Application of peptide ggf2 in treatment or prevention of heart failure in mammals

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology. Characterised is application of therapeutically efficient quantity of peptide GGF2, which contains domain, similar to epidermal growth factor (EGF-like), in treatment or prevention of heart failure in mammal by injection of said peptide every 48 hours in dose, which constitutes from approximately 0.001 mg/kg to approximately 10 mg/kg.

EFFECT: invention improves therapeutic effect with introduction of neuroregulin with minimisation of any potential side effects.

14 cl, 15 dwg, 11 tbl

 

The SCOPE of the INVENTION

The field of invention relates to the treatment of heart failure. More specifically, the invention relates to an improved dispensing, under which is stored and/or improves therapeutic effect of the introduction of neuregulin, such as a growth factor, glia 2 (GGF2) or its fragment, while minimizing any potential side effects.

BACKGROUND of the INVENTION

The fundamental problem associated with the administration of medicines to patients is the relationship between portability and efficiency. therapeutic index is the range, however, when the patient can enter the effective dose of the substance, and the dose at which the observed adverse effects. Generally, the greater the difference between the effective dose and the dose at which the start side effects, the more poorly the current is substance and more likely that it will transfer the patient.

Heart disease, especially congestive heart failure (CHF) is one of the leading causes of death in industrialized countries. Factors underlying congestive heart failure include high blood pressure, coronary heart disease, exposure to cardiotoxic compounds, such as ant is AllInOne antibiotics, radiation exposure, physical trauma, and genetic defects associated with an increased risk of heart failure. Thus, CHF is often a result of increased load on the heart due to hypertension, myocardial damage due to chronic ischemia, myocardial infarction, viral diseases, chemical toxicity, radiation, and other diseases, such as scleroderma. These conditions cause progressive decrease in the pumping function of the heart. First, the increased load resulting from high blood pressure or loss of contractile tissue, induces compensatory hypertrophy of cardiomyocytes and thickening of the wall of the left ventricle, thus increasing contractility and supporting heart function. However, over time the chamber of the left ventricle expands, systolic pump function deteriorates, cardiomyocytes undergo apoptotic cell death and function of the heart muscle is getting progressively worse.

Neuregulin (NRG) and receptors NRG constitute a system like growth factor receptor tyrosinekinase for intercellular signaling, which is involved in organogenesis and development of the cells in nerve, muscle, epithelial and other tissues (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Burden et al., Neuron 18:847-855, 1997). Family NRG consists of a couple, the ex genes coding many ligands including such epidermal growth factor (EGF), immunoglobulin (Ig) and other recognizable domains. Many secreted and membrane-bound isoforms function as ligand in this system, the signal transmission. The receptor ligand NRG are all members of the family of EGF receptors (EGFR) and EGFR (or ErbB1), ErbB2, ErbB3 and ErbB4, people also known under the names from HER1 to HER4, respectively (Meyer et al., Development 124:3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90:1867-71, 1993; Marchionni et al., Nature 362:312-8, 1993; Chen et al., J. Comp. Neurol. 349:389-400, 1994; Corfas et al., Neuron 14:103-115, 1995; Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; Pinkas-Kramarski et al., Oncogene 15:2803-2815, 1997).

Four gene NRG, NRG-1, NRG-2, NRG-3 and NRG-4, are mapped to different chromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA 91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci. USA 94:9562-9567, 1997), and together encode a diverse set of proteins NRG. For example, the gene products of NRG-1, include the group of about 15 different structurally related isoforms (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Peles and Yarden, BioEssays 15:815-824, 1993). First identified isoforms of NRG-1 included the factor of Neu differentiation (NDF; Peles et al., Cell 69, 205-216, 1992, and Wen et al., Cell 69, 559-572, 1992), heregulin (HRG; Holmes et al., Science 256:1205-1210, 1992), the inducer activity of the acetylcholine receptor (ARIA; Falls et al., Cell 72:801-815, 1993) and growth factors glial GGF1, GGF2,and GGF3 (Marchionni et al. Nature 362:312-8, 1993).

Gene NRG-2 identified through cloning by homology (Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; Higashiyama et al., J. Biochem. 122:675-680, 1997) and by genomic methods (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). cDNA NRG-2, also known as derived from nervous tissue and thymus activator ErbB-kinases (NTAK; inventory number GenBank AB005060), divergent variant neuregulin (Don-1) and obtained from cerebellar growth factor (CDGF; PCT application WO 97/09425). Experimental data demonstrate that, apparently, cells expressing ErbB4, or a combination of ErbB2/ErbB4, show a very strong response to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol. 18:6090-6101, 1998). It is also known that the product of the gene NRG-3 (Zhang et al., above) binds and activates ErbB4 receptors (Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998).

In the main part of all forms of NRG present EGF-like domain, and it is necessary for binding and activation of ErbB receptors. Installed amino acid sequence of the EGF-like domain encoded by three genes, approximately 30-40% identical (pairwise comparisons). In addition, NRG-1 and NRG-2, apparently, there are at least two subforms EGF-like domains, which can provide various kinds of biological activity and tissue-specific actions.

Cellular response to NRG mediated tyrosinase kinases receptors NRG EGFR, ErbB2, ErbB3 and ErbB4 family recipe is the moat epidermal growth factor. High-affinity binding of all NRG mediated mainly ErbB3 or ErbB4. Ligand binding NRG leads to dimerization with other subunits ErbB and TRANS-activation by phosphorylation on specific tyrosinase residues. In certain experimental conditions, apparently, almost all combinations of ErbB receptors can form dimers in response to the binding isoforms of NRG-1. However, apparently, ErbB2 is the preferred dimerization partner, which may play an important role in the stabilization of the complex ligand-receptor. Himself ErbB2 does not bind ligand, but should have been prepared to mate with one of the other receptor subtypes. ErbB3 has tyrosinekinase activity, but is a target for phosphorylation by other receptors. It is known that the expression of NRG-1, ErbB2 and ErbB4 is required for the formation of trabeculae of the myocardium of the ventricle in the ontogeny of the mouse

Neuregulin stimulate compensatory hypertrophic growth and inhibit apoptosis of myocardiocytes subjected to physiological stress. In accordance with these observations introduction neuregulin suitable to prevent, minimize, or reverse the development of congestive heart disease arising from such contributing factors as hypertension, ischemic heart disease and cardiotoxicity. See, for example, U.S. patent n the measures (USPN) 6635249, which fully included in the present description.

Due to the high prevalence of heart failure in the General population, continues to be an unmet need to prevent or minimize the progression of this disease, such as preventing loss of cardiac function or by improving cardiac function.

The INVENTION

The present invention includes a method of treating or preventing heart failure in a mammal. The method is based on the unexpected observation that therapeutic effects of the peptide, which contains a domain similar to epidermal growth factor (EGF-like), can be achieved by dosing schedules for the administration of neuregulin that do not support the stationary state, such as by administration of a therapeutically effective amount of the peptide to the mammal at intervals insertion through or for 48, 72, 96 or more hours. Thus, the present method provides for the intermittent or intermittent introduction (each lasts for 48-96 hours, or even with large intervals) to a mammal peptide containing EGF-like domain, where EGF-like domain is encoded by the genome of neuregulin, and where the introduction of the peptide produced in a quantity effective to treat or prevent genial, OEM is the second failure in a mammal. Dosing regimens for administration of neuregulin that do not support the steady-state concentrations are equally effective as dosing regimens with more frequent introduction, but without the hassle, cost or side effects, which can be a consequence of more frequent administration. In the framework of the invention, the term intermittent or intermittent introduction includes dosage at intervals of at least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 month, 3 month, 4 month, or any combination or increment, provided what interval/mode is at least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 month, 3 month, 4 month. In the framework of the invention, the term intermittent or intermittent introduction includes dosage at intervals of not less than 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months or any combination or increment, provided what interval/mode of composition is given not less than 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months.

In accordance with the present invention, intermittently, or intermittent administration to a mammal of a peptide containing the EGF-like domain, where EGF-like domain is encoded by the genome of neuregulin, aimed at achieving dosing schedules, where not supported stationary concentration of the injected peptide in a narrow range, thus reducing the likelihood that the mammal will develop adverse side effects that may arise from maintaining the levels of the input peptide above physiological in the long term. For example, side effects associated with the levels of exogenous NRG entered above physiological, include hyperplasia of nerve membranes, hyperplasia of mammary glands, renal nephropathy, hypospermia, elevated liver enzymes, changes in heart valves and skin changes at the injection site.

In a preferred embodiment, the present invention relates to the intermittent dosage regimen which causes or permits fluctuation of serum levels of a peptide containing the EGF-like domain encoded by the genome of N. Jigulina, and, thus, reduces the possibility of adverse side effects associated with more frequent introduction of the peptide. Thus, the intermittent dosing regimen of the present invention provides the mammal a therapeutic effect, but does not support the inpatient therapeutic levels of peptide containing EGF-like domain encoded by the genome of neuregulin. It is understood by experts in this field, there are different ways of carrying out the invention for the implementation of the intermittent dosing; the advantages of these embodiments can be specified in various ways, for example, this introduction does not support the inpatient therapeutic levels of the indicated peptide, introduction reduces the possibility of harmful side effects associated with more frequent introduction of peptide NRG, etc.

In specific embodiments of the invention, neuregulin can be a gene product of a gene or its corresponding subsequence or fragment, containing, essentially consisting of or consisting of: NRG-1, NRG-2, NRG-3 and NRG-4. In a preferred embodiment, a subsequence or fragment of NRG according to the invention contains a domain similar to epidermal growth factor (EGF-like) or its homologue. It is understood by experts in this field peptide, homologous peptide EGF-like domain determined by searching structural homology or by homologous peptide that acts as acts EGF-like peptide in functional assays, for example, linking and activating ErbB receptors. Preferably the length of the fragment is at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 of amino acids. The peptide of neuregulin according to the invention, in turn, can be encoded in any of these genes neuregulins (or subsequence). In a more specific embodiment, the peptide used in the method, is a recombinant human GGF2 or a fragment or subsequence. Relative to the amino acid sequence and nucleic acid full-sized human GGF2 see figures 8A-8D.

In one aspect of the invention suitable mammals include as non-limiting examples of mice, rats, rabbits, dogs, monkeys or pigs. In one of the embodiments of the invention the mammal is a human.

In other embodiments of the invention, heart failure can result from hypertension, ischemic heart disease, exposure to cardiotoxic compounds (e.g., cocaine, alcohol is, antibodies to ErbB2 or antibodies to HER, such as Herceptin®, or anthracycline antibiotics such as doxorubicin or daunomycin), myocarditis, thyroid disease, viral infection, gingivitis, drug addiction, alcoholism, pericardial effusion, atherosclerosis, vascular disease, hypertensive cardiomyopathy, acute myocardial infarction or previous myocardial infarction, systolic dysfunction of the left ventricle, surgery coronary artery bypass graft surgery, starvation, radiation, malnutrition or genetic defect.

In another embodiment of the invention, the antibody to ErbB2 or HER2, such as Herceptin®, is administered to the mammal before, during or after the introduction of anthracycline.

In other embodiments of the invention the peptide is administered before exposure to cardiotoxic compounds within the cardiotoxic effect of this connection, or after exposure to the specified cardiotoxic compounds; the peptide is administered before or after the diagnosis of congestive heart failure at the specified mammal. The method according to the invention can be applied after the specified mammal occurred compensatory hypertrophy of the heart; the method according to the invention includes that the outcome of this method is the preservation of left ventricular hypertrophy or preventing PR is dressirovannye thinning of the myocardium or the inhibition of apoptosis of cardiomyocytes. In the method according to the invention, the peptide may contain EGF-like domain encoded by the genome of neuregulin, essentially consist of or consist of it. The peptide according to the invention is administered before, during or after exposure to cardiotoxic compounds. In another embodiment, the peptide containing the EGF-like domain, administered for two or all three of these periods. In accordance with the present invention, the peptide containing the EGF-like domain encoded by the genome of neuregulin, introduced at intervals of every lasts for 48-96 hours. In one of the embodiments of the present invention, the peptide containing the EGF-like domain encoded by the genome of neuregulin is a GGF2. In other embodiments of the invention, the peptide is administered before or after diagnosis in a mammal of congestive heart failure. In yet another embodiment of the invention the peptide is administered to a mammal, in which there is compensatory hypertrophy of the heart. In other specific embodiments of the invention the introduction of the peptide retains the hypertrophy of the left ventricle, to prevent the progression of thinning of the myocardium and/or inhibits apoptosis of cardiomyocytes.

Embodiments of the invention include a method of treating heart failure in a mammal, where the method includes the introduction of e is saganaga peptide, contains a domain similar to epidermal growth factor (EGF-like) provided to the mammal, where the aforementioned introduction with the specified intervals reduces the adverse side effects associated with the introduction of the specified exogenous peptide specified mammal. A method of treating heart failure in a mammal, where the method includes the introduction of exogenous peptide that contains a domain similar to epidermal growth factor (EGF-like) provided to the mammal, where the specified EGF-like domain is encoded by the genome of neuregulin (NRG)-1 and the specified exogenous peptide is administered to the specified mammal in a therapeutically effective for the treatment of heart failure the number of intervals of at least 48 hours, where the aforementioned introduction with the specified intervals does not support fixed levels specified exogenous peptide at the indicated mammal. A method of treating heart failure in a mammal, where the method includes the specified mammal introduction of exogenous peptide that contains a domain similar to epidermal growth factor (EGF-like), or its homolog, and the specified exogenous peptide is administered to the specified mammal in a therapeutically effective for the treatment of heart failure the number of intervals on minicamera or not less than 48 hours, where specified, the introduction of these intervals allow deviations serum concentrations specified exogenous peptide at the indicated mammal between doses up to background levels or to levels before the introduction.

In the framework of the invention, the term "adverse" or "harmful side effects" refers to the unintended and undesirable consequence of medical treatment. In relation to the present invention adverse or harmful side effects resulting from the introduction of exogenous peptide may include one or more of the following: hyperplasia shell nerve hyperplasia of the breast, renal nephropathy and skin changes at the injection site.

In the framework of the invention, the term "deviation serum concentrations specified exogenous peptide at the indicated mammal between doses to levels before the introduction" refers to the difference between the levels of serum concentrations to doses of exogenous peptide.

In the framework of the invention, the term "stationary levels" refers to the level(s) of the exogenous means (e.g., peptide), which is enough to achieve equilibrium (within the range of variation between subsequent doses) between the introduction and elimination. "Maintaining stationary therapeutic levels" refers to maintaining concentric and exogenous means at the level sufficient to provide a therapeutic effect in an individual or patient.

BRIEF DESCRIPTION of DRAWINGS

Onfigure 1presents the histogram, which presents heart function, illustrated by the changes in ejection fraction and fraction shortening. As indicated, rats were treated with GGF2 at 0.625 mg/kg) or equimolar amounts of EGF-like fragment (fragment; EGF-id) intravenous (IV) daily (once per day).

Onfigure 2presents a line chart, which presents heart function, detected by changes in ejection fraction and fraction shortening. As indicated, rats were treated with GGF2 at 0.625 mg/kg or 3.25 mg/kg/once a day.

Onfigure 3presents a line chart, which presents cardiac function detected in significant improvement in end-systolic volume during the treatment period. As indicated, rats were treated with GGF2 at 0.625 mg/kg or 3.25 mg/kg/once a day.

Onfigure 4presents a line chart, which presents heart function, detected by changes in ejection fraction and fraction shortening. As indicated, rats were treated with GGF2 of 3.25 mg/kg intravenous (IV) every 24, 48 or 96 hours.

Onfigure 5presents a line chart, which presents heart function, identify the changes electr the cardiographic ejection fraction. As indicated, rats were treated with media or 3.25 mg/kg GGF2 intravenous (IV), with BSA or without it.

Onfigure 6line chart, which presents the half-life of recombinant human GGF2 (rhGGF2) after the on/in the introduction.

Onfigure 7line chart representing the period of the half-life of recombinant human GGF2 (rhGGF2) after subcutaneous injection.

Onfigures 8A-Dpresents nucleic acid sequence and amino acid full-GGF2. The nucleic acid sequence designated SEQ ID NO: 1 and the amino acid sequence designated SEQ ID NO: 2.

Onfigure 9presents nucleic acid sequence and amino acids similar to epidermal growth factor (EGFL) domain 1. The sequence of the nucleic acid EGFL domain 1 in the present description denoted by SEQ ID NO: 3 and amino acid sequence EGFL domain 1 in the present description denoted by SEQ ID NO: 4.

Onfigure 10presents nucleic acid sequence and amino acids similar to epidermal growth factor (EGFL) domain 2. The sequence of the nucleic acid EGFL domain 2 in the present description denoted by SEQ ID NO: 5, and amino acid sequence EGFL domain 2 in the present description denoted by SEQ ID NO: 6.

Onfigure 11presents the sequence nuclein the second acid and amino acids similar to epidermal growth factor (EGFL) domain 3. The sequence of the nucleic acid EGFL domain 3 in the present description denoted by SEQ ID NO: 7 and amino acid sequence EGFL domain 3 in the present description denoted by SEQ ID NO: 8.

Onfigure 12presents nucleic acid sequence and amino acids similar to epidermal growth factor (EGFL) domain 4. The sequence of the nucleic acid EGFL domain 4 in the present description denoted by SEQ ID NO: 9 and amino acid sequence EGFL domain 4 in the present description denoted by SEQ ID NO: 10.

Onfigure 13presents nucleic acid sequence and amino acids similar to epidermal growth factor (EGFL) domain 5. The sequence of the nucleic acid EGFL domain 5 in the present description denoted by SEQ ID NO: 11 and amino acid sequence EGFL domain 5 in the present description denoted by SEQ ID NO: 12.

Onfigure 14presents nucleic acid sequence and amino acids similar to epidermal growth factor (EGFL) domain 6. The sequence of the nucleic acid EGFL domain 6 in the present description denoted by SEQ ID NO: 13 and amino acid sequence EGFL domain 6 in the present description denoted by SEQ ID NO: 14.

Onfigure 15presents the amino acid sequence of the polypeptide containing epidermal growth factor (EGFL) domain, which in the us is oasam description denoted by SEQ ID NO: 21.

DETAILED description of the INVENTION

The authors of the present invention made an unexpected discovery that the discontinuous or intermittent introduction neuregulin with appropriately spaced time intervals provides a therapeutically effective amount of neuregulin the patient and such a regimen suitable to prevent, prevention, improvement, mitigation, treatment, or reverse the development of heart disease, such as congestive heart failure.

Despite the conventional wisdom and practice development in relation to the preparation of dosing schedules to maintain the narrow range of stationary concentrations, the authors of the present invention in the present description demonstrate that dosing regimens for the introduction neuregulin that do not support the stationary concentration in a narrow range, are equally effective as dosing regimens with more frequent introduction. In addition, the authors of the present invention demonstrated that the treatment of an individual with heart failure by neuregulins with dosing intervals of at least 48 hours, 72 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months is CA, 4 months, or any combination or increment, provided that the interval/mode is at least 48 hours, are as effective as daily dosing.

To assess the pharmacokinetics of exogenous NRG, the authors of the present invention showed that the half-life of neuregulin with intravenous ranges from 4 to 8 hours, and after subcutaneous administration is 11-15 hours. See, for example, tables 1 and 2 and figures 6 and 7. Thus, the dosage when the modes are not more often than every fourth day could not support any detectable levels for at least three days between doses. On the basis of these results to the present invention it was impossible to predict that such correlation peaks/dips will correlate with persistent therapeutic effect. It is noteworthy that compounds with a half-life of this order, usually administered in accordance with frequent dosing regime (for example, daily or multiple daily doses). In addition, on the basis of pharmacokinetic data available for GGF2 in accordance with traditional designs, we can assume that the optimal treatment can include daily subcutaneous dosing.

In accordance with the generally accepted point of view and practice the development of other medicinal cf is DSTV with CHF, usually injected at least on a daily basis. Suppose that the frequency of such dosing is necessary because CHF is a chronic condition, usually caused by impaired contraction and/or relaxation of the heart, rather than acute condition. Individuals with a weak heart, leading to impaired relaxation and CHF, medical drugs include drugs that block the formation or action of specific neurohormones (eg, angiotensin converting enzyme inhibitors (ACE inhibitors), antagonists of angiotensin receptor (ARB), aldosterone antagonists and blockers beta-adrenergic receptors). These and other drugs currently represent the standard in the treatment of chronic CHF, as they have been shown to lead to improved symptoms, life expectancy and/or to reduce the number of hospitalizations. In conditions of acute or chronic symptoms, patients are often treated with inotropic agents (eg, dobutamine, digoxin) to improve cardiac contractility together with vasodilator agents (e.g., nitrates, nesiritide) and/or diuretics (eg, furosemide) to reduce congestion. Patients with hypertension and congestive heart failure treat one or more of the likeme antihypertensive agents, such as beta-blockers, ACE inhibitors and ARB, nitrates (isosorbide dinitrate treatment), hydralazine and calcium channel blockers.

Thus, despite the common practice regarding the treatment of CHF, the authors present invention has demonstrated that a new dosage regimen leads to effective treatment of CHF, while avoiding unwanted side effects. Not wanting to contact theory, it is likely that this treatment neuregulin enhances the pumping function of the heart, stimulating hypertrophy of cardiomyocytes and partially or completely inhibiting the further destruction of the heart by inhibiting the apoptosis of cardiomyocytes.

As additional information, the basic principle of dosing is the definition of effective circulating concentration and development of dosage to maintain these levels. To forecast dosing regimen that will maintain a fixed level of a particular drug, combined Pharmacokinetic (PK) and pharmacodynamic (PD) studies. A typical plan is to minimize the differences between Cmaxand Cminand, thereby, reduce side effects.

Medications describe their "therapeutic index" which represents the ratio of the toxic dose or circulating levels of effective dose or the CID is lirousi concentrations. When therapeutic index is large, there is a safe range, in which you can enter the effective dose without approaching toxic levels. When adverse effects occur at concentrations very close to the effective concentrations of therapeutic index is defined as narrow, and the drug is difficult to use safely.

When designing dosing schedules PK/PD data are combined with information about therapeutic index for planning dose and frequency of injection so that the connection is maintained for the patient (e.g., human) at a concentration which is higher than the effective concentrations below toxic concentrations. If the effective concentration of the drug cannot be maintained without induction unsafe effects from medicines throughout the development of refuse. Additional comments related to drug development can be found in several sources, including: Pharmacokinetics in Drug Development: Clinical Study Design and Analysis (2004, Peter Bonate and Danny Howard, eds.), which fully included in the present description.

Neuregulin are growth factors related to epidermal growth factors that bind to erbB receptors. In many models of heart failure, cardiotoxicity and ischemia are shown, Thu whom they improve heart function. Also it is shown that they protect the nervous system in models of stroke, spinal cord injury, exposure to nerve toxic substances, damage to peripheral nerves and chemical toxicity.

However, it is shown that the maintenance of excessive levels of exogenous input neuregulin has adverse effects, including hyperplasia of the sheath of the nerve, hyperplasia of the mammary gland and kidney nephropathy. These effects were observed after daily subcutaneous introduction neuregulin. See, for example, table 10.

As indicated in the present description, subcutaneous administration investigated due to the longer half-life compared to intravenous and original beliefs that maintain constant levels of the ligand may be useful. Development of dosing schedules to reduce these effects could significantly increase the use of neuregulins in the quality of medicines and that this relates to the present invention. The demonstration that less frequent dosing, which does not remain constant levels, is also effective, provided this development.

Neuregulin: As indicated above, peptides, encoded by the genes NRG-1, NRG-2, NRG-3 and NRG-4, contain EGF-like domains, which allow them to bind and activate the recipe is ture ErbB. Holmes et al. (Science 256:1205-1210, 1992) showed that for binding and activation of receptor p185erbB2 only one EGF-like domain. Thus, for the prevention or treatment of congestive heart failure in the methods according to the invention can use any peptide product encoded by the genome of NRG-1, NRG-2, or NRG-3, or any similar neuregulin peptide, for example, a peptide with EGF-like domain encoded by a gene or cDNA of neuregulin (for example, EGF-like domain containing subdomains peptide NRG-1 C-C/D or C-C/D', as described in USPN 5530109, USPN 5716930 and USPN 7037888; or EGF-like domain, as described in WO 97/09425). The content of each of USPN 5530109; USPN 5716930; USPN 7037888 and WO 97/09425 in full included in this description.

Risk factors: risk factors that increases chance of developing congestive heart failure in an individual well-known. They include, but are not limited to, Smoking, obesity, high blood pressure, coronary heart disease, vascular disease, coronary bypass surgery, myocardial infarction, systolic dysfunction of the left ventricle, exposure to cardiotoxic compounds (alcohol, drugs, such as cocaine and anthracycline antibiotics such as doxorubicin and daunorubicin), viral infection, pericarditis, myocarditis, gingivitis, thyroid disease, the impact is adjali, genetic defects for which it is known that they increase the risk of heart failure (such as defects described in Bachinski and Roberts, Cardiol. Clin. 16:603-610, 1998; Siu et al., Circulation 8:1022-1026, 1999; and Arbustini et al., Heart 80:548-558, 1998), starvation, eating disorders such as anorexia and bulimia, heart failure family history and myocardial hypertrophy.

In accordance with the present invention neuregulin you can enter intermittently way to achieve prevention, such as preventing or reducing the rate of progression of congestive heart disease in individuals who have been diagnosed with the disease risk. For example, the introduction neuregulin patient with early compensatory hypertrophy maintains the state of hypertrophy and prevents the progression to heart failure. In addition, individuals who have identified risk can be cardioprotective treatment neuregulin to the development of compensatory hypertrophy.

Introduction neuregulin patients with malignant tumor before or during chemotherapy anthracyclines or combined treatment anthracyclines/antibody to ErbB2 (antibody to HER2) (e.g., Herceptin®) can prevent the occurrence of cardiomyocytes patient in apoptosis, thus preserving cardiac function. The patient is, who already suffer from loss of cardiomyocytes, also benefit from treatment with neuregulin, as the remaining myocardial tissue responds to the impact of neuregulins, showing hypertrophic growth and increased airway.

Treatment: neuregulin and peptides containing EGF-like domains, encoded by the genes of neuregulins, you can enter patients or experimental animals with a pharmaceutically acceptable diluent, carrier or excipient. The composition of the invention can be provided in a standard dosage form.

To provide suitable formulations or compositions for the introduction of such compositions to patients or experimental animals using conventional pharmaceutical practice. Although it is preferable to intravenous administration, you can use any suitable method, for example, parenteral, subcutaneous, intramuscular, transcutaneous, transvenous, intraperitoneal, intranasal, aerosol, oral, or topical (e.g., by applying adhesive tape containing the composition, is able to traverse the dermis layer and enter into the bloodstream) introduction.

Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, the compositions can be in the form of tablets or capsules; and for intran the screen compounds - in the form of powders, nasal drops or aerosols.

Methods that are well known in this area for obtaining compositions can be found, for example, in "Remington''s Pharmaceutical Sciences". The compositions for parenteral administration, for example, may contain excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Other potentially suitable systems for parenteral delivery for the introduction of molecules according to the invention include particles of a copolymer of ethylene-vinyl acetate, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example lactose, or they can be aqueous solutions containing, for example, a simple polyoxyethylene-9-lauric ether, glycocholate and dezoksiholatom, or they can be oily solutions for administration in the form of nasal drops or in the form of a gel.

In an additional aspect, the invention relates to compounds of the present invention for use as pharmaceuticals, particularly for the treatment or prevention of the above conditions and diseases. The invention relates to the use of compounds of the present invention to obtain medicines for treatment is, or prevent one of the above conditions and diseases.

Regarding intravenous injection dose levels are in the range of from about 0.001 mg/kg, 0.01 mg/kg to at least 10 mg/kg at regular intervals at least approximately every 24, 36, 48 hours to about every 96 hours and, in particular, every 48, 72 or 96 hours or more, as indicated in the present description. In a specific embodiment, levels of intravenous injection of doses are in the range of from about 0.1 mg/kg to about 10 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours and, in particular, every 48, 72 or 96 hours or more, as indicated in the present description. In another specific embodiment, levels of intravenous injection of doses are in the range of from about 1 mg/kg to about 10 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours and, in particular, every 48, 72 or 96 hours or more, as indicated in the present description. In another specific embodiment, levels of intravenous injection of doses are in the range of from about 0.01 mg/kg to about 1 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours and, in particular, every 48, 72 ili hours or more, as indicated in the present description. In another specific embodiment, levels of intravenous injection of doses are in the range of from about 0.1 mg/kg to about 1 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours and, in particular, every 48, 72 or 96 hours or more, as indicated in the present description.

Relative to subcutaneous injection dose levels are in the range of from about 0.01 mg/kg to at least 10 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours and, in particular, every 48, 72 or 96 hours or more, as indicated in the present description. In a specific embodiment, levels of injected doses are in the range of from about 0.1 mg/kg to about 10 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours or more, as indicated in the present description, and in particular, every 48, 72 or 96 hours. In another specific embodiment, levels of injected doses are in the range of from about 1 mg/kg to about 10 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours or more, as indicated in the present description, and frequent the spine, every 48, 72 or 96 hours. In another specific embodiment, levels of injected doses are in the range of from about 0.01 mg/kg to about 1 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours or more, as indicated in the present description, and in particular, every 48, 72 or 96 hours. In another specific embodiment, levels of injected doses are in the range of from about 0.1 mg/kg to about 1 mg/kg at regular intervals of approximately every 48 hours until approximately every 96 hours or more, as indicated in the present description, and in particular, every 48, 72 or 96 hours.

Transdermal dose, usually choose to provide similar or lower levels in the blood, which is achieved by the use of injectable doses.

Compounds according to the invention can be entered as the sole active agent or they can be administered in combination with other agents, including other compounds that demonstrate the same or similar therapeutic activity and that are defined as safe and effective for such a combined injection. Other such compounds are used to treat CHF include natriuretic peptide and brain (BNP), drugs that block is formirovanie or specific action of neurohormones (e.g., angiotensin converting enzyme inhibitors (ACE inhibitors), antagonists of angiotensin receptor (ARB), aldosterone antagonists and blockers beta-adrenergic receptors), inotropic tools (e.g., dobutamine, digoxin) to improve cardiac contractility, vasodilator (e.g., nitrates, nesiritide) and/or diuretics (eg, furosemide) to reduce congestion, and one or more antihypertensive drugs such as beta blockers, ACE inhibitors and ARB, nitrates (isosorbide dinitrate treatment), hydralazine and calcium channel blockers.

As stated above, medical intervention, including treatment for drug requires selection of a suitable medicinal product and its delivery with the appropriate measuring mode. The appropriate dosage regimen includes sufficient dose, route, frequency, and duration of treatment. The primary goal of treatment with the medication is to achieve the optimal concentration of drug at the site of action so as to ensure the treatment of the patient overcome the disease, for which treatment is required. In General, the basic knowledge of the fundamentals of pharmacokinetics of drugs facilitate the selection of appropriate dosing schedules. However, to assist the physician in determining effective and be the dangerous dosing schedules of selected medicines for medical treatment to the individual patient in this context as an additional tool you can use therapeutic drug monitoring tools (TDM).

The final concentration and therapeutic window: determination of the optimal concentration of the medicine varies depending on the pharmacodynamic properties of a particular drug. For example, the optimal therapy for time-dependent antibiotics such as penicillin, depends on the achievement of the relations of peak concentration to MIC (minimum inhibitory concentration) 2-4 and time above MIC equal to 75% of the dosing interval. Depending on the concentration of antibiotics, such, for example, gentamycin, effectiveness depends on achieving the relationship of peak concentration to MIC approximately 8-10. Regardless of the nuances associated with the introduction of a particular drug, treatment, medicine aims to achieve the final concentrations in the plasma (which often reflect the concentration at the site of action) within the "therapeutic window", which was previously determined on the basis of pharmacokinetic, pharmacodynamic and toxicity profile of the medicinal product in species targets. The width of this window for various medicines and species varies. When the difference between the minimum effective concentration and the minimum toxic concentration of small (2 to 4 times), therapeutic window is designated as narrow.In contrast, when there is a big difference between effective and toxic concentrations, the drug is considered as having a wide therapeutic window. An example of a drug with a narrow therapeutic window is digoxin, which is the difference between the average and toxic concentrations is 2 or 3 times. On the other hand amoxicillin has a wide therapeutic range and overdosing the patient, not usually associated with toxicity problems.

The variability in response to medicines: the variability in healthy individuals of the same species in relation to the response to medication is common. In addition, a painful condition have the ability to affect the system and the function of organs (e.g. kidney, liver, water content), which in turn may influence response to the drug. This in turn contributes to an increased difference in the response to the drug in patients individuals who enter drug. Another significant problem associated with the introduction of more than one drug at a time, which leads to pharmacokinetic interactions, which can lead to changes in response to one or both of Lech is only means. Thus, physiological (e.g., age), pathological (e.g., the effect of the disease) and pharmacological (e.g., drug interactions) factors that can alter the pharmacokinetics of drugs in animals. Increased variability among individuals, as a consequence, may lead to treatment failure or toxicity from drugs with a narrow therapeutic index.

The group of patients who may benefit from treatment according to the present invention, sufficiently different, for example, good candidates are patients with impaired renal function, since prolonged levels of protein drugs is often associated with deposits in the glomeruli of the kidneys. Thus, the use of a mode of treatment that is not supported constant plasma levels, as described in the present invention, in patients with impaired renal function, in which any reduction of the existing functions can be harmful, can be very significant. Similarly, short and intermittent exposure to a therapeutic agent, such as GGF2, as described in the present description, may be useful in patients with types of tumors that respond to constant and prolonged stimulation by the growth factor. Other patients, to whom that can extract the specific benefits of intermittent treatment, as described in the present description, are patients with schwannomas and other peripheral neuropathy. An advantage of the present invention is that the intermittent dosing may have significant advantages with no continuous associated with side effects stimulation of various tissues.

A suitable schedule for obtaining blood samples to determine serum level of the drug, and the interpretation of the obtained level need to take into account the pharmacokinetic properties measured medicines. In the following paragraphs defined the terms used in the discussion of these properties.

Half life: the time required to reduce serum concentrations present in the beginning of the interval by 50%. Knowledge of the approximate time the half-life is important for the practitioner, as it determines the optimal dosage oral means, fluctuations in serum concentration between doses, and the time required to achieve steady state.

In summary, for GGF2 conducted several pharmacokinetic studies. Typical elimination half-life GGF2 ranged from 4 to 8 hours for intravenous (IV) route, whereas the half-life of subcutaneously (s/C) input GGF2 was the t 11 to 15 hours. CmaxAUC, Tmaxand T1/2presented in tables 1 and 2 below. When the half was too long for a precise definition of these methods, instead of time put the dash.

Table 1
Average pharmacokinetic parameters provide 125I-rhGGF2 radioactivity in the plasma of rats Mab Sprague-Dawley after a single intravenous or subcutaneous dose of 125I-rhGGF2
Group 1 (n=2)Group 2 (n=1)
ParametersGeneralUSAID. TCAGeneralUSAID. TCA
Cmax(ág EQ/g)0,32890,29530,01570,01
AUC0-t(µg EQ-h/d)1,270,010,270,17
AUC back(µg EQ-h/d)1,370,960,390,26
Tmax(h)0,080,086,06,0
The half-life6,376,1113,2014,66
Group 1 - in/Group 2 - p/

Table 2
Average pharmacokinetic parameters provide 125I-rhGGF2 radioactivity in the plasma of rats Mab Sprague-Dawley after a single intravenous or subcutaneous dose of 125I-rhGGF2
Group 1 (n=2)Group 2 (n=1)
ParametersGeneralUSAID. TCA GeneralUSAID. TCA
Cmax(ág EQ/g)0,26110,22910,01970,0034
AUC0-t(µg EQ-h/d)1,4890,5670,3350,064
AUCback(µg EQ-h/d)1,6670,62--
Tmax(h)0,890,0812,012,0
The half-life7,75of 7.96--
Group 1 - in/Group 2 - p/

Plasma concentrations after administration presented on figures 6 and 7 for/and the/introduction, respectively. As shown in figures 6 and 7, Cmaxmean maximum plasma concentration (maximum concentration, measured in the plasma at any time after the introduction); AUCbackmean area under the curve of the dependence of concentration on time to infinity (where this method is used to determine that the analysis has limits of detection); AUC0-tmean area under the curve of plasma concentration curve from time from time zero to the last measurable concentration); AUC by any means evaluating the overall impact on the animal; and Tmaxmeans the average time of maximum plasma concentration.

As can be seen from the tables and figures to support inpatient therapeutic levels in any of the ways dosing with dosing every day, through the night or every fourth day is impossible. Through the day and even long before the levels become immeasurable, as reflected by the data presented in table 11.

Table 11
Pharmacokinetic parameters for GGF2 after intravenous*
Rats
Dose (mg/kg) AUC0-∞(hour·ng/ml)AUC0-∞/Dose ((hour·ng/ml)/mg/kg)AUC0-th(hour·ng/ml)AUC0-th/Dose ((hour·ng/ml)/mg/kg)CL (ml/min/kg)t1/2(h)Vss (ml/kg)
816100±205002010±256016800±223002100±279018,1±12,71,46± 1,841050±331
1639600±94402470±59038300±100002390±6257,00±1,331,69± 0,430532±145
Monkeys
815900±16901980±21215100±17301890±2178,48±0,9102,02±0,5831110±113
*taken from data obtained on the basis plazmaticeski the concentrations of GGF2, measured by ELISA. Data are presented as mean ± SD.

Stationary state: stationary serum concentrations represent those values that are repeated at each dose and represent the state of equilibrium between the amount of administered drug and the number of observations in a given time interval. During long-term dosing of any drug, the two main determining the average inpatient serum concentrations are the rate at which injected the drug and the total clearance of medicines in this patient.

Peak serum concentration: the Point of maximum concentration on the curve of plasma concentration against time. The exact time of peak serum concentrations are difficult to predict, as it reflects the complex dependencies between the speed of introduction and removal.

The minimum serum concentration: minimum serum concentration detected during the dosing interval. The minimum concentration theoretically present in the period immediately before the next dose is due.

Absorption: the process by which a drug enters the body. Intravascular injected drugs in anywayse fully, but extravascular introduction leads to various degrees and the rate of absorption. The relationship between the absorption rate and the removal rate is the main determinant of the concentration of the drug in the bloodstream.

Distribution: the distribution of systemically available drugs from the intravascular space into the extravascular fluids and tissues and, thus, to the location sites of the receptor target.

Therapeutic range: the range of serum concentrations of drugs associated with a high degree of effectiveness and low risk of dose-dependent toxicity. therapeutic range is a statistical concept: there is a range of concentrations associated with therapeutic response in most patients. As a consequence, some patients demonstrate a therapeutic response with serum levels below the lower limit of the range, while others are for therapeutic effect required serum levels above the upper limit.

The correct schedule sample is important because therapy drug often modify based on the definitions of serum concentrations. Phase absorption and distribution must pass completely and before receiving the sample should castigat the Xia stationary concentration. The levels obtained before the establishment of stationary concentrations may be falsely low; increase dosage on the basis of such a result can result in toxic concentrations. In addition, for comparative measurements, it is important that the time of receipt of samples has been agreed.

The schedule for obtaining blood samples in relation to the dosage is critical for correct interpretation of results of determination of serum concentrations. The timing of when you get the sample in relation to the administration of a medicinal product should be based on the pharmacokinetic properties of the drug, its dosage form and clinical studies for the evaluation of the sample (for example, the performance evaluation or identification of possible toxicity induced drug). For regular monitoring of serum levels of drugs with short elimination half-life for the characteristic profile of serum concentrations is possible to collect samples on a stationary peaks and dips; for drugs with a long half-life, as a rule, sufficient samples for stationary recession.

Under "congestive heart failure" refers to impaired cardiac function, which makes the heart unable to maintain nor the actual ejection of blood at rest or under load or to maintain normal cardiac output in normal pressure filling of the heart. Indicator of congestive heart failure is the ejection fraction of the left ventricle is approximately 40% or less (for comparison, the ejection fraction of approximately 60% is normal). Patients with congestive heart failure demonstrate the well-known clinical symptoms and signs such as tachypnea, pleural effusion, fatigue at rest or under load, contractile dysfunction and edema. Congestive heart failure easy to diagnose well-known methods (see, for example, "Consensus recommendations for the management of chronic heart failure". Am. J. Cardiol., 83(2A):1A-38-A, 1999).

The relative severity and disease progression appreciate the well-known methods, such as physical examination, echocardiography, radionuclide imaging, invasive hemodynamic monitoring, magnetic resonance angiography and testing with the load on the treadmill, combined with studies capture oxygen.

Under "ischemic heart disease" refers to any infringement resulting from the imbalance between the demand of oxygen and sufficient oxygen supply. Most cases of ischemic heart disease are the result of narrowing of the coronary arteries, as occurs in atherosclerosis or other vascular disorders.

The "heart attack is iocard" means the process by which coronary heart disease leads to the formation of the field with the replacement of myocardium with scar tissue.

Under "cardiotoxic" means a compound that reduces cardiac function through direct or indirect damage or destruction of cardiomyocytes.

Under "hypertension" mean arterial pressure, which is considered by medical professionals (e.g. doctor or nurse) as higher than normal and ensuring an increased risk of developing congestive heart failure.

Under "treatment" implies that the introduction of neuregulin or similar neuregulin peptide statistically significantly slows down or prevents the progression of congestive heart failure in the treatment relative to the progression of the disease, which can occur in the absence of treatment. For assessment of disease progression can make use of the well known indicators, such as the ejection fraction of the left ventricle, physical capacity and other clinical tests, as listed above, as well as survival rates and ratios of hospitalization. Slows down or prevents the treatment of the disease progression in a statistically meaningful way or not, you can define well-known in this is blasti ways (see, for example, SOLVD Investigators, N. Engl. J. Med. 327:685-691, 1992 and Cohn et al., N. Engl. J Med. 339:1810-1816, 1998).

Under the "prevention" means minimizing or partial or complete obstruction of the development of congestive heart failure in a mammal at risk of developing congestive heart failure (as defined in "Consensus recommendations for the management of chronic heart failure." Am. J. Cardiol., 83(2A):1A-38-A, 1999). Determining, minimizes or prevents the introduction neuregulin or similar neuregulin peptide congestive heart failure, carried out by known methods such as described in the SOLVD Investigators, above and Cohn et al., above.

The term "therapeutically effective amount" is intended to refer to that amount of a drug or pharmaceutical agent that causes a biological or medical response in a tissue, system, animal or human that is desired by the researcher, veterinarian, medical doctor or other Clinician. Therapeutic change is a change in the measured biochemical characteristics in the direction in which it is expected relief from the disease or condition. More specifically, a "therapeutically effective amount" is an amount sufficient to reduce the symptoms associated with the disease or indisposition, to normalize the function of the second body when the condition or violation, which lead to the violation of specific functions of the body, or to improve one or more of the clinically measured parameters of the disease.

The term "prophylactically effective amount" is intended to denote the number of pharmaceutical drug that prevents or reduces the risk of biological or medical event that the researcher, veterinarian, medical doctor or other Clinician wants to prevent the tissue, system, animal or human.

The term "therapeutic window" is intended to indicate the range of doses between the minimum number to achieve therapeutic change and the maximum, which leads to the answer that is the answer, directly in front of toxicity for the patient.

The phrase "risk of congestive heart failure" mean the individual who smokes, is obese (i.e., more than 20% or more relative to its ideal weight), is being or will be exposed to cardiotoxic compounds (such as anthracycline antibiotic) or has (or had) high blood pressure, coronary heart disease, myocardial infarction, genetic defect, for which it is known that it increases the risk of heart not is dostatochnosti, heart failure in family history, myocardial hypertrophy, hypertrophic cardiomyopathy, systolic dysfunction of the left ventricle, coronary bypass surgery, vascular disease, atherosclerosis, alcoholism, pericarditis, a viral infection, gingivitis or malnutrition (e.g., nervous anorexia or bulimia), or an alcoholic or cocaine addict.

The phrase "reducing the progression of thinning of the myocardium" imply the maintenance of hypertrophy of cardiomyocytes ventricle so that maintained or increased wall thickness of the ventricle.

The phrase "inhibits myocardial apoptosis" means that the processing neuregulin inhibits death of cardiomyocytes compared with untreated cardiomyocytes at least 10%, more preferably at least 15%, even more preferably at least 25%, even more preferably at least 50%, even more preferably at least 75%, and most preferably at least 90%.

Under the "neuregulin" or "NRG" means a peptide which is encoded by the genes or nucleic acid (e.g., cDNA) NRG-1, NRG-2, or NRG-3 and binds and activates receptors ErbB2, ErbB3 or ErbB4 receptors or combinations thereof.

Under the "neuregulin-1", "NRG-1", "heregulin", "GGF2" or "Li gang is ω p185erbB2" refers to a peptide which binds to the ErbB2 receptor, when he paired with another receptor (ErbB1, ErbB3 or ErbB4), and is encoded by the genome of p185erbB2 ligand as described in U.S. patent No. 5530109; U.S. patent No. 5716930 and U.S. patent No. 7037888, which are fully included in the present description by reference.

Under this neuregulin peptide" means a peptide that contains EGF-like domain encoded by the genome of neuregulin, and binds and activates ErbB2, ErbB3, ErbB4, or a combination of both.

Under the "domain similar to epidermal growth factor or EGF-like domain" refers to a peptide motif encoded by the genes NRG-1, NRG-2, or NRG-3, which binds and activates ErbB2, ErbB3, ErbB4, or combinations thereof, and has structural similarities with the receptor binding domain of EGF, as described in Holmes et al., Science 256:1205-1210, 1992; U.S. patent No. 5530109; U.S. patent No. 5716930; U.S. patent No. 7037888; Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998; Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; Higashiyama et al., J Biochem. 122:675-680, 1997; and WO 97/09425). Relative to the nucleic and amino acid sequences corresponding to the domains EGFL 1-6 encoded by the genome of NRG-1, see figure 9-14.

By "antibody to ErbB2 or antibody to HER2" refers to an antibody that specifically binds to the extracellular domain of the receptor ErbB2 (also known as HER2) and prevents dependent on ErbB2 (HER2) signal transmission initiated by St. what statements neuregulin.

By "transformed cell" means a cell (or a descendent of a cell), in which the methods of recombinant DNA or by known methods of gene therapy are introducing a DNA molecule encoding a neuregulin or peptide containing EGF-like domain neuregulin.

By "promoter" is meant a minimal sequence sufficient to control transcription. Also according to the invention is enabled promoter elements that are sufficient for implementation-dependent promoter gene expression, regulated on the basis of cell type or physiological status (e.g., hypoxic conditions compared with the conditions of normal oxygen content), or inducible by external signals or means; such elements may be located 5' or 3' or internal areas of the natural gene.

By "functionally linked" means that the nucleic acid encoding the peptide (e.g., cDNA) and one or more regulatory sequences are connected in such a way that ensures the expression of a gene when the regulatory sequence linked to the appropriate molecules (e.g., proteins activators of transcription).

Under "expressing vector" refers to a genetically engineered plasmid or virus, derived from, for example, a bacteriophage, adenovirus, retrovirus, dormancy is a virus herpes virus, or artificial chromosome, that is used for transfection coding sequence of the peptide (for example, neuregulin), functionally associated with the promoter in the cell host such that the encoded peptide or peptide is expressed in a host cell.

If not defined otherwise, all technical and scientific terms used in this description, have the same meaning as commonly understood by the expert in the field, belongs to the present invention.

Discussion of documents, acts, materials, devices, products, etc. included in the present description only for the purpose of providing context for the present invention. Do not assume or imagine that any or all of these materials were part of the prior art or were well-known in the field related to the present invention before the priority date of each of the claims of this application.

Other embodiments of the

Although the invention is described with reference to specific variants of its implementation, it should be understood that additional modifications and this application is intended to examine any variations, uses or adaptations of the invention, generally following the principles of the invention and including such departures from the present description is, located within known or customary practice in this area belongs to the invention, and can be applied to the essential features mentioned above in the present description, and they are included in the scope of the attached claims.

The following examples should help experts better understand the invention and its principles and advantages. It should be understood that these examples illustrate the invention and not limit its scope.

EXAMPLES

As described above in the present description, neuregulins are a family of growth factors structurally related to epidermal growth factor (EGF) and they are necessary for the normal development of the heart. Data show that neuregulin represent a potential drug for the treatment of heart disease, including heart failure, myocardial infarction, chemotherapeutic toxicity and viral myocarditis.

The research described in the present description, serve to determine dosing in a model of congestive heart failure in rats with legirovaniem the left anterior descending (LAD) artery. Cloned and produced several variants of splicing neuregulin. Slice neuregulin consisting of EGF-like domain (EGF-ld) previous publication (Liu et al., 2006), compared with moratorium neuregulins, known as a growth factor, glia 2 (GGF2), and EGF-like domain, Ig domain (EGF-Ig). Male and female rats, Sprague-Dawley podvergali legirovanie LAD artery. In 7 days after ligation in rats intravenous (IV) were daily treated with neuregulin. Cardiac function was monitored by echocardiography.

In the first study compared 10 days of dosing with equimolar amounts of EGF-ld or GGF2 (GGF2 they are computed as 0,0625 and 0,325 mg/kg). At the end of the dosing period GGF2 treatment resulted in significantly greater (p<0,05) improvement of ejection fraction (EF) and fraction shortening (FS) than the EGF-ld. In a second study compared 20 days GGF2 with EGF-ld and EGF-Ig in equimolar concentrations. The GGF2 treatment resulted in significantly improved EF, FS and LVESD (p<0,01). EGF-ld or EGF-Ig improvements physiology of the heart during this period was not provided. In the third study compared daily (every 24 hours) dosing, dosing in a day (every 48 hours) and every dosing every fourth day (96 hour) for 20 days using GGF2 (3,25 mg/kg). All three modes of GGF2 treatment led to significant improvements physiology of the heart, including EF, ESV and EDV, and the effects were maintained for 10 days after dosing. Presented in this study confirm that GGF2 is the main neuregulin connection and set on the optimum dosing regimens for its introduction.

As shown in the present description, in the studies of the present invention establish the relative effectiveness of GGF2 in comparison with the published fragments neuregulin (Liu et al., 2006), are conducting a study to determine the optimal dose and frequency of dose, and determine whether excipient BSA, as previously reported.

Methods and materials

Cloning, expression and purification of DNA domain IgEGF (Ig154Y) GGF2 (EGF-Ig): domain IgEGF amplified from an existing cDNA GGF2 and cloned into the vector pet-15b (Novagen, catalog No. 69661-3) using restriction sitesNdeI andBamHI. the Obtained protein is 21,89 kDa + ≈kDa His-tag (= ≈ 25 kDa).

The DNA sequence of clone IgEgf pet 15: the underlined sequences represent the primers used for amplification. Bold sequences represent the cloning sites for insertion of a sequence into the vector pet (NdeI andBamHI).

Below is the final translated from vector pet-15b protein. Part of the vector is underlined.

Expression of protein:A clone of transformed cells B121 for protein expression using the Overnight Express Autoinduction System (Novagen) in LB medium at 25°C for 24 cha is s.

Refolding of the protein:Adapted set Novagen Protein Refolding Kit, 70123-3.

The protein purification:His column TRAP - the manufacturer's instructions

Western blotting:the expression of the protein was assessed by Western blotting. The resulting band with His-tagged migrates to approximately 25 kDa.

To resolve protein used 4-20% criterion gel (Biorad) followed by transfer to nitrocellulose paper Protran (pore size of 0.1 μm from Schliecher and Schull). Blot blokirovki in 5% milk in TBS-T (0.1 per cent). Used primary antibody (affinity purified polyclonal Ab to EGF, NRG 1-alpha/HRG1-alpha man with catalogue number AF-296-NA from R&D systems) with a dilution of 1:1000 in 5% milk in TBS-T for 1 hour at RT (also works at 4°C during the night). Used secondary antibodies rabbit antibody goat HRP at a dilution of 1:10,000 in 5% milk in TBS-T for 1 hour at RT. All washing was performed in TBS-T.

The purification Protocol Ig154Y: Culture grown at 25°C in the system Overnight Express Autoinduction System 1 from Novagen (catalog No. 71300-4). The culture is centrifuged and the extraction is carried out from the precipitate, dissolved and subjected to refolding to get Ig154Y before cleaning.

Materials for extraction, dissolution and refolding:

10X wash buffer: 200 mm Tris-HCl, pH 7.5, 100 mm EDTA, 10% Triton X-100

10X buffer for dilution: 500 mm CAPS, pH of 11.0

50X buffer for dialysis: 1M Tris-HCl, pH 8.5

30% N-laurylsarcosine - obavljati in the form of a powder (Sigma 61739-5G)

1M DTT

Restored glutathione (Novagen 3541)

Oxidized glutathione (Novagen 3542)

A. lysis of the cells and obtaining Taurus enable

Cell sediments were thawed and resuspendable in 30 ml of 1X wash buffer.

To the suspension was added protease inhibitors (25 ál 10X 50 ml), Tnkase (200 μl of 1 mg/ml to 50 ml) and MgCl2(500 ál 1M, 50 ml).

Cells were literally by ultrasonic treatment with cooling on ice.

After ultrasonic treatment bullock inclusion collected by centrifugation at 10000g for 12 minutes.

The supernatant was removed and the residue carefully resuspendable in 30 ml of 1X wash buffer.

Repeated step 4.

- The precipitate thoroughly resuspendable in 30 ml of 1X wash buffer.

Taurus inclusion collected by centrifugation at 10000g for 10 minutes.

B. Dissolution and refolding

- From the total mass of Taurus enable to handle expected number of 1X buffer for dissolving required to resuspendable Taurus enable c concentration of 10-15 mg/ml If the calculated amount exceeds 250 ml, use 250 ml

At room temperature produces the calculated volume of 1X buffer for dissolving, supplemented with 0.3% N-laurylsarcosine (if needed for additional optimization can be used up to 2%) (300 mg/100 ml buffer) and 1 mm DTT.

To ablaut the calculated amount of 1X buffer for dilution of step 2 for the calves on and gently mix. Large particles can be destroyed by repeated pipetting.

- Incubated in a cooled shaker at 25°C, 50-100 rpm for 4-5 hours (or more if you want for additional optimization).

Is separated from impurities by centrifugation at 10000g for 10 minutes at room temperature

- Transfer the supernatant containing the dissolved protein in a clean test tube.

C. Protocol for dialysis refolding of protein

- Receive the required volume of buffer used for dialysis of the dissolved protein. Dialysis should be at least 2 changes of buffer volume 50 times larger volume of the sample. Diluted 50X buffer for dialysis to 1X until the desired volume and complement 0.1 mm DTT.

- Dialysis for at least 4 hours at 4°C. Replace the buffer and continue. Dialysis for an additional 4 hours or more.

- Get an additional buffer for dialysis, as defined in step 1, but do not add DTT.

- Continue dialysis with two additional replacements (at least 4 hours each) with buffer for dialysis without DTT.

D. redox buffer for refolding for stimulation of the formation of disulfide bonds

- Receive buffer for dialysis containing 1 mm restored glutathione (1.2 g/4 l) and 0.2 mm oxidized glutathione (0,48 g/4 l) in 1X buffer for dialysis. The volume should be 25 times more the eat volume of sample dissolved protein. Cooled to 4°C.

- Dialysis subjected to refolding of the protein from step 1 over night at 4°C.

Materials for cleaning

All procedures were performed at 4°C.

Chemical substances:

Trizma hydrochloride (Sigma T5941-500G)

5M solution of sodium chloride (Sigma S6546-4L)

10 N sodium hydroxide (JT Baker 5674-02)

The imidazole (JT Baker N811-06)

A. Purification on a column of HISPrep FF 16/10 - 20 ml (GE Healthcare)

Buffer A: 20 mm Tris-HCl + 500 mm NaCl pH 7.5

Buffer B: Buffer A + 500 mm imidazole pH 7.5

Trim column: buffer A - 5 column volumes buffer B - 5 column volumes buffer A - 10 column volumes

Make a 20 ml sample passage 20 ml column at 0.5 ml/min

Washed the column with 5 column volumes of buffer A

Column elute with 5 column volumes 280 mm imidazole.

Cleanse 10 column volumes of 100% buffer B.

Balance 15 column volumes of buffer A

Analyze fractions with a silver dye SDS-page

Collect fractions with Ig154Y

B. Remove the His-tag

Remove the His-tag is conducted through a set of Thrombin Cleavage Capture Kit from Novagen (catalog No. 69022-3). Based on previous testing, the best conditions are room temperature for 4 hours with thrombin with a 0.005 Units of the enzyme on μl of 10 μg protein Ig154Y. After four hours incubation, add 16 µl suspension of agarose with streptavidin per unit of the enzyme thrombin. Displacement is more of the sample for 30 minutes at room temperature. Restore Ig154Y by filtration, by centrifugation or sterile filtration (depending on volume).

Full splitting determined by Western blotting with EGF antibodies to His.

C. Concentration Ig154Y

Brought to the desired concentration using 15 ml of concentra Millipore Centriprep 3000 MWCO (Ultracel YM-3, 4320)

D. Storing in the destination buffer

Stored in 20 mm Tris +500 mm NaCl pH 7.5 and 1X PBS + 0.2% BSA.

Cloning, expression and purification of DNA 156Q (EGF-Id [domain EGF NRG1b2 (156Q)]: domain egf NRG1b2 cloned from cDNA of the human brain and cloned into the vector pet-15b (Novagen catalogue No. 69661-3) using restriction sitesNdeI andBamHI. the Obtained protein is 6,92 kDa + ≈3 kDa His-tag (= 9,35 kDa)

The DNA sequence of clone NRG1b2 egf pet15

The underlined sequences represent the areas of cloning (NdeI andBamHI)

Below is the final translated from vector pet-15b protein. Domain egf highlighted in green.

Calculated pI/Mw: 7,69/9349,58.

Expression of protein

A clone of transformed cells B121 for protein expression using the Overnight Express Autoinduction System (Novagen) in LB medium at 25°C for 24 hours. The expression occurs mainly in the insoluble Taurus included the Oia.

Refolding of the protein:Adapted set Novagen Protein Refolding Kit, 70123-3.

The protein purification:The protein was placed on uninominal DEAE column at 2.5 ml/min Fragment of EGF-ld remains in the eluate, whereas contaminants contact and suiryudan at high salt concentration. Boot and wash the snack bar represents 50 mm Tris pH of 7.9, and a buffer for elution represents 50 mm Tris pH to 7.9 with 1M NaCl. The eluate is collected and concentrated using Centriprep YM-3 from Millipore.

Western blotting:the expression of the protein was assessed by Western blotting. The resulting band migrates to approximately 10 kDa.

To resolve protein used 4-20% criterion gel (Biorad) followed by transfer to nitrocellulose paper Protran (pore size of 0.1 μm from Schliecher and Schull). Blot blokirovki in 5% milk in TBS-T (0.1 per cent). Used primary antibody (affinity purified polyclonal Ab to EGF, NRG 1-alpha/HRG1-alpha man with catalogue number AF-296-NA from R&D systems) with a dilution of 1:1000 in 5% milk in TBS-T for 1 hour at RT (also works at 4°C during the night). Used secondary antibodies rabbit antibody goat HRP at a dilution of 1:10,000 in 5% milk in TBS-T for 1 hour at RT. All washing was performed in TBS-T.

The purification Protocol for NRG-1560

Culture grown at 25°C in the system Overnight Express Autoinduction System 1 from Novagen (catalog No. 71300-4). There is a very small amount of dissolve the CSOs NRG-156Q (EGF-ld). The culture is centrifuged and the extraction is carried out from the precipitate, dissolved and subjected to refolding to get NRG-156Q before cleaning.

Materials for extraction, dissolution and refolding:

10X wash buffer: 200 mm Tris-HCl, pH 7.5, 100 mm EDTA, 10% Triton X-100

10X buffer for dilution: 500 mm CAPS, pH of 11.0

50X buffer for dialysis: 1M Tris-HCl, pH 8.5

30% N-laurylsarcosine - add in the form of a powder (Sigma 61739-5G)

1M DTT

Restored glutathione (Novagen 3541)

Oxidized glutathione (Novagen 3542)

A. lysis of the cells and obtaining Taurus enable

- Thawed and resuspended cellular precipitate in 30 ml of 1X wash buffer. Mix as needed to complete resuspendable.

To the suspension is added protease inhibitors (25 ál 10X 50 ml), Tnkase (200 μl of 1 mg/ml to 50 ml) and MgCl2(500 ál of 1 M, 50 ml).

Cells are lysed by sonication.

a. Throughout all stages of the cell is cooled on ice.

b. Using rectangular tip spend sonication for 30 seconds at level 6, 10 times until the suspension on the mill less viscous. Between each sonication, the suspension is allowed to cool on ice for 60 seconds. When the sonication support not more than 40 ml in a 50 ml conical tube.

- Upon completion of the transfer of each suspension 250 ml centrifuge to the foreheads with sloping necks for use in applying centrifuge F-16/250.

- Collect bullock inclusion by centrifugation at 10000g for 12 minutes.

- Remove supernatant (retain the sample for the analysis of soluble protein) and carefully resuspended residue in 30 ml of 1X wash buffer.

- Repeat the centrifugation as in step 4 and retain the sediment.

Again carefully resuspended residue in 30 ml of 1X wash buffer.

- Collect bullock inclusion by centrifugation at 10000g for 10 minutes. The supernatant is drained and removed the last traces of liquid, obstuctive inverted tube on a paper napkin.

B. Dissolution and refolding

- From the total mass of Taurus enable to handle expected number of 1X buffer for dissolving required to resuspendable Taurus enable c concentration of 10-15 mg/ml If the calculated amount exceeds 250 ml, use 250 ml

At room temperature produces the calculated volume of 1X buffer for dissolving, supplemented with 0.3% N-laurylsarcosine (if needed for additional optimization can be used up to 2%) (300 mg/100 ml buffer) and 1 mm DTT.

- Add the calculated amount of 1X buffer for dilution of step 2 for the calves on and gently mix. Large particles can be destroyed by repeated pipetting.

- Incubated in a cooled shaker at 25°C, 50-100 rpm than the s 4-5 hours.

Is separated from impurities by centrifugation at 10000g for 10 minutes at room temperature

C. Protocol for dialysis refolding of protein

- Receive the required volume of buffer used for dialysis of the dissolved protein. Dialysis should be at least 2 changes of buffer volume 50 times larger volume of the sample. Diluted 50X buffer for dialysis to 1X desired volume and complement 0.1 mm DTT.

- Dialysis for at least 4 hours at 4°C. Replace the buffer and continue. Dialysis for an additional 4 hours or more.

- Get an additional buffer for dialysis, as defined in step 1, but do not add DTT.

- Continue dialysis with two additional replacements (at least 4 hours each) with buffer for dialysis without DTT.

D. redox buffer for refolding for stimulation of the formation of disulfide bonds

- Receive buffer for dialysis containing 1 mm restored glutathione (1.2 g/4 l) and 0.2 mm oxidized glutathione (0,48 g/4 l) in 1X buffer for dialysis. The volume should be 25 times larger than the amount of sample dissolved protein. Cooled to 4°C.

- Dialysis subjected to refolding of the protein from step 1 over night at 4°C.

Materials for cleaning

All procedures were performed at 4°C.

Chemical substances:

Trizma hydrochloride (Sigma T5941-500G)

5M R is the target sodium chloride (Sigma S6546-4L)

10 N sodium hydroxide (JT Baker 5674-02)

E. Purification on anion DEAE column HiPrep 16/10 - 20 ml (GE Healthcare)

Buffer A: 50 mm Tris-HCl pH 8.0

Buffer B: 50 mm Tris-HCl with 1M NaCl pH 8.0

Trim column: buffer A - 5 column volumes buffer B - 5 column volumes buffer A - 10 column volumes

Put 50 ml of the sample passage 20 ml column at 2.0 ml/min (NRG-156 (EGF-ld) is located in the eluate).

- Washed with 20 ml column 5 column volumes of buffer A

20 ml column with a gradient to 100% B with 5 column volumes. This is carried out for the elution of contaminants.

- Clear 10 column volumes of 100% buffer B.

- Balance 15 column volumes of buffer A

- Analyze fractions with a silver dye SDS-page

- Collect fractions with NRG-156Q (10 kDa)

F. Concentration of NRG-156 (EGF-ld)

- Concentrated using 15 ml Centriprep concentrator 3000 MWCO (Ultracel YM-3, 4320)

- To determine the concentration using the modified analysis of protein by Lowry.

G. Removal of His-tag

Remove the His-tag is conducted through a set of Thrombin Cleavage Capture Kit from Novagen (catalog No. 69022-3). Based on previous testing, the best conditions are room temperature for 4 hours with thrombin with a 0.005 Units of the enzyme on μl of 10 μg protein NRG-156Q (EGF-ld). After four hours incubation, add 16 µl suspension of agarose with streptavidin per unit of the enzyme thrombin. Re eshivot the sample for 30 minutes at room temperature. Restore NRG-156Q by filtration, by centrifugation or sterile filtration (depending on volume).

Full splitting determined by Western blotting with EGF antibodies to His.

H. Storing in the destination buffer

Kept in 1X PBS with 0.2% BSA at 4°C.

Expression and purification GGF2

For cloning and basic information on GGF2, see USPN 5530109. Cell line described in USPN 6051401. The full contents of each of USPN 5530109 and USPN 6051401 in full included in the present description by reference.

Cell line CHO-(Alpha2HSG)-GGFThis cell line was designed to obtain sufficient quantities of fetuin (alpha2HSG person with maintaining high production speeds rhGGF2 in serum-free conditions.

Cells Cho (dhfr-) transfusional expressing vector, is presented below (pSV-AHSG). Stable cells were grown in the conditions of selection on ampicillin. The cell line identified (dhfr-/α2HSGP). Then cells dhfr-/α2HSGP was transfusional vector pCMGGF2 below, containing the coding sequence of human GGF2 using the cationic lipid reagent DMRIE-C (Life Technologies #10459-014).

Stable and vysokoplodorodnye cell lines were obtained according to standard protocols with methotrexate (100 nm, 200 nm, 400 nm, 1 μm) after 4-6 is edelenyi intervals. Cells gradually wean from containing serum environment. Clones were isolated by standard methods of limiting dilutions. Detailed descriptions of the requirements of environments are found in the above publications.

To enhance the transcription of the coding sequence of GGF2 was placed after insertion sequence (MIS) EBV BMLF-1. Cm. the chart below.

The sequence MIS (SEQ ID NO: 20)

The coding sequence of GGF2 (SEQ ID NO: 1) -

The sequence of the protein GGF2 (SEQ ID NO: 2) -

Getting GGF2: One tube with GGF2 from 2.2×106cells/ml were thawed in 100 ml of medium Acorda Medium 1 (see table 3) and were propagated to obtain sufficient quantities for sowing containers to obtain. The cells were inoculable on Wednesday to get Acorda Medium 2 (see table 4) c 1,0×105cells/ml in two-liter roller bottles with ventilation holes. Roller bottles were maintained at 37°C for 5 days, and then the temperature was lowered to 27°C for 26 days. In roller bottles was controlled by the number of cells and overall appearance, but they are not filled. After reaching viability below 10% of the cells were centrifuged and the conditioned medium was collected and spent sterile filtration is the situation.

Table 3
Wednesday 1
ManufacturerCatalog numberThe final concentration
CD-CHOInvitrogen10743-029- remove 50 ml, then add the following components
FeSO4•EDTASigmaF-05181× (10 ml/l)
L-glutamineCellgro25-005-CI4 mm, 20 ml/l)
Recombinant human insulinSigma1-9278290 U/l (1 ml/l)
Non-essential amino acidsCellgro25-025-CI1× (10 ml/l)
Peptone type 4 Soybean-HySoySigmaP0521Powder - received 20X CD-CHO (50 ml/l)
GentamicinInvitrogen15750-078100 mcg (2 ml/l)

Table 4
Wednesday 2
ManufacturerCatalog numberThe final concentration
CD-CHOInvitrogen10743-02950% (first -50 ml)
HyQ SFX-CHOHyCloneSH 30187,0250% (first -50 ml)
FeSO4•EDTASigmaF-05181× (10 ml/l)
L-glutamineCellgro25-005-CI4 mm, 20 ml/l)
Recombinant human insulinSigma1-9278290 U/l (1 ml/l)
Non-essential amino acidsCellgro 25-025-CI1× (10 ml/l)
Peptone type 4 Soybean-HySoySigmaP0521Powder - received 20X CD-CHO (50 ml/l)
GentamicinInvitrogen15750-078100 mcg (2 ml/l)

The purification Protocol GGF2

All procedures were performed at 4°C.

Chemical substances:

Sodium acetate

Glacial acetic acid to bring pH)

10 N NaOH (to bring pH)

NaCl

The sodium sulfate

L-arginine (JT Baker, catalogue number: 2066-06)

Mannitol (JT Baker, catalogue number: 2553-01)

Starting material: supernatant air-conditioned environments. To bring the pH to 6.5.

Stage 1:

Capture - cation-exchange chromatography

HiPrep 16/10 SP (Amersham Biosciences)

Trim column: buffer A - 5 column volumes buffer B - 5 column volumes buffer 15% B - 5 column volumes

Buffer A: 20 mm Na Acetate, pH 6,0

Buffer B: 20 mm Na Acetate, pH 6,0, 1M NaCl

Make the sample at 2 ml/min with continuous introduction during the night if possible. Linking is better with continuous inclusion.

The maximum capacity for the starting sample: 5 mg GGF2/ml environment

Flow rate: 3 ml/min

The first washing: 15% B, 10 column volumes

Second wash: 35% B, 10 the volume of the MOU speakers

Elution of GGF2: 60% B, 8 column volumes

The shaded columns: 100% B, 8 column volumes

Buffers:CompositionConductivityApplication
15% B20 mm Na acetate, pH of 6.0, 150 mm NaClPre-trim
First wash
35% B20 mm Na acetate, pH 6,0, 350 mm NaClThe second hillshade
60% B20 mm Na acetate, pH 6,0, 600 mm NaClElution of GGF2
100% B20 mm Na acetate, pH 6,0, 1000 mm NaCl88 MS/cmThe shaded column

Step 2:

Cleansing - gel-filtration chromatography

Sephacryl S200 26/60

Buffer for elution: 20 mm Na acetate, 100 mm sodium sulfate, 1% mannitol, 10 mm L-arginine, pH 6,5

The conductivity of the buffer:

Sample: suirvey pool SP GGF2, the concentration of the new to ≈AU280 1,0

Flow rate: 1.3 ml/min

Peak elution: if ≈0.36 volume of the column from the beginning of introduction sample

Step 3:Destruction of DNA and endotoxin - filtration through a membrane Intercept Q.

Buffer for pre-equilibration: 20 mm Na acetate, 100 mm sodium sulfate, 1% mannitol, 10 mm L-Arginine, pH 6,5

Collect the eluate

Step 4:The final composition and get sample

To the sample add a further 90 mm L-arginine

Concentrate

Sterile-filtered

Material carrier/control used in the present description represents 0.2% of bovine serum albumin (BSA), 0.1 M sodium phosphate, pH 7,6.

In this experiment, we use lines of rats CD®IGS [Crl:CD®(SD)/MYOINFARCT] and Naive Sprague Dawley. These lines were obtained from Charles River Laboratories. The age of the tested animals is approximately 6-7 weeks upon arrival, and the weight is approximately 160-200 g at the time of surgical intervention. Actual range may vary, and it is documented in the data.

The study took all animals received Naive Sprague Dawley and they were designated as group 1. Animals considered suitable for the study, before treatment were weighed.

All animals received CD®IGS [Crl:CD®(SD)/MYOINFARCT] was randomly assigned to different treatment groups (groups 2-5) using the simple randomization procedure based on the calculated ejection fraction based on echocardiographic studies performed on day 7 after surgery, held at Charles River Laboratories. Conducted a simple randomization with the results in each group of treatment (groups 2-5), consisting of suitable quantities of animals, having approximately equal to the average group ejection fraction (±3%) in groups of 2-5.

All animals in groups 2-6 acclimatized at Charles River Laboratories in accordance with Standard Operating Procedures of the laboratory. Then the animals were randomly distributed into groups of processing. All is not exposed to animals in group 1 to their initial echocardiographic studies acclimatized for about 24 hours after receipt.

Animals were individually kept in suspended wire-type cells made of stainless steel. Cage with a solid bottom is mostly not used, because rodents are copropagate and eating faeces containing the selected test drug and metabolic products, or eating the lining can complicate interpretation of results of toxicity studies.

Provided fluorescent illumination with automatic timer for about 12 hours a day. Sometimes, the daily cycle periodically interrupted due to related research activities. Controlled temperature and wet is here and every day they were recorded, and they were supported to the extent possible from 17,78 to 26,11°C and from 30 to 70%, respectively.

The basic diet was a Lab Diet® Certified Rodent Diet #5002 (PMI Nutrition International, Inc for blocks. Unless otherwise noted, this ration was available without restrictions. Each used a batch code recorded in the research journal. Unless otherwise noted, all animals without restriction provided tap water through an automatic water supply system.

The RESEARCH PLAN

Table 5
GGF2 relatively fragment of EGF-ld (Liu et al., 2006), dosed for 10 days, starting on day 7 after LAD
GroupProcessingThe duration of lifeDoseInterval dosing†Time ECHO (after surgery)
1 (n=5 males, n=5 females)Control (Native)17 days after surgeryOnly media24 hoursDay 6, 17
2 (n=6 M; n=6F)GGF2 17 days after surgery0,0625 mg/kg24 hoursDay 6, 17
3 (n=6 males, n=6 females)GGF217 days after surgery0.625 mg/kg24 hoursDay 6, 17
4 (n=6 males, n=7 females)EGF-ld17 days after surgeryEquimolar24 hoursDay 6, 17
5 (n=7 males; n=6 females)EGF-ld17 days after surgeryEquimolar24 hoursDay 6, 17

Table 6
High dose GGF2 in comparison with EGF-ld and EGF-Ig, dosed for 20 days starting on day 7 after the LAD. 10 days excretion
GroupProcessingThe duration of lifeDose Interval dosing†Time ECHO (after surgery)
1 (n=5 males, n=5 females)N. p.: matching for age controls without treatment30 days after the initial ECHOH. p.H. p.‡Day 1, 12, 22 and 32
2 (n=6 males, n=6 females)Control (Native)38 days after surgeryOnly media24 hours*Day 7, 18, 28 and 38
3 (n=6 males, n=6 females)GGF-238 days after surgery0.625 mg/kg24 hours*Day 7, 18, 28 and 38
4 (n=6 males, n=7 females)GGF-238 days after surgery3,25 mg/kg24 hours*Day 7, 18, 28 and 38
5 (n=7 males; n=6 females)EGF-ld38 days after surgeryEquimolar 24 hours*Day 7, 18, 28 and 38
6 (n=7 males; n=6 females)EGF-Ig38 days after surgeryEquimolar24 hours*Day 7, 18, 28 and 38

Table 7
The frequency of dosing GGF2
GroupProcessingThe duration of lifeDoseInterval dosing†Time ECHO (after surgery)
1 (n=5 males, n=5 females)N. p.: matching for age controls without treatment30 days after the initial ECHOH. p.H. p.‡Day 1, 12, 22 and 32
2 (n=6 males, n=6 females)Control (Native)38 days after surgeryOnly media24 hours*Day 7, 18, 28 and 38
3 (n=6 males, n=6 females)GGF-238 days after surgery3,25 mg/kg24 hours*Day 7, 18, 28 and 38
4 (n=6 males, n=7 females)GGF-238 days after surgery3,25 mg/kg48 hours*Day 7, 18, 28 and 38
5 (n=7 males; n=6 females)GGF-238 days after surgery3,25 mg/kg96 hours*Day 7, 18, 28 and 38
TA 1 - Test product 1; M = males; F = females.

Table 8
GGF2 with BSA and without him
GroupProcessingThe duration of lifeDoseInterval dosing†Time ECHO (after surgery)
1 (n=5 males, n=5 females)N. p.: owls is daysie age controls without treatment 17 days after surgeryH. p.H. p.Day 6 and 17
2 (n=6 males, n=6 females)Control (Native)17 days after surgeryOnly media24 hoursDay 6 and 17
3 (n=6 males, n=6 females)GGF-2 + BSA17 days after surgery3,25 mg/kg24 hoursDay 6 and 17
4 (n=6 males, n=7 females)GGF-2 without BSA17 days after surgery3,25 mg/kg24 hoursDay 6 and 17

The INTRODUCTION of the TEST AND CONTROL DRUGS

Introduction

The test and control drugs were injected by intravenous injection. Animals, distributed in group 1 were treated with media or test drugs; these animals served as the same age controls without treatment. The frequency, duration and dose of injection was the same as the op is Sano in tables 5-8. The amount of dose was approximately 1 ml per kg

The introduction of the test drug

The test and control drugs were injected through the tail vein. Specific doses were based on the most recent measurement of body weight. Unless otherwise specified Sponsor dose was administered by bolus injection.

Obtaining a test system

Surgery - Ligation of the left anterior descending artery

Surgical intervention was performed at Charles River Laboratories, as described in Charles River Laboratories Surgical Capabilities Reference Paper, Vol. 13, No. 1, 2005. In brief, the chest is conducted in the cranio-caudal incision, a little left of the sternal bone, through the skin and chest muscles. The third and fourth ribs are cut, and the intercostal muscles are separated with a blunt way. Quickly enter the cavity of the thorax and fully open the pericardium. Heart temporarily output to the surface through the incision. Define pulmonary cone and the left ear. Deletion of the site 5-0 silk thread under the left anterior descending coronary artery using a small curve of the needle. The ligature tie, and the heart is placed again in the chest. The air in the chest cavity gently squeeze until the chest wall and the skin incision is closed. The animal is brought into consciousness using supply-and-exhaust ventilation, and placed in a rich the Yu oxygen environment.

Postoperative recovery

In Charles River Laboratories conducted short-term postoperative monitoring and introduction of appropriate analgesics, as described in Charles River Laboratories Surgical Capabilities Reference Paper, Vol. 13, No. 1, 2005.

To assess the animal signs of pain or infection conducted long-term postoperative monitoring. Daily inspection of the site of the incision was continued for 7 days after receipt of the animals. As appropriate, implement the additional treatment of pain and have antimicrobial therapy.

Table 9
Prescribed medicines and dosages
MedicationINTERVAL, DOSE AND route of administration
DAILY AFTER SURGERYECHO ON DAY 1/7*ECHO NIGHT 12/18*ECHO ON DAY 22/28*ECHO ON DAY 32/38* and showdown
IsofluraneDepending on the effect, inhalationDepending on the effect, inhalation Depending on the effect, inhalationDepending on the effect, inhalation
Buprenorphine0.01 mg/kg, I/m (only as needed)----
*- The day of the procedure ECHO, defined by the distribution of animals in the group, as described below

EVALUATION IN VIVO STUDY

Observations in the cells

All animals were examined at least twice a day on morbidity, mortality, damage and availability of food and water. Any animal with poor health were identified for further observation and possible painless killing.

Weight

Body weight was measured and recorded at least once prior to random assignment and once a week during the study.

Food consumption

Food consumption was not measured, but the lack of appetite documented.

Echocardiographic studies

In all animals, distributed in group 1, echocardiographic studies were performed on day 1, 12, 22, and day 32 after admission (day 0). In all animals, distributed in groups of 2-5, echocardiographies the e studies were performed on day 7, 18, 28, and day 38 after the surgical intervention, conducted in Charles River Laboratories (day 0).

For echocardiographic evaluation, each animal was anestesiologi in accordance with table 5 and their fur with chest cut. On echocardiographic transmitter inflicted contact gel and got the image for measurement of cardiac function at several levels. Images were obtained for each animal in the direction of the short axis (srednetsenovom level, or another level depending on the location observed by echocardiography region of a heart attack).

Echocardiographic parameters

ECHO images were obtained at the level of srednelistovoy muscles or a different level, depending on the location observed by echocardiography area of infarction of the left ventricle. Was recorded by M-mode and 2-D images and stored on CD and/or MOD. Produced by ECHO measured parameters included: the wall thickness of the intraventricular septum (diastole); units = cm; wall thickness of the intraventricular septum (systole); units = cm; inner cross-section of the left ventricle (diastole); units = cm; inner cross-section of the left ventricle (systole); units = cm; the thickness of the wall of the left ventricle at the papillary level (diastole); units = cm; the thickness of the wall of the left ventricle at the papillary level is e (systole); units = cm; end-diastolic volume; units = ml, end-systolic volume; units = ml; ejection fraction; recorded in percent; systolic volume; units = ml; and fraction shortening, %; recorded in the form of a percentage.

PAINLESS KILLING

The agony

Any animal in agony, as determined in accordance with the Testing Facility Standard Operating Procedure, painless cut out of humanitarian considerations. All animals painlessly're killing in end-stage disease or found dead were subjected to conventional autopsy.

A painless way of killing

Painless killing was performed by injection of saturated potassium chloride in the Vena cava in accordance with an approved method guaranteed death, such as bloodletting.

The final outcome

All animals taken in the study were subjected to a painless death in accordance with the plan for them by opening or, if necessary, subjected painless death in end-stage disease.

RESULTS

Study 1 - Treatment of rats with GGF2 concentration of 0.625 mg/kg/once a day resulted in significant improvement in cardiac function, as shown in the present description by changes in ejection fraction and fraction shortening. Fragment of EGF-ld h is led to the same degree of improvement. Cm. table 5.

Study 2 - Treatment of rats with GGF2 concentration of 0.625 and 3.25 mg/kg/once a day resulted in significant improvement in cardiac function, as shown in the present description by changes in ejection fraction and fraction shortening. During the treatment period also saw significant improvements in end-systolic and end-diastolic volumes. Cm. table 6.

The results of study 3 - Treatment of rats with GGF2 concentration of 3.25 mg/kg in every 24, 48 or 96 hours resulted in significant improvement in cardiac function, as shown in the present description by changes in ejection fraction and fraction shortening. During the treatment period also saw significant improvements in end-systolic and end-diastolic volumes. Cm. table 7.

In previous publications (Liu et al.) it is shown that for optimal stability and activity neuregulin need a carrier protein such as BSA. GGF2 demonstrated stability without carriers such as BSA. This experiment was planned to test whether GGF2 stable and active in the treatment without BSA. After 10 days of processing and containing BSA and containing no BSA formulations with GGF2 was led to improvements in ejection fraction compared to controls with the carrier, similar to improvements observed in previous studies. In affect, the, from this study it is evident that the BSA or other protein-media compositions with GGF2 for the treatment of CHF are not required. Cm. table 8.

Table 10
Pathological data
DosingHyperplasia shell (NSH) sciatic nerveNSH breast cancerChange the injection site/skinEffects on heart
Daily p/C+++++++
Daily/++++/-
in/in every 48 hours+/---
in/in every 96 hours----
++ there often;
+ present;
+/- the viewers who have rarely,
- rarely or never see

As shown in table 10, the intermittent dosing GGF2 reduces the side effects associated with excessive levels of exogenous input GGF2. The authors of the present invention have found that this discovery is true regardless of injected whether GGF2 intravenously or subcutaneously.

In a day dosing sometimes see hyperplasia and the effect on the heart. The authors of this invention with less frequent dosing had ever seen it.

For a more complete description of the position to which belong the invention in this application are several publications and patent documents. All publications and patent applications mentioned in this description are included in the present description by reference to the same extent as if each independent publication or patent application was specifically and individually indicated as included in the reference.

1. The use of a therapeutically effective amount of GGF2, contains a domain similar to epidermal growth factor (EGF-like), in treating or preventing heart failure in a mammal by injection to the mammal a therapeutically effective amount of the peptide GGF2 every 48 hours dose comprising from about 0.001 mg/kg to about 10 mg/kg

2. Application under item 1, to the m injectable dose of peptide GGF2 is from about 0.01 mg/kg to about 10 mg/kg

3. Application under item 1, in which injectable dose of peptide GGF2 is from about 0.1 mg/kg to about 10 mg/kg

4. Application under item 1, in which injectable dose of peptide GGF2 is from about 1 mg/kg to about 10 mg/kg

5. Application under item 1, in which injectable dose of peptide GGF2 is from about 0.01 mg/kg to about 1 mg/kg

6. Application under item 1, in which injectable dose of peptide GGF2 is approximately 0.625 mg/kg

7. Application under item 1, in which injectable dose of peptide GGF2 is approximately 3,25 mg/kg

8. Application under item 1, in which the injection of a therapeutically effective amount of the peptide GGF2 is intravenous or subcutaneous injection.

9. Application under item 1, where the specified mammal is a human.

10. Application under item 1, where the specified peptide GGF2 is a recombinant human GGF2.

11. Application under item 1, where the peptide is:
SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO:22).

12. Application under item 1, where the peptide GGF2 is:
SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELY (SEQ ID NO:21).

13. Use one of the PP.1-12, in which the injection is intravenous.

14. Use one of the PP.1-12, in which the injection is subcutaneous.



 

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FIELD: medicine.

SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.

EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.

18 cl, 124 dwg, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula (1), which possess an affinity to the µ-opiod receptor and the ORL1-receptor. The invention also relates to the application of the said compounds for obtaining medications, which can be used in treatment of fear, stress and associated with stress syndromes, depressions, epilepsy, Alzheimer's disease, senile dementia, general cognitive dysfunctions, learning and memory disorders (as nootropic), withdrawal syndromes, alcohol and/or drug abuse and/or abuse of medications and/or alcohol, narcotic and medication addiction, etc. In general formula (1) (1) Y1, Y1', Y2, Y2', Y3, Y3', Y4 and Y4' in each case stand for -H; Q stands for -R0, -C(=O)-R0, -C(=O)OR0, -C(=O)NHR0, -C(=O)N(R0)2 or-C(=NH)-R0; R0 in each case stands for -C1-8-aliphate, -C3-12-cycloaliphate, -aryl, -heteroaryl, -C1-8-aliphate-C3-12-cycloaliphate, -C1-8-aliphate-aryl, -C1-8-aliphate-heteroaryl, -C3-8-cycloaliphate-C1-8-aliphate, -C3-8-cycloaliphate-aryl or -C3-8-cycloaliphate-heteroaryl; R1 and R2 independently on each other stand for -C-1-8-aliphate; R3 stands for -C1-8-aliphate, -aryl, -heteroaryl or -C1-8-aliphate-C3-12-cycloaliphate; n stands for 0; X stands for -NRA-;RA stands for -C1-8-aliphate; RB stands for -C1-8-aliphate; on condition that R1, R2, RA and RB simultaneously do not stand for the non-substituted-C1-8-aliphate.

EFFECT: increased efficiency of the application of the compounds.

9 cl, 11 tbl, 164 ex

FIELD: chemistry.

SUBSTANCE: invention relates to bi- and polycyclic substituted isoquinoline and isoquinolinones of formula (I), or to its stereoisomeric and/or tautomeric forms and/or to its pharmaceutically acceptable salts, where R1 represents OH; R3, R4, R5 and R8 represents H; R7 represents halogen or (C1-C6) alkyl; R6 represents one (C1-C4)alkylene, bound to a cycloalkyl ring, in which (C1-C4)alkylene forms the second bond with the other carbon atom of the cycloalkyl ring with the formation of a bicyclic ring system, where in the bicyclic ring system one carbon atom is substituted with a group, independently selected from O, S or SO2; and if m and s equal 2 or m equals 3 and s equals 1, R6 represents a group CH2-CH-(CH2)2, which via one group CH2 is bound to the cycloalkyl ring, and two other CH2 groups are bound to different carbon atoms of the cycloalkyl ring, and if m equals 3 and s equals 3, R6 represents two methylene groups, bound to different carbon atoms of the cycloalkyl ring, where the methylene groups or group CH2-CH-(CH2)2 are bound to the carbon atoms of the cycloalkyl ring and form an adamantane system of formula (XX) , where L can be bound to any secondary or tertiary carbon atom, or R6 together with R11 and an N atom form (C5) heterocycloalkyl, bound with the cycloalkyl residue in the form of a spirocyclic ring system, where the bicyclic ring system, or the adamantane system, or a ring system, containing (C5) heterocycloalkyl, represent non-substituted or optionally substituted with substituent R9; R9 represents (C1-C6)alkyl, (C2-C6)alkenyl, (C6)aryl or cyclopropyl R11 and R12 independently on each other represent H or (C1-C6)alkylene-(C6)aryl; n equals 0 or 1; m equals 2 or 3; s equals 1, 2 or 3; L represents O; its stereoisomeric and/or tautomeric forms and/or its pharmaceutically acceptable salts. The invention also relates to the application of a formula (I) compound.

EFFECT: novel bi- and polycyclic isoquinoline and isoquinolinone derivatives, useful as inhibitors of Rho-kinase, are obtained.

22 cl, 22 ex

FIELD: biotechnologies.

SUBSTANCE: peptide is characterised with sequence Lys-Leu-Lys-Gln-Lys-Leu-Ala-Glu-Leu-Leu-Glu-Asn-Leu-Leu-Glu-Arg-Phe-Leu-Asp-Leu-Val-Inp (SEQ ID NO: 16). The invention also relates to peptide/lipid complex based on the above peptide, in which phospholipid represents one or more of sphingomyelin, DPPC and DPPG, a pharmaceutical composition that contains it, and treatment methods of dyslipidemia, cardiovascular disease, endothelial malfunction, macrovascular illnesses and microvascular illnesses using it.

EFFECT: invention allows obtaining ApoA-I mimetic that is more stable in comparison to ApoA-I and that is easy to obtain.

14 cl, 21 dwg, 14 tbl, 24 ex

FIELD: medicine.

SUBSTANCE: on the first experimental day, cardiopathy is simulated by a single subcutaneous administration of equally portioned mixture of native egg albumin and Freund's complete adjuvant in rats. The mixture is administered at 0.2 ml of the mixture into 5 injection points: abdominally, into inguinal and axillary regions on the left and right. The cardiopathy is prevented by daily gastric administration of succinic acid 1.5 mmole/kg for 60 days through a probe.

EFFECT: higher clinical effectiveness.

2 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and medicine. What is presented is using treo-3-phenylglutamic acid hydrochloride of the following structural formula: as an agent possessing the cardioprotective, antiplatelet, anticoagulant and membrane-protective properties under stress stimulation.

EFFECT: compound possesses the high cardioprotective, antiplatelet, anticoagulant and membrane-protective activity.

9 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel compound of salvianolic acid L with general formula (I) , to its pharmaceutically acceptable salts and hydrolysable ethers, with compound of salvianolic acid L having one pair of protons of trans-form double bond and one proton of single-substituted double bond; and compound of salvianolic acid L is intended for treating cardiovascular disease, capture of free radicals and/or prevention of excessive oxidation.

EFFECT: invention also relates to method of its preparation, medication, which contains salvianolic acid L, and its application for preparation of drug for treatment of cardio-cerebral-vascular diseases.

13 cl, 13 dwg, 17 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: what is presented is using a conjugate of a granulocytic colony-stimulating factor immobilised on low-molecular polyethylene glycol prepared by electron-beam synthesis nanotechnology - the exposure to an accelerated electron flow at an energy of 2.5 MeV, absorbed dose 2-10 kGy and collection rate 1.65 kGy/h as agents for the correction of remote consequences of spermatogenesis caused by the cytostatic exposure.

EFFECT: due to the penetration through the blood-testis barrier, the agent corrects disturbed spermatogenesis caused by the cytostatic exposure by the early stimulation of reticular regeneration processes in the testicular tissues in the whole and rapid spermatogonium population recovery; the agent is almost non-toxic.

3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and represents toilet paper with the therapeutic properties containing a paper carrier with introduced therapeutic agents in the form of nanoparticles uniformly across the total volume in the weight relation of 1:50 to 1:1000; the therapeutic agents introduced into the dry paper substrate are presented either by a complex of Hamamelis Virginiana in the form of an infusion, or in homeopathic dilutions of 3-6-C, Aesculus Hyppocastanum in the form of an infusion, or in homeopathic dilutions of 3-6-C and Acidum Nitricum in dilutions of 6-12-C, or a complex of Hamamelis Virginiana in the form of an infusion or in dilutions of 3-6-C, Aesculus Hyppocastanum in the form of an infusion in dilutions of 3-6-C, Acidum Nitricum in dilutions of 6-12-C and camomile (Matricaria Chamomilla) in the form of an infusion or in dilutions of 3-6-C.

EFFECT: invention provides the improved preventive and therapeutic properties of the product with simplifying and cheapening the process.

1 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to factor of fibroblasts 21 (FGF21) growth, in particular, to FGF21 derivatives, which have covalently connected albumin binding agent of formula A-B-C-D-E-, and their pharmaceutical application, in particular, for treatment of diabetes, dyslipidemia, obesity, cardio-vascular diseases, metabolic syndrome and/or non-alcoholic fatty disease of liver (NAFDL). Derivatives by claimed invention have prolonged action, for instance, are able to supply low level of glucose during longer period of time, are capable of increasing time of FGF21 semiejection in vivo and/or result in lower clearance of FGF21.

EFFECT: derivatives by invention preferably have improved resistance to oxidation.

10 cl, 4 tbl, 58 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to cosmetology, and can be used for treatment of skin aging. For this purpose, used is a medication, which contains a basic fibroblast growth factor (bFGF) as a single active ingredient, which is introduced intracutaneously or subcutaneously into the place of a scar or into the surrounding region, for instance, a keloid, a hypertrophic scar and a scar contracture; in addition, the medication is also intended for treatment of one or more types of skin aging, selected from the following list: skin wrinkles, sagging skin, rough skin, skin thinning and reduction of skin resilience and elasticity because of rupture of dermal tissues or reduction of functions of fibroblast cells, with skin aging being photoaging, and a value of a dose of the basic fibroblast growth factor (bFGF) constituting from 0.1 mcg to 1 mg per 1 cm2 of skin, which represents the treatment target.

EFFECT: inventions ensure significant reduction of wrinkles, improvement of the skin structure, as well as due to an increase of its turgor and an increase of the volume of the subcutaneous adipose cellular tissue.

6 cl, 4 ex, 10 dwg

FIELD: biotechnology.

SUBSTANCE: composition is proposed which comprises stem cells of human amniotic fluid with the phenotype CD73+/CD90+/CD105+/CK19+, nutrient medium, erythropoietin, epidermal growth factor, and collagen taken in an effective amount.

EFFECT: invention enables to increase the proliferative potential and viability of the cells, while simultaneously providing cytoprotective effect on the cells of the transplant and stimulation of migration and proliferation of patient's own cells, and also to reduce significantly the concentration of injectable cells and to activate vascularisation and regeneration at the defect site and can be used in therapy for elimination of congenital and acquired defects of soft tissue arising as the result of injuries, after removal of tumors, congenital diseases, age-related changes or other damages.

2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and may be used for treating an individual suffering from a persistent, sluggish, partially healing, open or chronic wound. That is ensured by administering anti-connexin 43 polynucleotide approximately 0.001 to approximately 1 mg/kg of body weight into the wound of the individual in a pharmaceutically acceptable carrier. Stimulating and improving wound healing, improving wound healing rate in a diabetic patient and an individual suffering from a chronic skin wound is also suggested. Kits and finished products containing Pluronic F-127 and anti-connexin 43 polynucleotide presented by SEQ ID NO: 1 for wound healing.

EFFECT: group of inventions provides wound healing, relieved inflammation within the persistent wound, as well as minimised cicatrisation.

129 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to the field of biotechnology. Claimed is a method of purification of a factor, contributing to wound healing, which represents a hepatocyte growth factor (HGF). All stages of purification are carried out in the presence of antithrombin III (AT-III). In accordance with the claimed method carried out are: defrosting of the frozen HGF-containing source and removal of sediment from the defrosted source. After that, the obtained solution, which contains a supernatant and AT-III, is brought in contact with a carrier for affinity chromatography on an immobilised heparin. Then, the solution is separated from the carrier for affinity chromatography. The carrier is brought in contact with a desorption buffer with ionic strength sufficient for HGF desorption. The desorption buffer, containing HGF, AT-III and histidine-rich glycoprotein (HRGP) is collected. Also claimed are wound-healing compositions, which contain HGF, AT-III and/or HRGP, purified by the claimed method.

EFFECT: inventions make it possible to increase step-by-step output of the hepatocyte growth factor, with the hepatocyte growth factor being concentrated in eluate in the presence of AT-III.

26 cl, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to traumatology and orthopaedics, enabling preparing a biologically active preparation of autoblood for enhancing neogenesis processes. The presented technique involves sampling whole blood, centrifuging, selecting a middle layer of plasma so that to avoid the erythrocyte ingress. The centrifuged platelet concentrate is frozen in a cold room at temperature below minus 1 C°, dried for at least three minutes within the temperature range of 2 C° to 52 C°; the lyophilisate is sterilised before use.

EFFECT: technique of platelet-rich plasma lyophilisation enables preserving the TGF PDGF VEGF factor viability min 1,5 months from the moment of blood sampling.

1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to production of human pancreatic polypeptide analogues and can be used in medicine. The disclosed analogues differ from native human pancreatic polypeptide by replacement of amino acids on one or more residues, wherein position 0 is provided with an additional amino acid which is Gly.

EFFECT: obtained analogues provide effective treatment and prevention of obesity or diabetes in a patient, and can also be effective in reducing appetite, reducing food intake or reducing calorie intake in a patient.

26 cl, 2 dwg, 2 tbl,1206 ex

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

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