are obtained.

26 cl, 2 tbl, 40 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns a kit for treating diabetes comprising the first pharmaceutical composition and the second pharmaceutical composition, wherein the first pharmaceutical composition contains Gly(A21)-Arg(B31)-Arg(B32) human insulin, and the second pharmaceutical composition contains Gly(A21)-Arg(B31)-Arg(B32) human insulin and desPro36exendin-4(1-39)-Lys6-NH2; using the kit for preparing a medicated product for treating a patient suffering from diabetes for controlling the blood glucose concentration on an empty stomach, after meals and/or after absorption, or for improving the glucose tolerance.

EFFECT: group of inventions enables achieving the synergetic effect of the combination of the above compounds.

13 cl, 14 ex, 10 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely to novel analogues of insulin and can be used in medicine. The said insulin analogue is characterised by one of the following structures: Arg(A0), His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin; His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin; Arg(A0), Glu(A15), His(A8), Gly(A21), Arg(B31), Arg(B32)-NH2-insulin. The invention also relates to a method of obtaining the said analogue, a pharmaceutical composition and a medication, including its application for treatment of diabetes mellitus.

EFFECT: invention makes it possible to obtain the insulin analogue, characterized by the delayed release and steady and longer action, for instance, in comparison with human insulin or glargine insulin.

18 cl, 2 dwg, 24 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely to novel analogues of insulin and can be used in medicine. An insulin analogue, in which at least two hydrophobic amino acids are replaced with hydrophilic amino acids in comparison with a parent insulin, and where A-chain of the insulin analogue contains at least one mutation and B-chain contains at least one mutation in comparison with the parent insulin, and at least one mutation in A-chain is in one or more cleavage sites, selected from the group, consisting of A13-14, A14-15 and A19-20, and at least one mutation in B-chain is in one or more cleavage sites, selected from the group, consisting of B2-3, B6-7, B9-10, B10-11, B13-14, B14-15, B16-17, B22-23, B24-25, B25-26, and where amino acid in B30 position is deleted. The insulin analogue is used as a component of a pharmaceutical composition for treatment or prevention of hyperglycemia, type 1 or type 2 diabetes mellitus.

EFFECT: invention makes it possible to obtain the insulin analogue with an increased resistance with respect to one or more proteolytic enzymes in comparison with the parent insulin.

44 cl, 4 dwg, 5 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacy and medicine. What is presented is using a therapeutic agent containing a compound of the general formula

in a combination with at least one more therapeutic agent specified in biguanide and an α-glucosidase inhibitor for treating type 2 diabetes mellitus, diabetes complications, impaired glucose tolerance, insulin resistance or obesity, as well as the therapeutic agent based on the above combination for the same application.

EFFECT: technical effect consists in the synergetic action on an increase of the GLP-1 administration following administration of saccharose, a combination SK-0403 (a compound described by the general formula (I)) with Miglitol or metformin.

8 cl, 8 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of structural formula or a salt thereof, where each of Z1, Z2 and Z3 is independently selected from N and C(R9), where not more than one of Z1, Z2 and Z3 is N; each R9 is hydrogen; and is a second chemical bond between either W2 and C(R12), or W1 and C(R12); W1 is -N=, and W2(R14) is selected from -N(R14)- and -C(R14)=, such that when W1 is -N=, W2(R14) is -N(R14)- and is a second chemical bond between W1 and C(R12); R11 is selected from phenyl and a heterocycle which is selected from a saturated or aromatic 5-6-member monocyclic ring, which contains one or two or three heteroatoms selected from N, O and S, or an 8-member bicyclic ring which contains one or more heteroatoms selected from N, O and S, where R11 is optionally substituted with one or two substitutes independently selected from halogen, C1-C4 alkyl, =O, -O-R13, -(C1-C4 alkyl)-N(R13)(R13), -N(R13)(R13), where each R13 is independently selected from -C1-C4alkyl; or two R13 together with a nitrogen atom to which they are bonded form a 5-6-member saturated heterocycle, optionally containing an additional heteroatom selected from NH and O, where if R13 is an alkyl, the alkyl is optionally substituted with one or more substitutes selected from -OH, fluorine, and if two R13 together with the nitrogen atom to which they are bonded form a 5-6-member saturated heterocycle, the saturated heterocycle is optionally substituted on any carbon atom with fluorine; R12 is selected from phenyl, a 4-6-member monocyclic saturated ring and a heterocycle, which is selected from an aromatic 5-6-member monocyclic ring which contains one or two heteroatoms selected from N and S, where R12 is optionally substituted with one or more substitutes independently selected from halogen, -C≡N, C1-C4 alkyl, C1-C2 fluorine-substituted alkyl, -O-R13, -S(O)2-R13, -(C1-C4 alkyl)-N(R13)(R13), -N(R13)(R13); R14 is selected from hydrogen, C1-C4 alkyl, C1-C4 fluorine-substituted alkyl, C1-C4 alkyl-N(R13)(R13), C1-C4 alkyl-C(O)-N(R13)(R13); and X1 is selected from -NH-C(=O)-†, -C(=O)-NH-†, -NH-S(=O)2-†, where † denotes the point where X1 is bonded to R11. The invention also relates to a pharmaceutical composition having sirtuin modelling activity based on said compounds.

EFFECT: obtaining novel compounds and a pharmaceutical composition based on said compounds, which can be used in medicine to treat a subject suffering from or susceptible to insulin resistance, metabolic syndrome, diabetes or complications thereof.

18 cl, 2 tbl, 52 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, and concerns a dosage form of insulin containing insulin, a dissolving agent, and a zinc chelator, wherein the dosage form has a pH value varying within the range more than pH 7 and pH 7.6, represents a transparent aqueous solution; it also concerns an injection dosage form of insulin; a method of treating a diabetic patient involving the injection of the above dosage form into the patient.

EFFECT: group of inventions provides higher accumulation and transport through the epithelial cells as compared to insulin in a combination of HCl and zinc chelator above.

27 cl, 11 ex, 13 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of general formula (1) or salts thereof, where in formula (1) R1 is a lower C1-C6alkyl group, a lower C3-C6cycloalkyl group, a phenyl group, a heterocyclic group, which relates to a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic heterocyclic ring containing one, two or three heteroatoms in the ring, selected from a nitrogen atom, an oxygen atom and a sulphur atom, or a phenyl(C1-C6alkyl) group; in cases when R1 is a lower C1-C6alkyl group, that lower C1-C6alkyl group can have, as substitute(s), one, two or three groups selected from a halogen atom, a heterocyclic group which relates to a residue formed by removing a hydrogen atom from a saturated monocyclic heterocyclic ring containing one or two heteroatoms in the ring, selected from a nitrogen atom and an oxygen atom, a carboxyl group, a lower C1-C6alkoxycarbonyl group, a lower C1-C6alkylamino group, a lower C1-C6alkylamino group, substituted with a lower C1-C6alkylamino group, a lower C1-C6alkylamino group, substituted with a phenyl group; in cases when R1 is a phenyl group, a heterocyclic group which relates to a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic heterocyclic ring containing one, two or three heteroatoms in the ring, selected from a nitrogen atom, an oxygen atom or a sulphur atom, or a phenyl(C1-C6alkyl) group, that phenyl, heterocyclic or phenyl(C1-C6alkyl) group can contain, as substitute(s), one, two or three groups selected from a halogen atom, a lower C1-C6alkyl group, a hydroxyl group or a lower C1-C6alkoxy group; R2 is a hydrogen atom or a lower C1-C6alkyl group; R3 is a hydrogen atom or a lower C1-C6alkyl group; R4 and R5 can be identical or different and are a hydrogen atom or a lower C1-C6alkyl group; R6 is a hydrogen atom or a lower C1-C6alkyl group; R7 is a phenyl group or a heterocyclic group which relates to a residue formed by removing a hydrogen atom from a saturated monocyclic heterocyclic ring containing one heteroatom in the ring, selected from an oxygen atom and a sulphur atom; in cases where R7 is a phenyl group or a heterocyclic group which relates to a residue formed by removing a hydrogen atom from a saturated monocyclic heterocyclic ring containing one heteroatom in the ring, selected from an oxygen atom and a sulphur atom, that phenyl or heterocyclic group can contain, as substitute(s), one or two groups selected from a halogen atom, a lower C1-C6alkyl group, a hydroxyl group, a lower C1-C6alkoxy group and a nitro group; W is an oxygen atom or NR8; R8 is a hydrogen atom or a lower C1-C6alkyl group; X is an oxygen atom or a sulphur atom; Y is a lower C1-C6alkylene group; Z is an oxygen atom, a sulphur atom, NR9 or OCO; R9 is a hydrogen atom or a lower C1-C6alkyl group. The invention also relates to a pharmaceutical composition based on said compounds, having GR binding activity.

EFFECT: obtaining novel compounds and a pharmaceutical composition based on said compounds, which can be used in medicine as glucocorticoid receptor modulators.

10 cl, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely endocrinology, and concerns predicting the clinical effectiveness in diabetic polyneuropathy in the patients suffering type 2 diabetes and dyslipidemia. That is ensured by evaluating the intensity of diabetic polyneuropathy manifestations taking into account neuropathic dysfunctional score, evaluating triglycerides and glycolised blood hemoglobin before the treatment; the derived data are used to specify a therapeutic approach followed by further estimation of a probability of a successful correction of diabetic polyneuropathy by formula P=111+e(0,991,78Z), wherein e is the basis of hyperbolic logarithm e=2.72; Z is a regression coefficient calculated by formula 7=4.56-0.05*"ДлСД"-0.02*IntDPN-0.05*TG-0.53*HbAlc+0.14TherApp; if the value P is 0.7 or more, the successful correction of DPN is predicted in the patients, and the value P less than 0.7 requires the therapeutic approach to be changed.

EFFECT: method provides the improved clinical effectiveness ensured by the individual therapeutic approach based on the lipid and carbohydrate metabolism of a specific patient.

2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel N-containing heteroaryl derivatives of formula I or II or their pharmaceutically acceptable salts, which possess properties of JAK kinase, in particular JAK3, and can be applied for treating such diseases as asthma and chronic obstructive pulmonary disease (COPD). In formulae A represents carbon and B represents nitrogen or A represents nitrogen and B represents carbon; W represents CH or N; R1 and R2, independently represent hydrogen, C1-4alkyl, halogenC1-4alkyl, -CN; R3 represents C1-4alkyl, R9-C1-4alkyl, Cy1, where Cy1 is optionally substituted with one or several substituents R10; R4 represents hydrogen, C1-4alkyl, R12R7N-C0alkyl, where one of R7 and R12 represents hydrogen, and the other represents C1-4alkyl or group R13, which is selected from C1-5alkyl, Cy2-C0alkyl; R5 represents hydrogen; R6 represents hydrogen, C1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl, R12R7N-C1-4alkyl, R16CO-C0alkyl, Cy1; R7 represents hydrogen or C1-4alkyl; R9 represents halogen, -CN, -CONR7R12, -COR13, CO2R12, -OR12, -SO2R13, -SO2NR7R12, -NR7R12, -NR7COR12; R10 represents C1-4alkyl or R9-C0-4alkyl; R11 represents C1-4alkyl, halogen, -CN, -NR7R14; R12 represents hydrogen or R13; R13 represents C1-5alkyl, hydroxyC1-4alkyl, cyanoC1-4alkyl, Cy2-C0alkyl or R14R7N-C1-4alkyl; where Cy2 is optionally substituted with one or several constituents R11; R14 represents hydrogen or C1-4alkyl; R16 represents C1-4alkyl, halogenC1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl or cyanoC1-4alkyl; Cy1 represents monocyclic carbocyclic unsaturated or saturated ring, selected from C3-C6cycloalkyl, phenyl, or saturated monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or partially unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available C atom, and where one or several ring C or S atoms are optionally oxidised with formation of CO or SO2; and Cy2 represents monocyclic carbocyclic unsaturated ring, selected from C3-C6cycloalkyl, or aromatic monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available atom C or N.

EFFECT: obtaining novel heteroaryl derivatives.

27 cl, 41 ex

Transdermal plaster // 2553350

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine. Described is matrix layer, suitable for application in plaster for transdermal delivery, aimed at introduction of biologically active compounds, which includes phosphate compound of tocopherol and polymer carrier. Also described is transdermal plaster and method of its production.

EFFECT: plaster makes it possible to efficiency introduce biologically active compounds.

48 cl, 15 tbl, 13 dwg, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents clinical nutrition for prevention, treatment or relief of one or several symptoms, associated with impairment of metabolism or its disorder, which contains composition of polysaccharide high-viscosity dietary fibre, including viscous fibre mixture or its complex, consisting of from 48% to 90% in wt % of glucomannan, from 5 to 20 % in wt % of xanthan gum and from 5% to 30% in wt % of alginate, as well as, at least, one macroelement, selected from the group, consisting of protein carbohydrate and fat, where clinical nutrition is composed in order to provide dose of composition of polysaccharide high-viscosity dietary fibre from 20 g/day to 35 g/day for time period, effective for prevention, treatment and relief of one or several symptoms, associated with impairment of metabolism or its disorder.

EFFECT: invention ensures extension of arsenal of means, preventing, relieving or treating one or several symptoms, associated with impairment of metabolism or metabolic disease.

14 cl, 6 ex, 20 tbl, 48 dwg

FIELD: medicine.

SUBSTANCE: patients with diabetic microangiopathy are subjected to an examination which includes: general blood test, blood sugar, general urine analysis, ultrasonic examination of kidneys with the determination of indices of the kidney blood flow (Vmax, Vmin, S/D, PI, RI), basic ophthalmological parameters (vision acuity, examination of eye fundus vessels). Then, the intake of mildly-mineralised hydrocarbonate-chloride-sodium mineral water "Obyhovskaya" directly from the spring under sanatorium conditions is administered. Water is taken in heated to a temperature of 37°C in a dose of 3 ml per 1 kg of body weight 3 times per day 40 minutes before meal, the course constitutes 18 days.

EFFECT: application of the invention makes it possible to normalise the general blood test, blood sugar, general urine analysis, improve the condition of the visual analyser and indices of the kidney blood flow.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to 2-pyridone compounds, represented by general formula [1], , where A represents benzene ring or pyridine ring, X represents structure, represented by general formula [3], V represents single bond or lower alkylene, W represents single bond, ether bond or lower alkylene, which can include ether bond, or their tautomers or stereoisomers.

EFFECT: obtaining pharmaceutically acceptable salts, which possess excellent activating activity with respect to GK and can be applied as medications.

27 cl, 23 tbl, 371 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to endocrinology, and deals with stimulation of insulin secretion. For this purpose 2 ml of concentrated nitro-glycerine solution are diluted with distilled water, cotton swab is soaked with obtained solution, stretched to 10-12 cm long and 3-4 cm wide size, applied perpendicular to spine on the left at the level of Th12, covered with cellophane and sealed with self-adhering plaster, with patient being turned onto back with preservation of said position for 1 hour.

EFFECT: method provides enhancement of insulin secretion by pancreas due to improvement of its blood supply.

2 ex, 2 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new substituted aminotetrahydropyranes of structural formula or to their pharmaceutically acceptable salts , and , wherein V is specified in groups having the formulas below, Ar represents phenyl unsubstituted or substituted by one to five halogen atoms, each of R1 and R2 is independently specified in C1-C6alkyl; R3 is specified in a group consisting of C1-C6alkyl; cyano; tetrazolyl; -C(O)OC1-C6alkyl and -C(O)NH2; wherein C1-C6alkyl is substituted by 1-4 substitutes independently specified in a group consisting of OH; -C(O)NH2 and -CO2H. The declared compounds can be dipeptidylpeptidase-IV inhibitors and can be applicable in treating or preventing diseases involving the enzyme dipeptidylpeptidase-IV, such as diabetes, and especially type 2 diabetes mellitus.

EFFECT: invention also refers to a pharmaceutical composition containing the above compounds, and using the above compounds and compositions for preventing or treating the diseases involving the enzyme dipeptidylpeptidase-IV.

12 cl, 14 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to derivatives of diaza-spiro[4.5]decan-1-one of formula (I) or to their pharmaceutically acceptable salts, where. R1 is a substituted phenyl, which contains one substituent, selected from the group, including C1-4-alkyl, C3-6-cycloalkyl halo-C1-4-alkyl and halo -C1-4-alkoxy, and which can additionally contain one substituent, selected from a halogen; R2 is hydrogen, C1-4-alkyl, phenyl, substituted phenyl, with the substituted phenyl containing one substituent, selected from the group, including C1-4-alkoxy; R3 is -R4, -C(OH)R5R6 or -C(O)NR7R8; R4 is phenyl, phenyl-C1-4-alkyl, substituted phenyl, substituted phenylcarbonyl, with the substituted phenyl, substituted phenylcarbonyl containing from one to two substituents, selected from the group, including a halogen, halo-C1-4-alkyl; one of R5 and R6 is hydrogen, C1-4-alkyl, and the other is aminocarbonyl, phenyl, substituted phenyl or substituted phenyl-C1-4-alkyl, with the substituted phenyl or substituted phenyl-C1-4-alkyl containing from one to two substituents, independently selected from the group, including a halogen; one of R7 and R8 is hydrogen C1-4-alkyl, and the other is C1-4-alkyl, C3-6-cycloalkyl, C1-4-alkoxy-C1-4-alkyl, phenyl-C1-4-alkyl, substituted phenyl or substituted phenyl-C1-4-alkyl, with the substituted phenyl or substituted phenyl-C1-4-alkyl containing one substituent, selected from the group, including a halogen, halo-C1-4-alkyl; or R7 and R8 together with a nitrogen atom, which they are bound to, form pyrrolidinyl; n equals zero or 1/ The invention also relates to a pharmaceutical composition based on the compound of formula (I), application of the formula (I) compound and a method of treatment.

EFFECT: obtained are novel heterocyclic compounds, useful as an inhibitor of hormone-sensitive lipase.

17 cl, 57 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and represents a formulation specially adapted for insulin transformation into the aerosol state, containing insulin in water from 100 IU/ml to 1,200 IU/ml and 2 to 4 Zn2+ ions per the insulin hexamer, wherein the formulation is preservative-free and wherein the formulation is able to transform into the aerosol state as an aerosol spray when using a holed vibration plate with no foaming of the formulation, when the formulation is kept on a back surface of the holed plate by gravity, and the spray is ejected from the front surface of the holed plate entirely by vibrating the holed plate.

EFFECT: invention provides reducing the foaming of the formulation, the time of transformation into the aerosol state and providing the more effective administration of an insulin dose.

19 cl, 5 dwg

FIELD: veterinary medicine.

SUBSTANCE: composition comprises succinic acid, trace elements in the form of sulphates of iron, copper, cobalt, zinc and additionally comprises methionine and beet-root molasses at the following content of components in 1000 ml of an aqueous solution: succinic acid - 5.0 g, beet-root molasses - 150.0 ml, methionine - 2.0 g, ferrous sulphate - 10.0 g, copper sulphate - 0.1 g, cobalt sulphate - 0.5 g, zinc sulphate - 0.5 g.

EFFECT: use of the claimed composition has a positive effect on the immune-metabolic status and growth activity of piglets.

3 tbl, 2 ex

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