Method of treating visceral pain by administering antagonist antibodies against calcitonin gene-related peptide

FIELD: medicine.

SUBSTANCE: presented group of inventions refers to medicine. What is presented is a method of treating visceral pain and/or one or more symptoms of visceral pain, involving administering a therapeutically effective amount of an antagonist antibody against calcitonin gene-related peptide (CGRP) into an individual suffering visceral pain, or an individual suffering a risk of visceral pain, wherein the CGRP agonist antibody is applicable for peripheral administration. What is also presented is a pharmaceutical composition containing the CGRP agonist antibody and a pharmaceutically acceptable carrier, applicable for peripheral administration.

EFFECT: presented group of inventions provides the effective treatment of visceral pain with using no opiates.

18 cl, 3 dwg, 2 tbl, 3 ex

 

The SCOPE of the INVENTION

The invention relates to a method of treatment and/or prevention of visceral pain and/or symptoms of visceral pain using antibodies against CGRP and CGRP antibody against for use in the prevention and/or treatment of visceral pain and/or symptoms of visceral pain.

PRIOR art

Visceral pain is the leading cause of patient visits to the clinic, however, effective treatment of visceral pain is limited. Clinically the treatment of visceral pain is difficult and often requires the use of opiates. Despite their widespread use, heavy doselimiting adverse effects of opiates tend to degrade their effectiveness. In addition, opiates are associated with the risk of abuse and physical dependence and lead to constipation and other undesirable side effects that in many cases are contraindicated and reduces the quality of life.

Visceral pain associated with internal organs, including the internal organs of the body. These bodies include, for example, heart, lungs, reproductive organs, bladder, urinary tract, digestive system, liver, pancreas, spleen and kidneys. Visceral pain may occur with many conditions such as, for example, pancreatitis, childbirth, surgery in which Estella on the organs of abdominal cavity associated with intestinal obstruction, cystitis, menstruation or dysmenorrhea. Similarly, visceral pain can be considered renal pain, epigastric pain, chest pain and painful biliary colic, pain in appendicitis. Chest pain or pressure, due to early myocardial infarction, are also visceral. To visceral pain can cause diseases of the stomach, duodenum or colon. Common disorders of the gastrointestinal tract (GIT), leading to visceral pain include functional bowel disorder (PRK) and inflammatory bowel disease (IBD). These gastrointestinal disorders include a wide range of painful conditions, the effectiveness of the treatment which is currently limited, and includes, relative to PRK, gastroesophageal reflux, dyspepsia, irritable bowel syndrome (IBS) syndrome and functional abdominal pain (SFAB), and, regarding IBD, Crohn's disease, REIT and ulcerative colitis, each of which regularly leads to visceral pain.

IBS affects 10-20% of adults and adolescents worldwide (Longstreth et al., 2006, Gastroenterology 130(5):1480-91). The main reason for the treatment of these patients to the doctor is a chronic visceral pain, caused, as I believe, increased visceral sensitive is thew (Aziz, 2006, Gastroenterology 131(2):661-4). Shown that patients with IBS reduced threshold of visceral sensitivity to distension of the colon and rectum, and it is highly correlated with symptoms of visceral pain (Delafoy et al, 2006, Gut 55(7):940-5). Colitis induced in rats by stretching of the colon and rectum after the introduction of trinitrobenzenesulfonic (TNBS) is an animal model used by many researchers to study the mechanisms of visceral hypersensitivity (Gay et al, 2006, Neuroimmunomodulation 23;13(2):114-121; Delafoy et al, 2006; Adam et al., 2006, Pain 123(1-2):179-86).

Interstitial cystitis (IC) is a syndrome of pain in the bladder, clinical signs which are urgent urination, frequent urination and chronic pelvic pain. Clinical studies show that it is involved hypersensitivity visceral sensory afferent nerves, in which the sensation of bladder filling occurs at a lower than normal volume, and the perception of bladder filling becomes painful. In patients with IC shows the increase in the number of nerve endings in the submucosal layer of the bladder and the signs of neurogenic inflammation. Histopathology CI suggests the involvement in the pathological process of visceral afferent nerves.

Visceral pain may sniket in response to, for example, inflammation, tension or high blood pressure. It is not always caused by damage to internal organs. In addition, visceral pain is diffuse, can irrationality in other areas and may be associated with other autonomic and motor reflexes (e.g., nausea, muscle tension lumbar renal colic) (Lancet 1999, 353, 2145-48).

CGRP (peptide associated with the gene calcitonin) is a neuropeptide of 37 amino acids, belonging to the family of peptides including calcitonin, adrenomedullin and Amylin. In humans there are two forms of CGRP (α-CGRP and β-CGRP), having similar activities. They differ by three amino acids and differently distributed. Differences activities may also be influenced by at least two subtypes of receptors for CGRP. CGRP is a neurotransmitter in the Central nervous system, and shown that it acts as a strong vasodilator in the periphery, where neuronal processes, including CGRP, are closely associated with blood vessels. CGRP-mediated vasodilation is also associated with neurogenic inflammation as part of the chain of events leading to release of plasma from blood vessels and expansion of microvessels and what is happening with migraines.

It is shown that spinal introduction of selective antagonists of CGRP with small molecules useful in the treatment with which of Taani with neuropathic and nociceptive pain (Adwanikar et al, Pain, 2007, 132(1-2):53-66), suggesting that the elimination of alarms associated with endogenous CGRP in the spinal cord exerts an antinociceptive effect. In the message it is established that blockade of CGRP-alarm systems is effective in the elimination of visceral hypersensitivity (VH) by systemic injection of CGRP 8-37, receptor antagonist CGRP (Delafoy et al., 2006; Plourde et al., 1997, Am J Physiol. 273(1 Pt 1):G191-6; and Julia Bueno, 1997, Am J Physiol. 272(1 Pt 1):G141-6). However, CGRP 8-37 has a very short half-life in vivo and for this reason is not applicable as a drug. Thus, there is an urgent medical need for the establishment of new medicines for the treatment and prevention of visceral pain.

In this application there are links to various publications (including patents and patent applications). The contents of these publications are fully incorporated here by reference.

SUMMARY of the INVENTION

According to the present invention, a method for prevention and/or treatment of visceral pain and/or symptoms of visceral pain in an individual, comprising introducing a therapeutically effective amount of an antibody antagonist against CGRP individual suffering from visceral pain, or individual with the risk of visceral pain.

In some embodiments the antibody is an antagonist against CGRP enter the peripheral the ski. In other embodiments the antibody is an antagonist against CGRP administered orally, sublingually, by inhalation, transdermal, subcutaneously, intravenously, intraarterially, intraarticularly, periartikulyarno, locally and/or intramuscularly.

In some embodiments of visceral pain associated with functional bowel disorder (PRK). PRK can be a gastroesophageal reflux, dyspepsia, irritable bowel syndrome (IBS) syndrome and functional abdominal pain (SFAB). In some embodiments of visceral pain associated with inflammatory bowel disease (IBD). IBD may represent Crohn's disease, REIT or ulcerative colitis. In some embodiments of the visceral pain is associated with renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis or pancreatitis. In some embodiments of visceral pain associated with interstitial cystitis (IC).

In some embodiments the antibody is an antagonist against CGRP binds to CGRP; blocking CGRP binding to its receptor; blocks or reduces activation of CGRP receptors; inhibits, blocks, inhibits or reduces biological activity of CGRP; enhances clearance of CGRP and/or inhibits the synthesis, the formation and release of CGRP.

In some embodiments the antibody is an antagonist against CGRP is the Wallpaper of the human antibody or humanitariannet antibody. In some embodiments the antibody is an antagonist against CGRP is a monoclonal antibody. In some embodiments the antibody is an antagonist against CGRP may contact CGRP with KD50 nm or less (as measured by surface plasma resonance at 37°C) and/or has a half-life in vivo for at least 7 days.

In some embodiments the antibody is an antagonist against CGRP-specific binds to the C-terminal region of CGRP. In some embodiments the antibody is an antagonist against CGRP-specific recognizes the epitope defined by the sequence GSKAF (SEQ ID NO:39). In some embodiments the antibody is an antagonist against CGRP contains the variable domain of the heavy chain (VH), amino acid sequence which is at least 90% identical to SEQ ID N0:1 or 19.

In some embodiments the antibody is an antagonist against CGRP contains variable domain light chain (VL), amino acid sequence which is at least 90% identical to SEQ ID NO:2 or 20. In some embodiments the antibody is an antagonist against CGRP further comprises a domain VH, amino acid sequence which is at least 90% identical to SEQ ID NO:1 or 19. In other embodiments, the antibody against CGRP contains at least one hypervariable segment (CDR) selected from the group consisting of: (a) hypervariable participants is 1 and the heavy chain (CDR-H1), as set forth in SEQ ID NO:3, 21, 33, 34, 36 or 37; (6) a hypervariable site 2 of the heavy chain (CDR-H2), as set forth in SEQ ID NO:4, 22, 35 or 38; (b) hypervariable section 3 of the heavy chain (CDR-H3), as set forth in SEQ ID NO:5 or 23; (g) hypervariable region 1, light chain (CDR-L1), as set forth in SEQ ID NO:6 or 24; (d) hypervariable site 2 light chain (CDR-L2), as set forth in SEQ ID NO:7 or 25; and (e) a hypervariable site 3 light chain (CDR-L3), as set forth in SEQ ID NO:8 or 26; and (f) options L1, L2 and H2.

In some embodiments, the antibody against CGRP contains a full amino acid sequence of the heavy chain of antibody G1 shown in SEQ ID NO:11, with C-terminal lysine or without him. In some embodiments, the antibody against CGRP contains a full amino acid sequence of the light chain of the antibody G1 shown in SEQ ID NO:12. In some embodiments, the antibody against CGRP contains a full amino acid sequence of the heavy chain of antibody G1 shown in SEQ ID NO:11, with C-terminal lysine or without it; and a full-length amino acid sequence of the light chain of the antibody G1 shown in SEQ ID NO:12.

In some embodiments, the antibody against CGRP contains a full amino acid sequence of the heavy chain of the antibody G2 shown in SEQ ID NO:29, with C-terminal lysine or without him. In some embodiments, the antibody against CGRP contains the full amino acid sequence of the light chain of the antibody G2, shown in SEQ ID NO:30. In some embodiments, the antibody against CGRP contains a full amino acid sequence of the heavy chain of the antibody G2 shown in SEQ ID NO:29; and a full-length amino acid sequence of the light chain of the antibody G2 shown in SEQ ID NO:30.

In some embodiments the antibody is an antagonist against CGRP contains a domain VH, amino acid sequence which is at least 90% identical to SEQ ID NO:1, and the domain VL, amino acid sequence which is at least 90% identical to SEQ ID NO:2. In some embodiments the antibody is an antagonist against CGRP contains a heavy chain obtained using the expression vector with the registration number in the American type culture collection (ATSS) MOUTH-6867. In some embodiments the antibody is an antagonist against CGRP contains a light chain, obtained using the expression vector with the registration number in ATSC MOUTH-6866. In some embodiments the antibody is an antagonist against CGRP produced using expression vectors with registration numbers in ATSC MOUTH-6867 and MOUTH-6866.

In some embodiments the antibody is an antagonist against CGRP is administered by subcutaneous or intravenous injection of one, two, three or four times a month. In some embodiments the antibody is an antagonist against CGRP is administered at a concentration of 5-100 mg/ml In some wapl is the use of antibody antagonist against CGRP is administered at a concentration of 1-100 mg/kg body weight/

In some embodiments the antibody is an antagonist against CGRP does not associated with Central nervous system (CNS) disorder of coordination or attention. In some embodiments the antibody is an antagonist against CGRP does not enter the Central, spinal or intrathecal. In some embodiments the antibody is an antagonist against CGRP is not Central, spinal or intrathecal penetrating molecule.

In some embodiments the antibody is an antagonist against CGRP is administered separately, sequentially or simultaneously in combination with one or more than one additional pharmacologically active compound. In some embodiments one or more additional pharmacologically active compound selected from: opioid analgesic, e.g. morphine, heroin, hydromorphone, Oxymorphone, Levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, Dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorfina, naloxone, naltrexone, buprenorphine, butorphanol, nalbufina or pentazocine; non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indometacin, Ketoprofen, Ketorolac, meclofenamic acid, mefenamovoy is islote, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindaka, tolmetin or zomepirac or their pharmaceutically acceptable salts; barbiturates sedatives, such as amobarbital, aprobarbital, butabarbital, butalbital, mephobarbital, metharbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutal, thiamylal or thiopental or their pharmaceutically acceptable salts; benzodiazepine with sedative effects, such as hlordiazepoksida, clorazepate, diazepam, flurazepama, lorazepam, oxazepamum, temazepam, or triazolam or their pharmaceutically acceptable salts; H1-antagonist with a sedative action, e.g. diphenhydramine, pyrilamine, prometazina, chlorpheniramine or chlorcyclizine or their pharmaceutically acceptable salts; sedatives, such as glutethimide, meprobamate, methaqualone or dichloralphenazone or its pharmaceutically acceptable salt; a means of relaxing skeletal muscles, such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, Methocarbamol or grenadine or their pharmaceutically acceptable salts; receptor antagonist N-methyl-D-aspartate (NMDA), e.g. dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, of memantine, Pirro is kinoleninianu or CIS-4-(phosphonomethyl)-2-piperidinecarboxylic acid or their pharmaceutically acceptable salts; alpha-adrenergic tools, such as doxazosin, tamsulosin, clonidine or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydrothieno-2-yl)-5-(2-pyridyl)-hintline; tricyclic antidepressant such as desipramine, imipramine, amitriptyline or nortriptyline; anti-convulsants such as carbamazepine or valproic; antagonist tachykinin (NK), in particular an antagonist of NK-3, NK-2 or NK-1, e.g. (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-were)-7H-[1,4]thiazocine[2,1-g][1,7]naphthiridine-6-13-dione (TAK-637), 5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifloromethyl)phenyl]ethoxy-3-(4-forfinal)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazole-3-one (MK-869), linepithema, capitant or 3-[[2-methoxy-5-(triptoreline)phenyl]methylamino]-2-phenylpiperidine (2S,3S); antagonist of muscarinic receptors, such as oxybutynin, tolterodine, propiverine, chloride tropica or darifenacin, inhibitor of cyclooxygenase-2 (COX-2), such as celecoxib, rofecoxib or valdecoxib; non-selective inhibitor of COX (preferably with a protective effect on the stomach), for example nitrofluorene (HCT-1026); an analgesic, a derivative of aniline, in particular paracetamol; neuroleptic such as droperidol; agonist (e.g., resiniferatoxin) or antagonist (e.g., capsazepine) vanilloid receptors; beta-adrenal the practical tools, such as propranolol; local anesthetic, such as meksiletin; corticosteroid, such as dexamethasone; agonist or antagonist of serotonin receptors; cholinergic (nicotinic) analgesic; Tramadol®; inhibitor of phosphodiesterase-5 (PDE-5), such as sildenafil, vardenafil or taladafil; alpha-2-Delta ligand such as gabapentin or pregabalin; and cannabinoid.

According to the present invention has also proposed the use of antibody-antagonist against CGRP for the manufacture of a medicine for the prevention and/or treatment of visceral pain and/or symptoms of visceral pain. In some embodiments, the drug is made for peripheral administration. In some embodiments the antibody is an antagonist against CGRP has with the introduction of peripheral action.

According to the present invention also proposed a pharmaceutical composition for the treatment and/or prevention of visceral pain and/or symptoms of visceral pain from the individual containing the antibody antagonist against CGRP and a pharmaceutically acceptable carrier. In some embodiments, the composition is made for peripheral injection.

According to the present invention also offered a kit comprising the pharmaceutical composition for treatment and/or prevention of visceral pain and/or symptoms the s visceral pain in an individual, and instructions for peripheral injection of a therapeutically effective quantity of a specified pharmaceutical composition to an individual for the treatment and/or prevention of visceral pain and/or symptoms of visceral pain.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

In Fig.1A and 1B shows a model of visceral pain. (A) Antibody 4901 ("4901") or PBS control ("filler") was intravenously injected animals received injections of trinitrobenzenesulfonic (TNBS) after laparotomy. Visceral pain threshold in animals was investigated using a balloon stretching. Pain threshold is specified in mm RT. Art. (y-axis). "Simulation" means that after laparotomy instead of TNBS animals were injected with the control solution (30% ethanol). (B) receptor Antagonist CGRP CGRP 8-37 or control PBS ("filler") was intravenously injected animals received TNBS after laparotomy. As in (A), visceral pain threshold in animals was investigated using a balloon, stretching, and pain threshold is specified in mm RT. Art. (y-axis).

In Fig. 2 shows a model of visceral pain. The animal was intravenously injected antibody 4901 ("4901") or PBS control ("filler"). The motility of bladder, measured as the number of contractions (y axis) was determined after 1 h, 3 h and 5 h after turpentine-induced inflammation of the bladder.

DETAILED description of the INVENTION

According to the invention, opened here, the proposed treatment and/or prevention of visceral pain in an individual by introducing the individual a therapeutically effective amount of the antibodies and the of tagonist against CGRP.

According to the invention, opened here, also suggested that antibody antagonists against CGRP and polypeptides originating from G1 or its variants shown in Table 6 WO 2007/054809, fully incorporated here by reference. According to the invention is also proposed methods of making and using these antibodies and polypeptides.

General methods

In the practical use of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), Microbiology, cell biology, biochemistry and immunology, which are included in the competence of the experts in this field of technology. Such techniques are described in detail in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et ai., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approah (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Definition

"Antibody" is an immunoglobulin molecule capable of specific contact with a target, such as carbohydrate, polynucleotide, lipid, polypeptide, and so on, through at least one site of antigen recognition, located in the variable regions of immunoglobulin molecules. When using this term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain fragments (ScFv), their mutant variants, fused proteins containing part of the antibody (such as a single domain antibody)? and any other modified form of the molecules of the immunoglobulin that contains the site of antigen recognition. The antibody includes the antibody of any class such as IgG, IgA or IgM (or their subclasses), and it is not necessary that the antibody was an antibody of any particular class. Depending on the amino acid sequence of the constant domain of the heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can b shall be further divided into subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of the heavy chains, corresponding to different classes of immunoglobulins are called alpha, Delta, Epsilon, gamma and mu, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.

When used here "monoclonal antibody" refers to an antibody obtained from a population essentially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally mutations that may be present in small quantities. Monoclonal antibodies of vysokospetsifichnymi and directed against a single antigenic site. In addition, unlike drugs polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody was obtained from an essentially homogeneous population of antibodies, and it should not be interpreted as requiring receipt of antibodies specific way. For example, monoclonal antibodies for use in the present invention can be obtained hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or could the t can be obtained by means of recombinant DNA, such as described in U.S. patent No. 4816567. For example, monoclonal antibodies may also be isolated from phage libraries derived using the techniques described in McCafferty et al., 1990, Nature, 348:552-554.

When used here "humanized" antibodies are forms of antibodies from a source that is non-human (e.g., mice) that are specific chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding subsequences of antibodies) that contain minimal sequence, with the origin of the immunoglobulin from a source, not a person. The most part, humanized antibodies are human immunoglobulins (antibodies-recipients), in which the remains of the hypervariable segment (CDR) of the recipient are replaced by residues CDR species that are not human (antibody-donor), such as a mouse, rat or rabbit having the desired specificity, affinity and biological activity. In some cases, remnants of the framework region (FR) of a human immunoglobulin are replaced by corresponding residues from a source, not a person. In addition, humanitariannet antibody may contain residues that are not on the antibody-recipient or insertion posledovatel the particular CDR or frame areas but included to further improve and optimize the properties of the antibody. In General, humanitariannet antibody will contain essentially all of at least one, and typically two, variable domains (for example, the variable domain of the heavy chain and the variable domain of the light chain), where all or substantially all of the CDR-plots correspond to the CDR portions of immunoglobulin from a source, not a person, and all or substantially all of the FR-region represent the FR-region consensus sequence of human immunoglobulin. In the optimal case humanitariannet antibody will also contain at least a portion of a constant region or domain of immunoglobulin (Fc), typically constant region or domain of a human immunoglobulin. Antibodies can be Fc-region, modified as described in WO 99/58572. Other forms of humanized antibodies have one or more than one CDR (one, two, three, four, five, six), the modified relative to the original antibody, which are also referred to as one or more than one CDR having origins from one or more than one CDR of the original antibody.

When used here "human antibody" means an antibody having the amino acid sequence corresponding to the amino acid sequence of antibodies formed by the individual is CA and/or obtained using any of the methods of obtaining human antibodies, known in the art or disclosed here. This definition of human antibodies includes an antibody that contains at least one polypeptide of a human heavy chain or at least one polypeptide of a human light chain. One such example is the antibody containing polypeptides murine light chain and human heavy chain. Human antibodies can be obtained using a variety of techniques known in the art. In one embodiment, the human antibody is selected from ragovoy library that expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et ai, 1991, J. Mol. Biol., 222:581). Human antibodies can also be obtained by introducing loci of human antibodies in transgenic animals, such as mice, have their own immunoglobulin genes have been partially or completely inactivated. This method is described in U.S. patent№5545807; 5545806; 5569825; 5625126; 5633425 and 5661016. Alternatively, a human antibody can be obtained by the immortality of the human b-lymphocytes producing an antibody directed against a target antigen (such b cells can be obtained from the individual or may be immunized in vitro. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p. 77, 1985; Boerer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. patent No. 5750373.

When used herein, the term "peptide associated with the gene calcitonin and CGRP" refers to any form of the peptide linked to the genome of calcitonin and its variants, preserving, at least in part, the activity of CGRP. For example, CGRP can be an α-CGRP or β-CGRP. When you use here CGRP includes native sequence CGRP all kinds of mammals, such as humans, dogs, cats, horses and cows.

When used here "antibody antagonist against CGRP" (interchangeably called "antibody against CGRP") refers to an antibody able to bind with and inhibit CGRP biological activity of CGRP and/or follow-up(s) metabolic(s) path (path), indirect(s) CGRP signaling. Antibody antagonist against CGRP includes antibodies, blocking, antagonizers, suppresses or reduces (including significantly reducing) the biological activity of CGRP, including subsequent metabolic pathways, mediated by CGRP signaling, such as binding to receptors and/or the induction of cellular response to CGRP. For the purposes of the present invention clearly imply that the term "antibody antagonist against CGRP covers all of the above terms, titles, and functional States and properties, essentially eliminating, reducing or neutralise the matter of CGRP itself, the biological activity of CGRP (including, without limitation, its ability to mediate any aspect of visceral pain) or the consequences of its biological activity to any significant extent. In some embodiments the antibody is an antagonist against CGRP binds to CGRP and prevents the binding of CGRP with the CGRP receptor. In other embodiments, the antibody against CGRP binds to CGRP and prevents activation of the CGRP receptor. Here are examples of antibody antagonists against CGRP.

When used here, the terms "G1" and "antibody G1" are used interchangeably to refer to antibodies produced using expression vectors having custody of the rooms ATS-MOUTH-6867 and ATS-MOUTH-6866. Amino acid sequences of the variable regions of the heavy chain and light chain shown in SEQ ID NO:1 and 2. CDR-sites of antibody G1 (including CDR by Chothia and Kabat) is schematically shown in Fig.5 WO 2007/054809, the contents of which are fully incorporated here by reference. Polynucleotide encoding the variable regions of the heavy and light chain shown in SEQ ID NO:9 and 10. Properties of antibody G1 described in the Examples of WO 2007/054809.

When used here, the terms "G2" and "antibody G2" are used interchangeably to refer to mouse monoclonal antibodies against rat CGRP, as described in Wong NA et al. Hybridoma 12:93-106, 1993. Amino acid sequence VA is iabeling regions of the heavy chain and light chain shown in SEQ ID NO:19 and 20. Polynucleotide encoding the variable regions of the heavy and light chain shown in SEQ ID NO:27 and 28. CDR-areas G2 antibodies are shown in SEQ ID nos:21-26.

The terms "polypeptide", "Oligopeptide", "polypeptide" and "protein" are used here interchangeably to refer to chains of amino acids of any length, preferably relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, it may contain modified amino acids and/or can be interrupted components that are not amino acids. The terms also encompass a chain of amino acids, modified naturally or by intervention; for example, the formation of disulfide bonds, glycosylation, lepidosauria, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with component label. The definition also includes, for example, polypeptides containing one or more than one equivalent of amino acids (including, for example, synthetic amino acids, and so forth), as well as other modifications known in the art. Assume that the polypeptides may be separate circuits or associated circuits.

As is known in the art, polynucleotide" or "nucleic acid", as they are interchangeably used here relate to the circuits NUS is Evtimov of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into DNA or RNA polymerase. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. In the presence of modified nucleotides modification of the structure of nucleotides may be performed before or after Assembly of the circuit. The nucleotide sequence can be interrupted components, non-nucleotides. Polynucleotide can be further modified after polymerization, such as by conjugation with component label. Other types of modifications include, for example, "copy", substitution of one or more than one naturally occurring nucleotide analogue, mezhnukleotidnyh modifications, such as modifications with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamide, carbamates, etc.) and with charged linkages (e.g., phosphorothioate, phosphorodithioate and so on), the modification comprising side groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and so forth), modification intercalators (e.g., acridine, psoralen, etc.), option is the ratification, including chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), modifications, including alkylating agents, versions with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotide (polynucleotides). In addition, any of the hydroxyl groups normally present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, protected by standard protective groups or activated for a education additional links to additional nucleotides, or may be conjugated to a solid substrate. 5'- and 3'-terminal Oh-group can be phosphorylated or substituted amines or organic kapinowski groups of 1-20 carbon atoms. Other hydroxyl groups may also be derivatization using standard protective groups. Polynucleotide can also contain ribose analogues or desoxyribose generally known in the art, including, for example, 2'-O-methyl, 2'-O-allyl-, 2'-fluoro - or 2'-isidoros, carbocyclic analogues of sugars, alpha - or beta-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and nucleoside analogues is without reason, such as methylribose. One or more than one fosfomifira connection can be replaced by alternative linking groups. These alternative linking groups include, without limitation, the embodiment where the phosphate is replaced by P(O)S ("tiat"), P(S)S ("ditial"), (O)NR2(amidate"), P(O)R, P(O)OR', CO or CH2(formatall"), where each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C) may contain ether linkage (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. It is not necessary that all communication polynucleotide were identical. The preceding description applies to all polynucleotides named here, including RNA and DNA.

"Variable region" of an antibody refers to the variable region of the light chain of the antibody or variable region of the heavy chain of the antibody, by itself or in combination. Each variable region heavy and light chain consists of four frame regions (FR), connected by three areas, complementarity determining (CDR), also known as hypervariable sites. In each chain FR retain CDR together in close proximity, and these CDR with CDR of the other circuit involved in the formation of the antigen-binding site of antibodies. There are at least two methods of determining CDR: (1) the method based on inter is iloveu sequence variability (i.e Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) the method based on crystallographic studies of complexes of antigen-antibody (Al-lazikani et al (1997) J. Moiec. Biol. 273:927-948)). When using CDR here can refer to CDR identified by any method or combination of both.

"Constant region" of an antibody refers to the constant region of the light chain of the antibody or the constant region of the heavy chain of the antibody, by itself or in combination.

When used here "immunologically specific" binding of the antibody refers to antigen-specific binding interaction that occurs between the antigen-binding site of antibody and specific antigen recognized by this antibody (the antibody interacts with the protein in an ELISA or other immunological analysis and does not interact detectable manner with unrelated proteins).

The epitope "selectively binding" or "specific binding" (used here interchangeably) with the antibody or polypeptide, is a term well known in the art, and methods to determine such specific or selective binding are also well known in the art. They say that the molecule shows a "specific binding" or "selective binding" if it maintains deistvuet or associated with a specific cell or substance more often faster, for a longer period of time and/or with greater affinity than with alternative cells or substances. Antibody "specific binds" or "selectively binds" to a target if it binds with greater affinity, avidity, faster and/or for a longer period of time than with other substances. For example, an antibody specific or selective binding epitope CGRP, is an antibody binding to this epitope with greater affinity, avidity, faster and/or for a longer period of time than with other epitopes CGRP or epitopes of antigens, non-CGRP. When using this definition also implies that, for example, the antibody (or grouping, or epitope) that are specific or selective binding to the first target may be specific or selective contact with the second target or not to contact specific or selective, with the second target. As such, "specific binding" or "selective binding" does not necessarily imply (although it may enable) exclusive binding. Usually, but not necessarily, the reference to the selective binding means binding.

When used here "essentially pure" refers to a substance, the degree of purity which is at least 0% (i.e., free from contaminants), more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%.

"A host cell" includes an individual cell or cell culture which can be or are the recipient of a vector (vectors) for the introduction of polynucleotide inserts. Cell hosts include progeny of a single host cell, and this offspring is not necessarily completely can be identical (in morphology or chromosomal DNA) of the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells, transfetsirovannyh polynucleotides (polynucleotide) this invention.

The term "Fc region" is used to denote the C-terminal region of the heavy chain of immunoglobulin. "Fc region" may be a native sequence Fc region or Fc region. Despite the fact that the boundaries of the Fc region of the heavy chain of immunoglobulin may vary, Fc-region of the heavy chain of human IgG is usually determined by the segment from amino acid residue in position Cys226 or from Rho to-end of the heavy chain. The numbering of residues of the Fc-region corresponds to the numbering of the EU index in Kabat. Kabat et al., Sequences of Proteins of Imunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. Fc-region of immunoglobulin is and usually contains two constant domain, CH2 and CH3.

When used here "Fc receptor" and "FcR" is used to describe a receptor, to bind to the Fc region of antibodies. Preferred FcR is a native sequence human FcR. Moreover, a preferred FcR FcR is communicating with an IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants of these receptors and variants of these receptors formed by alternative splicing. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Review of FcR are shown in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor FcRn, providing transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; Kirn et al., 1994, J. Immunol., 24:249).

"Complement-dependent cytotoxicity" and "CDC" refers to the lysis of the target in the presence of complement. Initiation path activation of complement provides the binding of the first component of the complement system (C1q) with a molecule (e.g. antibody) in combination with cognatum antigen. To assess activation of complement may be the analysis of the CDC, for example, as described in Gazzano-three-bet et al., J. Immunol. Methods, 202:163 (996).

"Functional Fc-region performs at least one effector function of native sequence Fc region. Typical "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); the binding of Fc-receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; lower regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions usually require a combination of the Fc-region with a binding domain (e.g., variable domain antibodies), and can be assessed using various assays known in the art to assess such effector functions of antibodies.

"Native sequence Fc region" comprises the amino acid sequence identical to the amino acid sequence of Fc-field, discovered in nature. "Variant Fc region" comprises the amino acid sequence differing from the amino acid sequence of a native sequence Fc region of at least one amino acid modification, but keeping at least one effector function of native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence is Yu Fc region or Fc region of the original polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or Fc region of the original polypeptide. Preferably, the sequence variant Fc region is at least about 80% identical to a native sequence Fc region and/or the Fc-region of the original polypeptide, and most preferably at least about 90% identical to a native sequence Fc region and/or the Fc-region of the original polypeptide, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to a native sequence Fc region and/or the Fc-region of the original polypeptide.

When used here "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer cells (m-cells), neutrophils, and macrophages), recognize bound antibody on the target cell and then lead to lysis of the target cells. ADCC activity systems is emnd molecules can be estimated using analysis of ADCC in vitro, such as analysis, described in U.S. patent No. 5500362 or 5821337. Effector cells applicable for such analyses include the mononuclear cells of peripheral blood (RVMS) and MC-cells. Alternative or additionally, ADCC activity of the considered molecules can be assessed in vivo, e.g., in animal models, such as model disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

When used here "treatment" is a method of obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, without limitation, one or more of the following: relief of any aspect of visceral pain, including reducing the severity of, reducing the severity of pain and other related symptoms, reduce the frequency of relapses, improving the quality of life of individuals suffering from visceral pain, and reduce the dose of other medications required to treat visceral pain. Other related pain symptoms include, without limitation, cramps, prolonged pain, diffuse pain, pressure, feeling of fullness, sensation of constriction, nausea, vomiting and sensitivity to light, sound and/or movement.

"Reducing incidence" of visceral pain refers to any reducing severity (which can include reducing the need for and/is whether the decrease in the number (for example, exposure to) other drugs and/or therapies generally used for the treatment of this condition, including, for example, opiates (e.g., oxycodone, morphine, butorphanol, nalbuphine and so forth), duration and/or frequency. Specialists in the art it is clear that the response of individuals to treatment may vary, and, in fact, for example, "method of reducing the incidence of visceral pain in the individual" means the introduction of antibody antagonist against CGRP, based on the reasonable expectation that such introduction could lead to such a reduction in incidence in that particular individual.

"Reducing the intensity of visceral pain and/or symptom associated with visceral pain, means the reduction or attenuation of one or more than one symptom of visceral pain and/or symptom associated with visceral pain compared with no introduction-antibody antagonist against CGRP. "Reduced intensity" also includes shortening or reducing the duration of symptoms.

"Relief" visceral pain and/or symptom associated with visceral pain, means reducing the severity of one or more than one undesirable clinical manifestations of visceral pain in an individual or group of individuals receiving treatment with antibody-antagon the place against CGRP according to the invention.

When using "control of visceral pain" refers to maintaining or reducing the severity or duration of one or more than one symptom of visceral pain or frequency of visceral pain compared to the level before treatment. For example, the duration or severity of visceral pain or frequency of visceral pain in an individual can be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60% or 70% compared to the level before treatment.

When using the "delay" development of visceral pain is delay, deter, deceleration, braking, stability and/or delay of progression of visceral pain. The duration of this delay may vary depending on the medical history and/or individuals receiving treatment. As is obvious to a person skilled in the art, a sufficient or significant delay can, in fact, encompass prevention, in which case the individual is not the development of visceral pain. The way in which "delays" development of a symptom, is a way that reduces the likelihood of symptom in a given period of time and/or reducing the severity of symptoms in a given period of time compared to the absence of the application of the method. Such comparisons are usually based on clinical research is ovadiah, where the number of subjects is sufficient to demonstrate statistically significant differences between subjects receiving treatment and not receiving treatment.

"Development" or "progression" visceral pain means initial manifestations and/or further progression of the disorder. The development of visceral pain can be identified and assessed using standard clinical techniques well known in the art. However, the development also refers to the progression, which may be undetectable. For purposes of this invention, the development or progression refers to the biological course of the symptoms. "Development" includes the occurrence, recurrence and start. When used here "beginning" or "origin" visceral pain includes the primary occurrence and/or recurrence.

"Biological sample" encompasses a variety of sample types obtained from an individual and can be used in the analysis for the diagnosis or monitoring. This definition encompasses blood and other liquid samples of biological origin, samples of soft tissue such as a biopsy specimen or tissue cultures or cells originating from them, and their offspring. This definition also includes samples that after receiving them were carried out any manipulation, such as clicks the processing reagents, solubilization or the selection of certain components, such as proteins or polynucleotide, or confinement in semi-solid or solid matrix in order to prepare slices. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, supernatant cells, cell lysates, serum, plasma, biological fluids and tissue samples.

When used here, the "effective dose" or "effective amount" of the drug, compound or pharmaceutical composition is an amount sufficient for obtaining beneficial or desired results. For preventive use is beneficial or desired results include results such as eliminating or reducing the risk, reducing the severity, or delay the onset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes arising from the development of the disease. For therapeutic applications beneficial or desired clinical results include outcomes such as reduction of pain intensity, reducing the duration or frequency of attacks visceral pain and reducing the intensity of one or more than one symptom resulting from visceral pain (biochemical, g is ecologicheskogo and/or behavioral), including its complications and intermediate pathological phenotypes arising from the development of pain, improving the quality of life of individuals suffering from pain, reduce the dose of other medications required to treat the pain, enhancing effect of another drug and/or delay of progression of pain in patients. Effective dose can be introduced by one or more than one introduction. For purposes of this invention, an effective dose of the drug, compound or pharmaceutical composition is an amount sufficient for preventive or therapeutic treatment, directly or indirectly. In a clinical context implies that the effective dose of the drug, compound or pharmaceutical composition can be achieved or may not be achieved in combination with other drug, compound or pharmaceutical composition. Thus, the "effective dose" can be seen in the context of the introduction of one or more than one therapeutic agent, and we can assume that one agent is administered in an effective amount if, in conjunction with one or more than one other agent, this leads to the possibility of reaching or achieving the desired result.

"Individual" or "subject" is the first mammal, more preferably human. Mammals include, without limitation, farm animals, sport animals, Pets, primates, horses, dogs, cats, mice and rats.

When used here "vector" means a construct that allows you to deliver and it is preferable to Express the one or more than one gene of interest or one or more than one sequence of interest in a cell host. Examples of vectors include, without limitation, viral vectors, expression vectors of deproteinizing DNA or RNA, plasmid, cosignee or phage vectors, DNA or RNA expression vectors, associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as cells-producers.

When using the "sequence control expression" means a nucleic acid sequence that controls transcription of the nucleic acid. The sequence control expression can be a promoter such as a constitutive or inducible promoter or enhancer. The sequence controlling the expression of functionally associated with the transcribed sequence of the nucleic acid.

When used here "pharmaceutically PR is acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance, which in combination with the active ingredient allows you to preserve the biological activity of this ingredient and does not interact with the immune system of the subject. Examples include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions, such as the emulsion of the type oil-in-water, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9 percent saline solution. Compositions containing such carriers, made well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

When using mean here that the term "kon" refers to the rate constant for the Association of the antibody with the antigen.

When using mean here that the term "koff" refers to the rate constant for the dissociation of antibodies from complex antibody/antigen.

When using mean here that the term "KD" refers to the equilibrium dissociation constant of the interaction of antibody-antigen.

Methods of preventing or treating visceral pain

There are disclosed methods is the avoiding and/or treatment of visceral pain and/or symptoms of visceral pain and medicine for the prevention and/or treatment of visceral pain and/or one or more than one symptom of visceral pain from the individual.

In some embodiments according to the invention, a method for prevention and/or treatment of visceral pain and/or one or more than one symptom of visceral pain in an individual, comprising peripheral introduction to the individual an effective amount of an antibody antagonist against CGRP.

In other embodiments according to the invention, a method for reducing intensity, controlling, reducing incidence or delay the development or progression of visceral pain and/or one or more than one symptom of visceral pain in an individual, comprising peripheral introduction to the individual an effective amount of an antibody antagonist against CGRP.

In some embodiments according to the invention proposed the use of antibody-antagonist against CGRP for the manufacture of a medicine for the prevention and/or treatment of visceral pain and/or one or more than one symptom of visceral pain, where the drug is made for peripheral injection or where the drug is injected perifericheskie.

In other embodiments according to the invention proposed an antibody antagonist against CGRP for use in the prevention and/or treatment of visceral pain and/or symptoms of visceral pain, where the specified antibody receive for peripheral introduction the treatment or where the specified antibody is administered perifericheskie.

In other embodiments according to the invention proposed the use of antibody-antagonist against CGRP for the manufacture of drugs to reduce the intensity, controlling, reducing incidence or delay the development or progression of visceral pain and/or symptoms of visceral pain, where the drug is made for peripheral injection or where the drug is injected perifericheskie.

In some embodiments, the individual is preferably a mammal, such as a domestic animal such as a horse, cat or dog, or farm animals such as sheep, cow or pig. Most preferably, the mammal is a human.

In some embodiments, the drug and/or antibody antagonist against CGRP made for oral, sublingual, transbukkalno, local, rectal, inhalation, transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intracardial, intraosseous, intradermal, intraperitoneal, transmucosal, vaginal injection, injection into the vitreous body, intra-articular, periarticular, Central, local or cutaneous injection.

In some embodiments, the drug is made on the peripheral I introduction to, and/or during and/or after the development of visceral pain.

In some embodiments the antibody is an antagonist against CGRP has with the introduction of peripheral action. In some embodiments the antibody is an antagonist against CGRP does not enter the Central, spinal or intrathecal.

In some embodiments of the visceral pain is associated with and/or caused by disease, such as, for example, functional bowel disorder (PRK) or inflammatory bowel disease (IBD). In embodiments where the visceral pain is associated with PRK, PRK may represent, for example, without limitation, gastroesophageal reflux, dyspepsia, irritable bowel syndrome (IBS) or syndrome, functional abdominal pain (SFAB). Most preferably the disease is an IBS. In embodiments where the visceral pain associated with inflammatory bowel diseases, IBD may represent, for example, without limitation, Crohn's disease, REIT or ulcerative colitis. Other types of visceral pain include the pain associated with, for example, cancer, renal colic, dysmenorrhea, cystitis, including interstitial cystitis (IC), surgical interventions associated with intestinal obstruction, menstruation, childbirth, menopause, bone fracture, diverticulitis, peritonitis, pericarditis, hepatitis, appendicitis, colitis, cholecystitis, what endometriosis, chronic and/or acute pancreatitis, myocardial infarction, renal pain, pleural pain, prostatitis, pelvic pain and trauma.

In some embodiments of the methods and applications of the invention can be designed to reduce the intensity of visceral pain and/or one or more than one symptom associated with visceral pain, the individual with PRK, IBD or CI.

Diagnosis or assessment of visceral pain are well known in the art. The assessment may be conducted on the basis of methods known in the art, such as the description of the pain patient using different scales for pain assessment. See, for example, Katz et al., Surg Clin North Am., 1999, 79(2):231-52; Caraceni et al. J Pain Manage Option, 2002, 23(3):239-55. For example, for pain evaluation and assessment of response to treatment can be used verbal descriptive scale (verbal descriptor scale (VDS), a visual analogue scale (visual analog scale (VAS)), scale for assessment of pain in the hospital. Prince Henry Prince Henry Hospital Pain Scale (PHHPS)), numeric rating scale (numeric rating scale (NRS) and the scale for assessment of pain by the choice of images with different facial expressions (Faces Pain Scale) and their variants. There are also a widely used scale to assess the course of the disease, such as an indicator of the severity of functional bowel disorders (Functional Bowel Disorder Severity Index (FBDSI)) (Drossman et al., 1995, Digestive Diseases and Sciences 40(5):986-995) and si is the topic of assessing the severity of IBS (IBS Severity Scoring System (Francis et al., 1997, Aliment Oil Displayed Pure Pharmacol Ther., 11(2):395-402). Such scales can be used to assess response to treatment.

In some embodiments, the decrease in the intensity, controlling, reducing incidence or delay the development or progression of pain associated with PRK, and/or symptoms of pain associated with PRK, evaluate one or more of the FBDSI, VDS, VAS, PHHPS, NRS and scales for pain assessment selection of images with different facial expressions. In another embodiment, the decrease in the intensity, controlling, reducing incidence or delay the development or progression of pain associated with IBS, and/or symptoms of pain associated with IBS, evaluate one or more of the system for assessing the severity of IBS, VDS, VAS, PHHPS, NRS and scales for pain assessment selection of images with different facial expressions.

In some embodiments, the decrease in the intensity, controlling, reducing incidence or delay the development or progression of pain associated with IC, and/or symptoms of pain associated with IC, evaluate one or more of the VDS, VAS, PHHPS, NRS and scales for pain assessment selection of images with different facial expressions.

Antibody antagonists against CGRP

In some embodiments the antibody is an antagonist against CGRP binds to CGRP. Preferably, the antibody is an antagonist against CGRP binds to CGRP and inhibits the ability of CGRP to contact the CGRP receptor. In some embodiments the antibody and tagonist against CGRP associated with human CGRP, and CGRP rodent, preferably human and mouse CGRP. More preferably, the antibody binds to human CGRP. In preferred embodiments the antibody is an antagonist against CGRP binds to human α-CGRP or human α-CGRP and/or β-CGRP. Most preferably, the antibody is an antagonist against CGRP is an antibody, demonstrating any one or more of the following functional properties: (a) binds to CGRP; (b) blocks the binding of CGRP to its receptor (the receptor); (C) blocks or reduces activation of CGRP receptors, including activation with formation of cyclic adenosine monophosphate (camp); (g) inhibits, blocks, inhibits or reduces biological activity of CGRP, including subsequent metabolic pathways, mediated by CGRP signaling, such as the binding of receptors and/or the induction of cellular response to CGRP; (d) provides for the prevention, reduction intensity or treatment of any aspect of visceral pain; (e) increases the clearance of CGRP; and (g) inhibits (reduces) the synthesis, the formation and release of CGRP.

In some embodiments the antibody is an antagonist against CGRP binds to a fragment of CGRP, more preferably a fragment of CGRP and full-CGRP. Preferably, the antibody is an antagonist against CGRP binds to the C-terminal region or fragment of CGRP. C-terminal about the art or a fragment of CGRP preferably include amino acids 19-37, or 25-37, or 29-37, or, alternatively, amino acids 30-37, or, alternative, amino acids 31-37 CGRP. In another embodiment of the C-terminal region or fragment of CGRP preferably include amino acids 32-37, most preferably amino acids 33 to 37 CGRP. Preferably, CGRP is an α-CGRP or β-CGRP, more preferably human CGRP or CGRP rodent, more preferably a human or rat CGRP, more preferably human CGRP, more preferably human α-CGRP or β-CGRP, most preferably human α-CGRP.

In some embodiments the antibody is an antagonist against CGRP associated with specific amino acid sequence GSKAF (SEO ID NO:39). Preferably, the sequence GSKAF (SEQ ID NO:39) CGRP is an epitope which binds an antibody antagonist against CGRP.

In some embodiments proposed antibody antagonist against CGRP-specific binding to the epitope defined by amino acids with G33 on F37 CGRP. Antibody antagonist against CGRP may be specific to contact the epitope defined by the amino acid sequence GSKAF (SEQ ID NO:39). In some embodiments according to the present invention proposed the use of such antibodies in applications and methods defined in various aspects of the present invention.

In some embodiments the antibodies of the antagonist against CGRP inhibits or prevents activation of the CGRP receptor. Preferably, the antibody against CGRP has an average inhibitory concentration (IC50) from about 0,0001 (0.1 nm) to about 500 μm. In some preferred embodiments of the IC50is from about 0,0001 μm to about 250 μm, 100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 250 nm, 100 nm, 50 nm, 20 nm, 15 nm, 10 nm, 5 nm, 1 nm or 0.5 nm, as measured in the analysis of binding in vitro. In some more preferred embodiments of the IC50is less than about 500 PM, or about 100 PM, or about 50 PM, as measured in the analysis of binding in vitro. In another more preferred embodiment of the IC50approximately 1.2 nm and 31 nm.

In some embodiments of the used antibody antagonist against CGRP able to compete with the antibody described above for binding to CGRP, or with a fragment of CGRP, or with a fragment of CGRP and with full CGRP, preferably the C-terminal region or fragment of CGRP. In preferred embodiments of the C-terminal region or fragment of CGRP include amino acids 19-37, 25-37, 29-37, 30-37 or 31-37 CGRP. In another embodiment of the C-terminal region or fragment of CGRP preferably include amino acids 32-37, most preferably 33 to 37 CGRP.

In some embodiments the antibody is an antagonist against CGRP binds to CGRP, area CGRP or a fragment of CGRP with a binding affinity of (KDfrom approximately 0,00001 microns (0.01 nm) to about 500 μm. In some embodiments the binding affinity of (KD) is from about 0,00001 μm to about 250 μm, 100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 250 nm, 100 nm, 50 nm, 20 nm, 15 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 1 nm to 0.05 nm or 0.01 nm, as measured in the analysis of binding in vitro. In some embodiments the binding affinity of (KD) is less than about 500 PM, 100 PM, 50 PM or 10 PM, as measured in the analysis of binding in vitro. In other preferred embodiments the binding affinity of (KD) is roughly 0.04 nm or 16 nm.

Antibody antagonist against CGRP used in the present invention may be selected from the group including monoclonal antibodies, polyclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, Fv, Fc, ScFv, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single-chain (ScFv) antibodies, their mutant variants, fused proteins containing part of the antibody (such as a single domain antibody), humanized antibodies, and any other modified form of the molecules of the immunoglobulin that contains the site of the recognition of the desired antigen specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibody antagonist against CGRP may have produced the circulation of the mouse, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments the antibody is an antagonist against CGRP can be humanized, but more preferably is a human. In some embodiments the antibody is an antagonist against CGRP is selected. In some embodiments the antibody is an antagonist against CGRP is essentially pure. Where the antibody is an antagonist against CGRP is a fragment of the antibody, the fragment preferably retains the functional properties of the original antibody, i.e. the binding of CGRP and/or antagonistic activity against CGRP, as described in the functional properties described above.

Examples of antibody antagonists against CGRP is known in the art. Therefore, according to preferred embodiment of the present invention the antibody antagonist against CGRP used in the present invention, preferably is an antibody against CGRP, revealed, in General or specifically in any of (1) WO 2007/054809, (2) WO 2007/076336, (3) Tan et al., Clin. Sci. (Lond). 89:565-73, 1995, (4) Sigma (Missouri, US), product serial number C (clone #4901), (5) PIourde et al., Peptides 14:1225-1229, 1993, or containing or consisting of:

a) a fragment of the indicated antibodies (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv, and so forth);

b) the light chain of the indicated antibodies;

C)the heavy chain of the specified antibody;

g) one or more than one variable region of the light chain and/or heavy chains of the indicated antibodies;

d) one or more than one CDR (one, two, three, four, five or six CDRs) of the specified antibody;

(e) CDR H3 of the heavy chain of the indicated antibodies;

g) CDR L3 light chain of the indicated antibodies;

C) three light chain CDR specified antibody;

I) three CDRs of the heavy chain of the indicated antibodies;

K) three CDRs of a light chain and three CDRs of the heavy chain of the indicated antibodies;

l) any one or more of (a)-(K).

In some embodiments the antibody is an antagonist against CGRP is an antibody G2 or antibody G1. According to the most preferred embodiment of the present invention used antibody antagonist against CGRP is an antibody G1 against CGRP, as specifically disclosed in PCT publication patent application no WO 2007/054809, or antibody containing its variants shown in Table 6 WO 2007/054809, also including antibodies that are functionally equivalent to G1, that is, containing conservative substitutions of amino acid residues or one or more than one amino acid deletions or insertion, no significant impact on their functional properties, such as binding to CGRP or antagonistic activity against CGRP, and variants with increased or decreased activity and/or binding. When is ispolzovanie here the terms "G1" and "antibody G1" are used interchangeably to refer to antibodies, obtained using the expression vectors having custody of the rooms ATS MOUTH-6867 and ATSS MOUTH-6866, as disclosed in the application WO 2007/054809. Functional properties of antibody G1 described in PCT patent application no PCT/IB2009/050849 and PCT/IB2009/050852, filed March 3, 2009, and fully incorporated here by reference.

According to another embodiment of the present invention the antibody antagonist against CGRP contains or consists of a polypeptide selected from:

(a) antibody G1 or its variants shown in Table 6 WO 2007/054809; (b) a fragment or region of antibody G1 or its variants shown in Table 6 WO 2007/054809; (b) the light chain of antibody G1 or its variants shown in Table 6 WO 2007/054809; (g) the heavy chain of antibody G1 or its variants shown in Table 6 WO 2007/054809; (d) one or more than one variable region of the light chain and/or heavy chain of antibody G1 or its variants, shown in Table 6 WO 2007/054809; (e) one or more than one CDR (one, two, three, four, five or six CDRs) of antibody G1 or its variants shown in Table 6 WO 2007/054809; (g) CDR-H3 of the heavy chain of antibody G1 or its variants shown in Table 6 WO 2007/054809; (C) CDR L3 light chain of antibody G1 or its variants shown in Table 6 WO 2007/054809; (and) three light chain CDR of antibody G1 or its variants, shown in Table 6 WO 2007/054809; (K) three CDRs of the heavy chain of antibody G1 or its VA is Ianto, shown in Table 6 WO 2007/054809; (K) three CDRs of a light chain and/or the three CDRs of the heavy chain of antibody G1 or its variants shown in Table 6 WO 2007/054809; and (m) antibody, containing any of the (b)-(l). According to the invention also suggested polypeptides containing any one or more of the above. In some embodiments at least one, two, three, four, five, or six CDR of at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one, two, three, four, five, or six CDR G1 or its variants shown in Table 6 WO 2007/054809. The definition of CDR-plots included in the competence of the specialist in the art. Assume that in some embodiments the CDR can be a combination of CDRs by Kabat and Chothia. In some embodiments CDR represent a CDR according to Kabat. In other embodiments CDR represent CDR by Chothia.

Antibody antagonist against CGRP preferably contains or consists of a fragment or antibody G1 (for example, Fab, Fab', F(ab')2, Fv, Fc, ScFv, etc.) or its variants shown in Table 6 WO 2007/054809. Preferably, the specified fragment or region possesses functional properties of the antibody antagonist against CGRP, such as, for example, binding activity against CGRP and/or antagonistic activity against CGRP, and contains or consists of state is t from one or more of: (1) the light chain; (2) the heavy chain; (3) a fragment containing one or more than one variable region light chain and/or heavy chain; and (4) one or more than one CDR of the light chain and/or heavy chain antibodies G1.

In some embodiments the antibody is an antagonist against CGRP contains a variable region light chain (LCVR) containing a peptide with a sequence selected from the group consisting of SEQ ID nos:28-32, and/or variable region of the heavy chain (HCVR) containing a peptide with a sequence selected from the group consisting of SEQ ID nos:34-38, according to the patent application WO 2007/076336.

More preferably, the antibody is an antagonist against CGRP polypeptide contains L. CVR with SEQ ID NO, as shown in Table 1 of patent application WO 2007/076336, and further comprises a HCVR polypeptide with SED ID NO, as shown in Table 1 of patent application WO 2007/076336.

According to another embodiment of the invention used antibody antagonist against CGRP contains the CDR of the light chain (CDRL) selected from the group consisting of SEQ ID nos:8-13, and/or CUR heavy chain (CDRH) selected from the group consisting of SEQ ID NO:14-22 patent application WO 2007/076336.

Methods of preparation and selection of antibody antagonists against CGRP from the application WO 2007/076336 and data showing their binding to CGRP and antagonistic properties against CGRP described in the application WO 2007/076336.

In some embodiments the antibody is an antagonist against CGRP to use is isawanya in the present invention contains a domain V H, amino acid sequence which is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:1 or SEQ ID NO:19, is presented here.

In some embodiments the antibody is an antagonist against CGRP contains a domain VL, amino acid sequence which is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least priblisitelno%, at least about 99%, or 100% identical to SEQ ID NO:2 or SEQ ID NO:20, is presented here.

In some embodiments the antibody is an antagonist against CGRP contains a domain VHand domain VL, amino acid sequence which is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical represented here by SEQ ID NO:1 and 2, respectively, or SEQ ID NO:19 and 20, respectively.

In some embodiments the antibody is an antagonist against CGRP contains a domain VH, amino acid sequence which is at least 90% identical to SEQ ID NO:1, and the domain VL, amino acid sequence which is at least 90% identical to SEQ ID NO:2 represented here.

Alternatively, an antibody antagonist against CGRP may contain domain VH, amino acid sequence which p is at least 90% identical to SEQ ID NO:19, and domain VL, amino acid sequence which is at least 90% identical to SEQ ID NO:20, is presented here.

In some embodiments the antibody is an antagonist against CGRP contains at least one CDR selected from the group consisting of: (a) CDR-H1 as set forth in SEQ ID NO:3, 21, 33, 34, 36 or 37; (b) CDR H2 as set forth in SEQ ID NO:4, 22, 35 or 38; (b) CDR H3, as set forth in SEQ ID NO:5 or 23; (g) CDR-L1 as set forth in SEQ ID NO:6 or 24; (e) CDR L2, as set forth in SEQ ID NO:7 or 25; (e) CDR L3, as set forth in SEQ ID NO:8 or 26; and (g) variants CDR L1, CDR L2 and CDR-H2, as shown in Table 6 WO 2007/054809.

In some embodiments of the constant region of the heavy chain of the antibody antagonist against CGRP may be a constant region of any type, such as IgG, IgM, IgD, IgA and IgE; and any isotype, such as IgG1, IgG2, IgG3 and IgG4.

In some embodiments the antibody is an antagonist against CGRP contains a heavy chain obtained using the expression vector with the registration number in ATSC MOUTH-6867. More preferably, the antibody is an antagonist against CGRP contains a light chain, obtained using the expression vector with the registration number in ATSC MOUTH-6866. In some embodiments the antibody is an antagonist against CGRP contains a full amino acid sequence of the heavy chain of antibody G1 (including modified IgG2, as described here), shown in SEQ ID NO:11, with concave is lysine or without him. Antibody antagonist against CGRP also includes antibody without terminal lysine of the heavy chain, as is usually the part of the antibody is lost in the process of obtaining them. In some embodiments the antibody is an antagonist against CGRP contains a full amino acid sequence of the light chain of the antibody G1 shown in SEQ ID NO:12. C. some embodiments the antibody is an antagonist against CGRP contains a full amino acid sequence of the heavy chain of the antibody G2 shown in SEQ ID NO:29. In some embodiments the antibody is an antagonist against CGRP contains a full amino acid sequence of the light chain of the antibody G2 shown in SEQ ID N0:30. In some embodiments the antibody is an antagonist against CGRP produced using expression vectors with registration numbers in ATSC MOUTH-6867 and MOUTH-6866.

In some embodiments the antibody is an antagonist against CGRP for use in the present invention is an antibody antibody G1 or G2 defined here. In preferred embodiments the antibody is an antagonist against CGRP for use in the present invention is an antibody G1 or its antigen-binding fragment.

According to other embodiments of the invention the antibody is an antagonist against CGRP contains a modified constant region, such as described in WO 2007/054809. PR is doctitle, modified constant region immunologically inert, including partial immunological inertness, so that it does not lead to complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), does not activate the microglia. Preferably, one or more of these activities modified constant region is reduced. Most preferably, the constant region is modified, as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT application no PCT/GB99/01441; and/or the application for patent of great Britain No. 8809951.8. In some embodiments the antibody is an antagonist against CGRP contains a constant region of the heavy chain of human lgG2, containing the following mutations: A330, R at S330, S331 (numbering of amino acids relative to the sequence lgG2 wild type). Eur. J. Immunol., 1999, 29:2613-2624.

Methods of preparation and selection of antibody antagonists against CGRP from the application WO 2007/054809 and data showing their binding to CGRP and antagonistic properties against CGRP described in the application WO 2007/054809. Sequence SEQ ID NO:1-14 said application presented here as SEQ ID NO:1-14, respectively.

Dose and introduction

In some embodiments the antibody is an antagonist against CGRP is administered perifericheskie, for example, from about one times to about 7 times per week, more preferably from the roughly one times to about four times a month, more preferably from about one times to about six times in 6 months, more preferably from about once to about twelve times a year. Preferably, the antibody is an antagonist against CGRP is administered perifericheskie frequency selected from: about once a day, once in two, three, four, five or six days, once a week, once in two weeks, every three weeks, once a month, once in two months once in three months once in every four months, once in five months, once every six months, once in seven months, once in eight months, once in nine months, one time in ten months, once in eleven months or once a year. According to preferred embodiments the antibody is an antagonist against CGRP is administered by way of introduction, selected from one or more of the oral, sublingual, transbukkalno, local, rectal, inhalation, transdermal, subcutaneous, intravenous, intraarterial, or intramuscular, intracardial, intraosseous, intradermal, intraperitoneal, transmucosal, vaginal injection, injection into the vitreous body, transdermal, intra-articular, periarticular, or local injection.

what according to another embodiment of the present invention, the drug is made for peripheral injection of from about one times to about 7 times a week, more preferably from about once to about four times a month, more preferably from about one times to about six times in 6 months, more preferably from about once to about twelve times a year. Preferably, the drug is manufactured for peripheral injection frequency selected from: about once a day, once in two, three, four, five or six days, once a week, once in two weeks, every three weeks, once a month, once in two months once in three months once in every four months, once in five months, once every six months, once in seven months, once in eight months, once in nine months, one time in ten months, once in eleven months or once a year. According to preferred embodiments, the drug is made for the peripheral introduction by way of introduction, selected from one or more of the oral, sublingual, transbukkalno, local, rectal, inhalation, transdermal, subcutaneous, intravenous, intraarterial, or intramuscular, intracardial, intraosseous, intradermal, intraperitoneal, transmucosal is about, vaginal injection, injection into the vitreous body, transdermal, intra-articular, periarticular, or local injection.

According to another embodiment of the present invention the concentration of the antibody ranges from about 0.1 to about 200 mg/ml, preferably approximately 0,5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml +/- 10% tolerance, most preferably about 50 mg/ml

According to another embodiment of the present invention, the drug is made for peripheral injection when the antibody concentration is from 0.1 to 200 mg/kg body weight, preferably approximately 0,5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/kg body weight +/- 10%tolerance most preferably about 10 mg/kg

According to a preferred embodiment of the present invention the antibody antagonist against CGRP has a half-life in vivo more about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208 or 210 days +/- 1 day, more preferably more than about 7, 8, 9, 10, 11, or 12 m the months.

Preferably, the antibody is an antagonist against CGRP has a half-life in vivo than 6 days.

According to another preferred embodiment of the present invention, the drug and/or antibody antagonist against CGRP does not affect the Central nervous system and/or cognitive disorder. Preferably, the drug and/or antibody antagonist against CGRP does not lead to any one or more of the following; amnesia, confusion, depersonalization, gipestesia, abnormal thinking, trism, vertigo, akathisia, apathy, ataxia, perioral paresthesia, CNS stimulation, emotional lability, euphoria, hallucinations, aggressiveness, hyperesthesia, hyperkinesia, hypotension, disturbances of coordination, increase libido, manic reaction, myoclonus, neuralgia, neuropathy, psychosis, epileptic seizure, abnormal speech, stupor, suicidal thinking, dizziness, drowsiness, insomnia, anxiety, tremor, depression, or paresthesias. Most preferably, the drug and/or antibody antagonist against CGRP does not lead to the breakdown of coordination or attention.

According to another embodiment of the present invention, the drug and/or antibody antagonist against CGRP does not lead to dysfunction of the respiratory system the volumes, liver, kidney or gastrointestinal tract.

According to another embodiment of the present invention, the drug and/or antibody antagonist against CGRP does not affect the physical and/or psychological dependence. Preferably, the drug and/or antibody antagonist against CGRP shows no affinity for opiate, benzodiazepine, phencyclidine (PCP) receptors or receptors N-methyl-D-aspartic acid (NMDA), or is not a Central nervous system stimulant, or has no sedative or euphoric effect.

In some embodiments the antibody is an antagonist against CGRP in the introduction provides a reduction in intensity, controlling, reducing incidence or delay the development or progression of Central pain perception.

In other embodiments, the effect of the antibody antagonist against CGRP equals and/or exceeds the effects of NSAIDs and/or opioids in the same models of visceral pain. In one embodiment the antibody is an antagonist against CGRP effective in the treatment of individuals with refractory pain.

According to other embodiments of the present invention proposed the use or method according to any other aspect of the invention, where the antibody is an antagonist against CGRP is administered separately, sequentially or simultaneously in combination with one or more other pharmacologically and the active compound or agent, preferably the compound or agent, useful for the treatment of visceral pain. Preferably, the additional(s) agent(s) selected is/are selected from one or more of:

1) opioid analgesic, e.g. morphine, heroin, hydromorphone, Oxymorphone, Levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, Dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorfina, naloxone, naltrexone, buprenorphine, butorphanol, nalbufina or pentazocine;

2) non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indometacin, Ketoprofen, Ketorolac, meclofenamic acid, mefenamovoy acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindaka, tolmetin or zomepirac, inhibitors of cyclooxygenase-2 (COX-2), celecoxib, rofecoxib, meloxicam, JTE-522, L-745337, NS398 or their pharmaceutically acceptable salts;

3) barbiturates sedatives, such as amobarbital, aprobarbital, butabarbital, butalbital, mephobarbital, metharbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutal, thiamylal or thiopental or their pharmaceutically acceptable salts;

4) benzodiazepine, possessing the th sedative effect, for example hlordiazepoksida, clorazepate, diazepam, flurazepama, lorazepam, oxazepamum, temazepam, or triazolam or their pharmaceutically acceptable salts;

5) Hi-antagonist, having a sedative action, e.g. diphenhydramine, pyrilamine, prometazina, chlorpheniramine or chlorcyclizine or their pharmaceutically acceptable salts;

6) sedatives such as glutethimide, meprobamate, methaqualone or dichloralphenazone or their pharmaceutically acceptable salt;

7) funds, relaxing skeletal muscles, such as baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, Methocarbamol or orphenadrine or their pharmaceutically acceptable salts;

8) antagonist of NMDA receptors, e.g. dextromethorphan ((+)-3-hydroxy-M-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-M-methylmorphinan), ketamine, memantine, pyrroloquinoline or CIS-4-(phosphonomethyl)-2-piperidinecarboxylic acid or their pharmaceutically acceptable salts;

9) alpha-adrenergic tools, such as doxazosin, tamsulosin, clonidine or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydrothieno-2-yl)-5-(2-pyridyl)-hintline;

10) tricyclic antidepressant such as desipramine, impramine, amitriptyline or nortriptyline;

11) anti-convulsants, such as carbamate is on or valproate;

12) antagonist tachykinin (NK), in particular an antagonist of NK-3, NK-2 or NK-1, e.g. (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-geragera-9-methyl-5-(4-were)-7H-[1,4]thiazocine[2,1-d][1,7]naphthiridine-6-13-dione (TAK-637), 5-[[(2R,38)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-forfinal)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazole-3-one (MK-869), linepithema, capitant or 3-[[2-methoxy-5-(triptoreline)phenyl]methylamino]-2-phenylpiperidine (2S,3S);

13) an antagonist of muscarinic receptors, such as oxybutynin, tolterodine, propiverine, chloride tropica or darifenacin;

14) of the COX-2 inhibitor such as celecoxib, rofecoxib or valdecoxib;

15) non-selective inhibitor of COX (preferably with a protective effect on the stomach), such as nitrofluorene (HCT-1026);

16) analgesic, a derivative of aniline, in particular paracetamol;

17) neuroleptic such as droperidol;

18) agonist (such as resiniferatoxin) or antagonist (e.g capsazepine) vanilloid receptors;

19) beta-adrenergic tools, such as propranolol;

20) local anesthetic, such as meksiletin;

21) the corticosteroid, such as dexamethasone;

22) agonist or antagonist of serotonin receptors;

23) cholinergic (nicotinic) analgesic;

24) tramadol;

25) inhibitor PDE-V, such as sildenafil vardenafil or taladafil;

26) alpha-2-Delta ligand such as gabapentin or pregabalin;

27) cannabinoid; and

28) antidepressant such as amitriptyline (Elavil®), trazodone (Desyrel®) and imipramine (Tofranil®), or anticonvulsants, such as phenytoin (Dilantin®) or carbamazepine (Tegretol®).

According to another aspect of the present invention proposed a pharmaceutical composition for prevention and/or treatment of visceral pain and/or symptoms of visceral pain or to reduce the intensity, controlling, reducing incidence or delay the development or progression of visceral pain and/or symptoms of visceral pain from the individual containing the antibody antagonist against CGRP and a pharmaceutically acceptable carrier and/or excipient made for peripheral injection.

Sets

According to another aspect of the present invention proposed a kit comprising a pharmaceutical composition as defined above, and instructions for peripheral introducing an effective quantity of a specified pharmaceutical composition to an individual for the prevention and/or treatment of visceral pain and/or symptoms of visceral pain or to reduce the intensity, controlling, reducing incidence or delay the development or progression of visceral pain and/or symptoms of visceral pain.

Set in locate one or more than one container, containing antibody antagonist or polypeptide against CGRP described herein, and instructions for use in accordance with any of the methods and uses according to the invention. The kit may additionally include a description of the choice of the individual suitable for treatment based on the detecting of the presence of visceral pain or the risk of visceral pain in an individual.

How peripheral the introduction of the pharmaceutical composition can include information about the dose scheme introduction and routes of administration for the intended treatment.

Mutatis mutandis, the preferred features of each aspect of the invention applies equally to every other aspect.

EXAMPLES

The following examples are for illustrative purposes only, and imply that they in no way limit the scope of the present invention. Indeed, on the basis of the above description specialists in the art will be apparent various modifications of the invention included in the scope of the attached claims, in addition to the modifications shown and described herein.

Example 1: a Model of visceral pain

This example shows the effect of introducing antibody antagonist against CGRP in a model of visceral pain.

Shown that patients with IBS reduced threshold visceral coustical the surface to stretch the colon and rectum, and that it is highly correlated with symptoms of visceral pain (Delafoy et al., 2006). Stretching of the colon and rectum after TNBS-induced colitis in rats is an animal model used by many researchers to study the mechanisms of visceral hypersensitivity (Gay et al, 2006, Delafoy et al., 2006, Adam et al., 2006). In this example, the model of TNBS-colitis in rats was used to evaluate the effect of antibodies against CGRP, blocking the functional activity of CGRP. In this model, as in studies of IBS in humans, visceral pain threshold was measured in response to balloon distension of the colon.

During the night before surgery, rats were fed and then held them under anesthesia with ketamine (80 mg/ml)/xylazine (12 mg/ml) at a dose of 1 ml/kg laparotomy was Performed and a solution of TNBS (50 mg in a concentration of 1.5 ml/kg in 30% ethanol, "group introduction TNBS) or 30% solution of ethanol (group"simulation") was inserted in the proximal part of the colon 1 cm distal to the cecum. Group simulation was used as control without colitis. On the fifth day after the surgery group introduction TNBS were divided into two groups. One group was intravenously injected monoclonal antibody antagonist against CGRP 4901 (commercially available from Sigma (Missouri, US), product number C (clone #4901)) at a concentration of 10 mg/kg Other group was injected filler (PBS, 0.01% of Tween 20) as the negative is part of the control.

Before sedmimi days after surgery, rats again not fed during the night and then rats, which were administered TNBS, with loss of body weight not more than 11% was determined visceral pain threshold using a balloon stretching. In the distal part of the colon was injected latex balloon size 5 cm, connected to a catheter, positioning the base of the cylinder and 5 cm from the anus. To prevent displacement of the balloon catheter was fixed to the tail of the tape. After an adaptation period of 30 minutes, the balloon was inflated sequentially from 5 mm RT. Art. up to 80 mm RT. Art. at intervals of 30 seconds. Balloon distension was stopped upon reaching the threshold pressure at which the animals were taken typical of visceral pain in rodents pose, known as the alpha-position (repeated waves of contraction of the oblique muscles with the preload of the rear legs), and this pressure is recorded as visceral pain threshold.

Rats who carried out the procedure of the simulation, the threshold accounted for 36.8 +/- 2,6 (N = 5, mean +/- standard error) on day 7 after surgery (Fig. 1A). In rats that were administered TNBS and, on the 5th day, the antibody 4901 (10 mg/kg), the threshold accounted for 32.3 +/- 4,1 (N=9) on day 7 that were statistically significantly different (one-factor analysis of variance with a posteriori criterion for multiple comparisons of Dunnet) from the threshold on day 7 in rats that were administered the TNBS and on the 5th day, the filler (21,0 +/- 3,0, N=10) (Fig.1A). The effect of antibody 4901 was comparable with the effect of receptor antagonist CGRP CGRP 8-37 (Fig. 1B). This result demonstrated that the antibody antagonist against CGRP effectively and meaningfully aligned visceral pain threshold threshold group simulation, i.e. reversible pain in a model of visceral pain.

Example 2: analysis of the interaction and the analysis of binding

The interaction analysis was carried out at 25°C and at 37°C in the system Biacore 3000™, equipped coated with streptavidin (SA) sensor chip (Biacore AB, Uppsala, Sweden) using standard buffers Biacore for analysis (HBS-P or HBS-EP). N-LC-biotinylation human and rat α - and β-CGRP was immobilized in separate flow cells at low concentrations (typically 100 units of the response) with the formation of reaction surfaces, while the raw flow cell was used as a control channel. Received purified Fab fragments of antibody G1 and G2. Usually Fab-fragments were obtained in the form of a twofold serial dilution with a maximum concentration of 0.5 µm and brought in for 1 minute at a speed of 100 MCP/min at the time of dissociation to two hours. The surface was restored with a mixture of 50%.about. ethanol and 25 mm NaOH for Fab-fragment G1 and 2:1 vol./about. buffer Pierce Gentle Eiution Buffer/4 M NaCl for Fab-fragment G2. Make Fab-fragment is in repeated to demonstrate the reproducibility of the analysis. Answers binding was compared with two controls with the use of the software BiaEvaluation v. 4.0, and they were generally consistent with a simple model binding. The affinity was obtained as the ratio of the kinetic rate constants (KD=koff/kon).

Results for antibody G1 shown in Table 1 below. Antibody G1 is associated with human α - and β-CGRP with similar high affinity (KD=163 and 155 PM, respectively, in the analysis of "side by side" on the same chip at 37°C and time of dissociation of 20 minutes). The sequence of the human CGRP and CGRP cynomolgus macaque identical; therefore, data binding with human CGRP also applicable for CGRP cynomolgus macaque. G1 is also associated with rat CGRP, but with different affinity against α - and β-isoforms (KD=2.57 m nm <150 PM, respectively, at 37°C).

Table 1
KDantibody G1, measured at 25°C and 37°C, against human and rat CGRP
N-Biotin-CGRP on a chipTemperature (°C)kon(1/(M×C))koff(1/c)T1/2(h)KD(nm)
Human α-CGRP/α-CGRP cynomolgus macaque25of 1.86×1057,80×10-624,680,042
Human α-CGRP/α-37by 5.87×1053,63×10-5and 5.300,063
CGRP cynomolgus macaque
Human β-CGRP/β-CGRP cynomolgus macaque374,51×105<6,98×10-5was 2.76<0,155
Rat α-CGRP25to 5.08×1046,18×10-53,121,22
Rat α-CGRP37of 1.55×105of 3.99×10-40,48to 2.57
Rat β-CGRP375,16×105<a 7.85×10-52,450,152

Dissociation of Fab fragments of G1 from the human α - and β-CGRP is very slow. Accurate measurement of the speed of dissociation per se (koffit is impossible without monitoring phase dissociation for a very long time. According to the basic rule-of-thumb to determine the exact rate of dissociation is a need to reduce response binding during dissociation on at least 5%. However, monitoring of dissociation for a long time on Biacore prevents the base drift indicators, representing a particularly serious problem when working with low concentrations of molecules on the surface that is necessary for kinetic analyses. In this study, phase dissociation was observed within two hours for α-CGRP, but only for 20 minutes for β-CGRP. In the rate of dissociation of β-CGRP cannot be determined with the same accuracy as the rate of dissociation for α-CGRP. However, in the analysis of "side by side" on the same chip in identical conditions at the time of dissociation of 20 minutes, the kinetics of binding of G1 with human α-CGRP/α-CGRP cynomolgus macaque, human β-CGRP/β-CGRP cynomolgus macaque and cu is Sinim β-CGRP was almost the same (K D=150 PM at 37°C).

Results for antibodies G2 shown in Table 2 below. Antibody G2 is associated with mouse α-CGRP with greater affinity (KD=0,9 nm at 25°C) than with human α - and β-CGRP (Kβ=19 nm and 20 nm, respectively, at 25°C). Linking G2 with mouse β-CGRP in this analysis was not investigated, but in the analysis with opposite orientation with mouse β-CGRP G2 demonstrated binding properties comparable to the binding properties of the human α - and β-CGRP (data not shown).

Table 2
KDantibodies G2, measured at 25°C and 37°C, against human and rat CGRP
N-Biotin-CGRP on a chipTemperature (°C)kon(1/(M×c))koff(1/c)Tl/2(min)KD(nm)
Rat α-CGRP252,31×1052,14×10-453,980,9
Rat α-CGRP375,0×105 1,7×10-3to 6.803,4
Human α-CGRP256,03×1041,15×10-3of 10.0519,1
Human α-CGRP379,3×1043,9×10-32,9641,9
Human β-CGRP258,14×104of 1.62×10-37,13to 19.9

The analysis of binding was performed to measure the IC50antibody G1 against CGRP in the blockade of binding to human α-CGRP with CGRP1 receptor. Membranes (25 μg) cells SK-N-MC were incubated for 90 minutes at 25°C in incubation buffer (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 0.1% of bovine serum albumin (BSA) containing 10 PM of human α-CGRP, labeled125I, and various concentrations of antibodies against CGRP (0.015 nm to 33 nm) in a total volume of 1 ml Incubation was ended by filtering through a glass microfibre filter (GF/B, 1 μm), blocked with 0.5% polyethylenimine. Associated with the protein is radioactively was determined on the count of gamma radiation. Built curves dose-response and the values of Kiwas determined using the equation: Ki=IC50/(1+([ligand]/KD); where the equilibrium dissociation constant KDwhen binding to human α-CGRP with CGRP1-receptor on the cells SK-N-MC was 8 PM. For comparison, the values obtained IC50(relative to IgG molecules with affinity (KD) defined on Biacore, it was adjusted based on the number of binding sites (multiplying by 2, because Biacore were analyzed by Fab-fragments). The observed IC50(1.8 nm) was 23 times more ToDobserved on Biacore (42 PM) at the same temperature. This discrepancy reflects the possible lack of sensitivity of the assay of the binding.

Example 3: a Model of visceral pain

This example shows the effect of introducing antibody antagonist against CGRP in a model of visceral pain.

In this example, the model of interstitial cystitis in rats was used to evaluate the effect of antibodies against CGRP, blocking the functional activity of CGRP. In this model of visceral hypersensitivity was measured on the motility of the bladder in response to his irritation turpentine oil.

During cystometry female rats were kept under continuous urethane anesthesia. Maintained body temperature 37°C using a rectal probe, thermostatically United with g is a tree with controlled temperature. One group of rats (n=7) was intravenously injected monoclonal antibody antagonist against CGRP 4901 (commercially available from Sigrna (Missouri, US), product number C (clone #4901)) at a concentration of 10 mg/kg Other group (n=7) were injected filler (PBS, 0.01% of Tween 20) as a negative control.

Twenty-four hours after injection 4901 or filler rats spent anesthesia and transurethral catheterization of the bladder tube RE (outer diameter 1 mm), which allowed to fill the bladder of normal saline, with a speed of 0.05 ml/min (using a syringe pump). Proximal bladder tube was located T-shaped connection, allowing to monitor the pressure in the bladder pressure sensor. To determine the motility of bladder pressure monitoring and abbreviations was performed during the interval with a duration of 14 minutes (total 0,84 ml). After receiving the original cystometrogram conducted irritation of the bladder by introducing 0.5 ml of 50% turpentine oil for 1 hour. Then the bladder was trenirovki and subsequent analysis of the motility of the bladder performed right after (after 1 h) introduction of turpentine oil, after 3 h and 5 h after injection of turpentine oil.

In rats, which were injected antibody 4901 (10 mg/kg) 24 hours before held the eating of cystometrogram, there were fewer contractions of the bladder at all time points when measured in comparison with rats, which were injected filler (Fig.2). This result demonstrated that the antibody antagonist against CGRP effectively reduced the motility of the bladder in response to stimulation turpentine oil, that is reversible pain in a model of visceral pain.

The Deposit of biological material

The following materials have been deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA (ATCC).

MaterialNo. antibodiesRegistration number in ATSDate Deposit
pDb.CGRP.hFcGIHeavy chain G1MOUTH-6867July 15, 2005
pEb.CGRP.hKGILight chain G1MOUTH-6866July 15, 2005

Vector pEb.CGRP.hKGI is polynucleotide encoding the variable region of the light chain and the constant region of the light chain is a Kappa G1; and the vector of the pDb.CGRP.hFcGI is polynucleotide encoding the variable region of the heavy chain G1 and con tantou region of the heavy chain lgG2, containing the following mutations: AN on S330S331 (numbering of amino acids relative to the sequence lgG2 wild type; see Eur. J. immunol., 1999, 29:2613-2624).

These deposits were made in accordance with the provisions of the Budapest Treaty on the international recognition of the Deposit of microorganisms for purposes of patent procedure and adopted on the basis of normative documents (Budapest Treaty). This ensures the viability of the deposited culture for 30 years from the date of Deposit. ATCC will provide access to the deposited cultures and materials under the terms of the Budapest Treaty and in accordance with the agreement between Rinat Neuroscience Corp. and ATCC, which provides a permanent and unlimited free access to the offspring of the deposited culture after the issuance of the corresponding U.S. patent or after the publication of any relevant application for a U.S. patent or patent of another state, regardless of which of these conditions will be executed first, and provides access to the offspring for a person whose rights of access determined by the Commissioner of patents and trademarks U.S. under article 122, section 35, United States Code (35 USC Section 122) and its associated regulations, regulating the activities of the commissioners (including article 1.14 of section 37 of the Code of Federal regulations AK is s (37 CFR Section 1.14) with particular reference to 886 OG 638 in the Official Bulletin of the Patent office of the USA).

The assignee of this application agrees that if the deposited culture or materials shall perish or be lost or destroyed when cultivated in suitable conditions, they will be immediately replaced by another specimen of the same culture or material upon notification. Availability of the deposited material should not be construed as a license to practice of the invention in contravention of the rights granted by the authorities of any state according to their patent laws.

Below is the sequence of antibodies applicable in the various embodiments disclosed here.

Sequence antibodies

Amino acid sequence of the variable region of the heavy chain of antibody G1 (SEQ ID NO:1)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIRSESDA SATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGLAIQNYWGQG TLVTVSS

Amino acid sequence of the variable region of the light chain of antibody G1 (SEQ ID NO:2)

ElVLTQSPATLSLSPGERATLSCKASKRVTTYvswyqqkpgqaprlliygasnrylgip ARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIK

CDR H1 antibody G1 (extended CDR) (SEQ ID NO:3)

GFTFSNYWIS

CDR H2 antibody G1 (extended CDR, same as CDR according to Kabat) (SEQ ID NO:4)

EIRSESDASATHYAEAVKG

CDR H3 antibody G1 (SEQ ID NO:5)

YFDYGLAIQNY

CDR L1 antibody G1 (SEQ ID NO:6)

KASKRVTTYVS

CDR 12 antibody G1 (SEQ ID NO:7)

GASNRYL

CDR 13 antibody G1 (SEQ ID NO:8)

SQSYNYPYT

The nucleotide sequence of the variable region of the heavy chain and is tutela G1 (SEQ ID NO:9)

GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCC

CTGCGTCTGTCCTGCGCTGCTTCCGGTTTCACCTTCTCCAACTACTGGATCT

CCTGGGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCC

GTTCCGAATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTC

GTTTCACCATCTCCCGTGACAACGCTAAGAACTCCCTGTACCTGCAGATGAA

CTCCCTGCGTGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGAC

TACGGTCTGGCTATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTT

TCCTCC

The nucleotide sequence of the variable region of the light chain of antibody G1 (SEQ ID NO:10)

GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAA

CGTGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCT

GGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTT

CCAACCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGG1A

CCGACTTCACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTA

CTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAA

CTGGAAATCAAA

Full amino acid sequence of the heavy chain of antibody G1 (including modified lgG2, as described herein) (SEQ ID NO:11)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIR

SESDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGL

AIQNYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT

VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSN

TKVDKWERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDV

SHEDPEVQFNWVVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKE

YKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKT7PPMLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGK

Full amino acid sequence of the light chain of the antibody G1 (SEQ ID NO:12)

EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPRLUYGASNR YLGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Full nucleotide sequence of the heavy chain of antibody G1 (including modified lgG2, as described herein) (SEQ ID NO:13)

AAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCC

CTGCGTCTGTCCTGCGCTGCTTCCGGTTTCACCTTCTCCAACTACTGGATCT

CCTGGGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCC

GTTCCGAATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTC

G1TTCACCATCTCCCGTGACAACGCTAAGAACTCCCTGTACCTGCAGATGAA

CTCCCTGCGTGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGAC

TACGGTCTGGCTATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTT

TCCTCCGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCC

CGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTA

CTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGG

CGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAG

CAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCT

GCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAG

AGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGA

CCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCA

GAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCA

GAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAA

GACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGT

GCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTA

AGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGA

CCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCCCCATCCAGA

GAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTC

TATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAA

CAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTG

TATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTC

TCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGC

CTGTCCCTGTCTCCAGGAAAGTAA

Full nucleotide sequence of the light chain of the antibody G1 (SEQ ID NO:14)

GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAA

CGTGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCT

GGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTT

CCAACCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTA

CCGACTTCACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTA

CTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAA

ACTGGAAATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCA

TCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATA

ACTTCTATCCGCGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC

AATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC

ACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAA

CACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCAGTC

ACAAAGAGCTTCAACCGCGGTGAGTGCTAA

Comparison of amino acid sequences of human and rat CGRP (human α-CGRP (SEQ ID NO:15); human β-CGRP (SEQ ID NO:16): rat α-CGRP (SEQ ID NO:17) and rat β-CGRP (SEQ ID NO:18))

NH2-ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-CONH2(SEQ ID

NO:15)

NH2-ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-CONH2(SEQ ID

NO:16)

NH2-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-CONH2(SEQ ID

NO:17)

NH2-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKAF-CONH2(SEQ ID

NO:18)

Amino acid sequence of the variable region of the heavy chain of the antibody G2 (SEQ IP NO:19)

EVQLQQSGPELVKPGASVKMSCKASGYTFTSSVMHWVKQKPGQGLEWIGYIN

PYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAKGGNDGY

WGQGTTLTVSS

Amino acid sequence of the variable region of the light chain of the antibody G2 (SEQ ID NO:20)

EIVLTQSPTTMAASPGEKITITCSASSSISSIYLHWYQQKPGFSPKVLIYRASNLA SGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSTIPFTFGSGTKLEIK

CDR H1 antibodies G2 (CDR no Kabat) (SEQ ID NO:21)

SSVMH

CDR H2 antibodies G2 (extended CDR) (SEQ ID NO:22)

YINPYNDGTKYNEKFKG

CDR H3 antibodies G2 (SEQ ID NO:23)

GGNDGY

CDR L1 antibodies G2 (SEQ ID NO:24)

SASSSISSIYLH

CDR-L2 antibodies G2 (SEQ ID NO:25)

RASNLAS

CDR-L3 of the antibody G2 (SEQ ID NO:26)

QQGSTIPFT

The nucleotide sequence of the variable region of the heavy chain of the antibody G2 (SEQ ID NO:27)

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTC

AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTCTGTTATG

CACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT

AATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCA

CACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCAGCC

TGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAAAGGGGGTAACGATG

GCTACTGGGCCAAGGCACTACTCTCACAGTCTCCTCA

The nucleotide sequence of the variable region of the light chain of the antibody G2 (SEQ ID NO:28)

GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAG

AAGATCACTATCACCTGTAGTGCCAGCTCAAGTATAAGTTCCATTTACTTGC

ATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAAGTCTTGATTTATAGGGC

ATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGG

GACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACT

TACTACTGCCAGCAGGGTAGTACTATACCATTCACGTTCGGCTCGGGGACA

AAGTTGGAAATAAAA

Full amino acid sequence of heavy chain antibodiesG2 (not including the Fc domain) (SEQ IP NO:29)

EVQLQQSGPELVKFGASVKMSCKASGYTFTSSVMHWVKQKP(3QGLEWIQYIN

PYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSSLTSEDSAVYYCAKGGNDGY

WGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWN

SGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDK

KIVPRD

Full amino acid sequence of the light chain of the antibody G2 (SEQ ID NO:30)

E1VLTQSPTTMAASPGEKITITCSASSSISSIYLHWYQQKPGFSPKVLIYRASNLA SGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSTIPFTFGSGTKLE1KRA DAAPTVS1FPPSSEQLTSGGASVVCFLNNFYPRD1NVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Full nucleotide sequence of heavy chain antibodies G2 (not including the Fc domain) (SEQ ID NO:31)

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTC

AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTCTGTTATG

CACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATT

AATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCA

CACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCAGCC

TGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAAAGGGGGTAACGATG

GCTACTGGGGCCAAGGCACTACTCTCACAGTCTCCTCAGCCAAAACGACAC

CCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTRCCCAAACTAACTCCAT

GGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGT

GACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGT

CCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAG

CACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCA

GCACCAAGGTGGACAAGAAAATTGTGCCCAGGGAT

Panoreserve the nucleotide sequence of the light chain of the antibody G2 (SEQ ID NO:32)

GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAG

AAGATCACTATCACCTGTAGTGCCAGCTCAAGTATAAGTTCCATTTACTTGC

ATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAAGTCTTGATTTATAGGGC

ATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGG

GACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACT

TACTACTGCCAGCAGGGTAGTACTATACCATTCACGTTCGGCTCGGGGACA

AAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCAC

CATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGA

ACAACTTCTACCCCAGAGACATCAATGTCAAGTGGAAGATTGATGGCAGTG

AACGACAAAATGGTGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACA

GCACCTACAGCATGAGCAGCACCCTCACATTGACCAAGGACGAGTATGAAC

GACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCAT

CGTCAAGAGCTTCAACAGGAATGAGTGTTAA

CDR H1 antibody G1 (CDR no Chothia) (SEQ ID NO:33)

GFTFSNY

CDR H1 antibody G1 (CDRs according to Kabat) (SEQ ID NO:34)

NYWIS

CDR H2 antibody G1 (CDR no Chothia) (SEQ ID NO:35)

RSESUASA

CDR H1 antibodies G2 (extended CDR) (SEQ ID NO:36)

GYTFTSSVMH

CDR H1 antibodies G2 (CDR no Chothia) (SEQ ID NO:37)

GYTFTSS CDR H2 antibodies G2 (CDR no Chothia) (SEQ ID NO:38)

NPYNDG

Although the disclosed invention has been described with reference to various applications, methods, kits and compositions, it should be understood that various changes and modifications can be made without deviation from the uncovered here of the invention and the following claims. The above examples are given for better illustration of the disclosed invention, and imply that they do not limit the scope of the invention presented here. Although the present invention has been described through these typical embodiments, a specialist in the art will clear the opportunity sets of the variations and modifications of these typical embodiments without undue experimentation. All such variations and modifications are included in the scope of the present invention.

The section headings used here are given only for organizational purposes, and should not be construed as in any way limiting the described object of the present invention. It should be understood that before the temperatures, concentrations, intervals and so on, indicated in the present invention mean "approximately", i.e. minor and insignificant deviations from these values are included in the scope of the present invention. In this application the use of the singular includes the plural unless specifically stated otherwise. In addition, assume that the terms "include", "includes", "including", "include", "contain" and "contains" are not limiting. It should be understood that both the foregoing General description and the following detailed description is provided only as examples and explanation and do not limit the invention.

All the links provided here, including patents, patent applications, publications, manuals and the like, and the references are fully incorporated here by reference. If one or more of the incorporated literature and similar materials differs from this proposal or contrary to it, including, without limiting the tion, certain terms, use of terms, described techniques, or the like, the preferred variants, listed in this application.

In the foregoing description and Examples are described in detail specific embodiments of the invention, at best, from the point of view of the authors of the invention, the form. However, it should be understood that, regardless of the details of the above descriptions and examples of many possible ways of practical use of the invention, and the invention should be considered according to the appended claims and any equivalents.

1. The method of treatment of visceral pain and/or one or more than one symptom of visceral pain in an individual, comprising introducing a therapeutically effective amount of an antibody antagonist against a peptide linked to the genome of calcitonin (CGRP), an individual suffering from visceral pain, or individual with the risk of visceral pain, where the antibody is an antagonist against CGRP produced for peripheral injection.

2. The method according to p. 1, where visceral pain associated with functional bowel disorder (PRK).

3. The method according to p. 2, where PRK is selected from the group consisting of gastroesophageal reflux, dyspepsia, irritable bowel syndrome (IBS) syndrome and functional abdominal pain (SFAB).

4. The method according to p. 1, where visceral pain with asana with inflammatory bowel disease (IBD).

5. The method according to p. 4, where inflammatory bowel diseases selected from the group consisting of Crohn's disease, ileitis and ulcerative colitis.

6. The method according to p. 1 wherein the visceral pain is associated with renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis or pancreatitis.

7. The method according to p. 6, where visceral pain associated with interstitial cystitis (IC).

8. The method according to p. 1, where the antibody is an antagonist against CGRP binds to CGRP with KD50 nm or less (as measured by surface plasma resonance at 37°C) and/or has a half-life ofin vivoat least 7 days.

9. The method according to p. 1, where the antibody is an antagonist against CGRP-specific binds to the C-terminal region of CGRP.

10. The method according to p. 9, where the antibody is an antagonist against CGRP-specific recognizes the epitope defined by the sequence GSKAF (SEQ ID NO: 39).

11. The method according to p. 1, where the antibody against CGRP contains the variable domain of the heavy chain (VH), having the amino acid sequence shown in SEQ ID NO:1 or 19.

12. The method according to p. 1, where the antibody against CGRP contains variable domain light chain (VL), having the amino acid sequence shown in SEQ ID NO:2 or 20.

13. The method according to p. 1, where the antibody against CGRP contains at least one hypervariable segment (CDR) selected from the group consisting of:
a) hypervariable uchastka the heavy chain (CDR-H1), as set forth in SEQ ID NO:3, 21, 33, 34, 36 or 37;
b) hypervariable section 2 of the heavy chain (CDR-H2), as set forth in SEQ ID NO:4, 22, 35 or 38;
b) hypervariable section 3 of the heavy chain (CDR-H3), as set forth in SEQ ID NO:5 or 23;
g) hypervariable region 1, light chain (CDR-L1), as set forth in SEQ ID NO:6 or 24;
d) a hypervariable site 2 light chain (CDR-L2), as set forth in SEQ ID NO:7 or 25; and
e) a hypervariable site 3 light chain (CDR-L3), as set forth in SEQ ID NO:8 or 26.

14. The method according to p. 1, where the antibody against CGRP contains a domain VHhaving the amino acid sequence shown in SEQ ID NO:1, and the domain VLhaving the amino acid sequence shown in SEQ ID NO:2.

15. The method according to p. 1, where the antibody against CGRP produced using expression vectors with registration numbers in the American type culture collection (ATSS) MOUTH-6867 and/or MOUTH-6866.

16. The method according to p. 1, where the antibody against CGRP contains:
full amino acid sequence of the heavy chain of antibody G1 shown in SEQ ID NO:11, with C-terminal lysine or without it; and
full amino acid sequence of the light chain of the antibody G1 shown in SEQ ID NO:12.

17. The method according to p. 1, where the antibody against CGRP contains:
full amino acid sequence of the heavy chain of the antibody G2 shown in SEQ ID NO:29; and
full amino acid the complete sequence of the light chain of the antibody G2, shown in SEQ ID NO:30.

18. Pharmaceutical composition for treatment and/or prevention of visceral pain and/or symptoms of visceral pain from the individual containing the antibody antagonist against CGRP and a pharmaceutically acceptable carrier, made for peripheral administration.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to using an anti-CGRP antagonist antibody for producing a therapeutic agent for preventing and/or treating chronic pain and/or chronic pain symptoms, and wherein the therapeutic agent is produced for peripheral administration.

EFFECT: using the above therapeutic agent for peripheral administration is effective for preventing and/or treating chronic pain.

3 cl, 9 dwg, 2 tbl, 10 ex

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: what is presented is a method for producing a polyclonal antiserum which is immunospecifically bound to a biologically active parathyroid hormone (PTH) in a part of three or four N-terminal amino acid residues of (1-84) PTH. The first peptide antigen is introduced in a host animal other than a human. A titre of produced antibodies is monitored. Antiserum is extracted. At least one antibody is separated and recovered from antiserum by affine chromatography with using the second peptide antigen. It is followed by removing at least one antibody possessing specificity with respect to the peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH by affine chromatography with using the third peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH respectively. There is recovered polyclonal serum possessing binding affinity with respect to no more than the first three or four amino acid residues of the N-terminal part of PTH and possessing no affinity with respect to any amino acid residues in the fifth amino acid residue or after the fifth amino acid residue of the N-terminal of PTH.

EFFECT: determining the level of biologically active intact PTH in serum, plasma or cell culture medium.

6 cl, 4 dwg

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: biotechnology, in particular production of corticotropin-releasing factor 2 receptor (CRF2R) agonists.

SUBSTANCE: CRF2R agonist protein is produced as derivative of CRF, urocortine I, urocortine II urocortine III, sauvagine, urotensine I and other connate peptides by amino acid displacement in protein sequences. Obtained CRF2R agonist is useful in pharmaceutical composition for prophylaxis and treatment of disorders associated with CRF2R in host.

EFFECT: effective agent for prophylaxis and treatment of disorders modulated with CRF2R, such as muscle dystrophy.

25 cl, 3 tbl, 3 ex

FIELD: biotechnology, medicine, pharmacy.

SUBSTANCE: invention proposes peptides as agonists of corticotropin-releasing factor CRF2R prepared by solid-phase synthesis. Synthesized agonists of CRF2R are used as components of pharmaceutical composition used in prophylaxis or treatment of diseases modulated by CRF2R. Proposed invention provides enhancing effectiveness of therapeutic effect and avoiding by-side responses in treatment of disorders modulated by CRF2R. Invention can be used in synthesis of novel peptides possessing activity of agonists of corticotropin-releasing factor-2 (CRF2R).

EFFECT: improved method of synthesis, valuable medicinal properties of agonists.

3 tbl, 3 ex

The invention relates to medicine, namely to a method for detection of N-terminal Pro-MNE, recombinant N-terminal Pro-MNE, its use as a standard in detection of N-terminal Pro-MNE, as well as antibodies that recognize the recombinant N-terminal Pro-MNEs, and their reception

The invention relates to biotechnology, in particular to antibodies against Rtln, obtained by genetic engineering methods

FIELD: medicine.

SUBSTANCE: present group of inventions refers to medicine, namely to neurology and cardiology, and concerns treating a complication related to an aneurysmal subarachnoidal haemorrhage. That is ensured by determining the patient's plasma citrulline, and if observing a decrease thereof, an effective amount thereof is administered.

EFFECT: method provides effective treatment of such complication as the vasospasm by increasing the nitrogen oxide level.

16 cl, 1 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention represents a coronarodilating medication in the form of a tablet, which contains a solution of levomenthol in menthyl isovalerate (validol), isomalt, and calcium or magnesium stearate.

EFFECT: obtaining the coronarolitating medication by a method of direct pressing, which does not contain sugar, is characterised by dosage homogeneity, storage stability, high bioavailability.

2 cl, 11 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves simulating endothelial dysfunction in Wistar male rats by daily intraperitoneal introduction of L-nitro-arginine-methyl ester 25 mg/kg/day for 7 days. Endothelial dysfunction is corrected by intragastric introduction of an api-phytocomposition in the form of an aqueous suspension of honey, royal jelly, bee bread, Resveratrol in the ratio of 10:1:1:2. The composition is introduced in a dose of 200 mg/kg of an animal's body weight once a day for 7 days.

EFFECT: method provides synergetic endothelioprotective effects with no side effects observed when treating with synthetic preparations.

1 ex

FIELD: medicine.

SUBSTANCE: method involves simulating gestosis in Wistar rats by the daily intraperitoneal introduction of L-nitro-arginine-methyl ester 25 mg/kg for 7 days. That is followed by the intragastric introduction of azithromycin 30 mg/kg once a day in the setting of the model above from 14th to 20th day of pregnancy.

EFFECT: method provides the manifested endothelial dysfunction correction in the special pathogenesis environment in the pregnant females in a combination with eliminating acute infectious diseases or aggravated chronic infectious processes that can also lead to the formation of gestosis.

1 ex

FIELD: medicine.

SUBSTANCE: nitrogen oxide deficiency is simulated by the daily 7-day intraperitoneal introduction of N-nitro-L-arginine methyl ester 25 mg/kg into experimental Wistar male rats. The nitrogen oxide deficiency is corrected by the simultaneous 7-day intragastric introduction of a combination of tadalafil 0.09 mg/kg and L-norvaline 10 mg/kg once a day.

EFFECT: effective management of the nitrogen oxide deficiency stimulated as described above by the therapeutic combination of the preparations first identified to this effect.

1 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: endothelial dysfunction is induced in experimental Wistar male rats for 28 days by daily intraperitoneal introduction of N-nitro-L-arginine methyl ester (L-NAME) 12.5 mg/kg a day. An agent for dysfunction correction is a mixture of solutions of homoeopathic dilutions of angiotensin II receptor C-terminal fragment antibodies - C12, C30, C200. This mixture is introduced at the same time for 28 day intragastrically 2 times a day in a dose of 4.5 ml/kg.

EFFECT: method provides effective endothelial dysfunction correction with eNOS increase.

1 tbl

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to medicine, namely to angiology, and deals with treatment of diseases of peripheral vessels. For this purpose pharmaceutical composition, containing efficient amount of 11-deoxy-prostaglandin compound, characterised by general formula III, is introduced.

EFFECT: introduction of said composition ensures enhancement of peripheral blood flow, reduction of intraocular pressure, recovery of barrier function and increase of ATP level in endothelial cells with absence of impact on arterial pressure and heart rate.

17 cl, 8 ex, 5 tbl, 22 dwg

FIELD: medicine.

SUBSTANCE: endothelial dysfunction is experimentally simulated in male white Wistar rats by daily intraperitoneal introduction of the endothelial synthetase inhibitor L-nitro-L-arginine-methyl ester 25 mg/kg for 7 days A degree of dysfunction development is estimated by the relation of endothelium-dependent and endothelial-independent vasodilation. Endothelial dysfunction is corrected by daily inhalations of mixed helium and oxygen in the amount of 150 ml and 50 ml respectively, for 7 days.

EFFECT: method provides evident endothelial dysfunction correction.

1 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: endothelial dysfunction is induced in experimental Wistar male rats for 28 days by daily intraperitoneal introduction of N-nitro-L-arginine methyl ester (L-NAME) 12.5 mg/kg a day. An agent for dysfunction correction is a mixture of solutions of homoeopathic dilutions of tumour necrosis factor-alpha (TNF-α) antibodies - C12, C30, C200. This mixture is introduced at the same time for 28 day intragastrically 2 times a day in a dose of 4.5 ml/kg.

EFFECT: effective endothelial dysfunction correction.

1 tbl

FIELD: medicine.

SUBSTANCE: endothelial dysfunction is induced in experimental Wistar male rats for 28 days by daily intraperitoneal introduction of N-nitro-L-arginine methyl ester 12.5 mg/kg a day. An agent for dysfunction correction is a mixture of solutions of homoeopathic dilutions of interleukin-6 antibodies - C12, C30, C200; it is introduced at the same time for 28 day intragastrically 2 times a day in a dose of 4.5 ml/kg.

EFFECT: method provides effective endothelial dysfunction correction.

1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to lambertianic acid amides of formula (Ia, b), which have expressed analgesic activity and stimulating action, manifested in increase of motor and investigation activity of animals, absence of anxiety, etc. In formula I ; (Ib).

EFFECT: improvement of compound properties.

6 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: in general formula

A represents optionally substituted aminocarbonyl group -N-C(O)-, in which amino group can be substituted and substituents can be selected from hydrogen, C1-C5alkyl, possibly substituted with C1-C3alkoxy, C3-C6cycloalkyl, 5-6-membered heteroaryl, in which heteroatoms are selected from oxygen or nitrogen; aryl, selected from phenyl, possibly substituted with hydroxy, C1-C5alkyl, C1-C5alkoxy, halogen, C1-C5acylamino group, or naphthyl; or amino group is selected from C3-C7heterocyclyl, containing 1-2 heteroatoms in cycle, selected from nitrogen, oxygen or sulphur, possibly substituted with hydroxy, C1-C3alkyl, benzyl, phenyl, which can be substituted with halogen, and said heterocyclyl can be condensed with benzene ring; acylamino group, in which acyl is selected from C1-C6alkylcarbonyl, where alkyl can be substituted with phenyl, substituted with phenyl, in which substituents are selected from C1-C5alkoxy; 5-membered heteroaryl with heteroatom, selected from atom of oxygen or sulphur; benzoyl, possibly substituted with C1-C5alkyl, C1-C5alkoxy, C1-C5alkylthio or halogen, methylenedioxy; heterocyclylcarbonyl, in which heterocyclyl is selected from 5-6-membered heterocyclyl, with 1-2 heteroatoms, selected from nitrogen, oxygen or sulphur, possibly condensed with benzene ring and possibly substituted with C1-C5alkyl, halogen; or ureido group, in which one of substituents of terminal amido group represents hydrogen, and the second substituent is selected from: C1-C3alkyl, substituted with phenyl, 5-membered saturated or aromatic heterocyclyl, in which heteroatoms are selected from oxygen or sulphur; C2-C6alkenyl; aryl, selected from phenyl, substituted with C1-C5alkyl, C1-C5alkoxy, ethylenedioxy, methylenedioxy, halogen, C1-C3alkylcarbonyl; 5-membered heterocyclyl, in which heteroatoms are selected from sulphur or oxygen atom, and possibly substituted with alkyloxycarbonyl group; B represents non-aromatic cyclic substituent, selected from C4-C6cycloalkyl; and has other values, given in the invention formula. Values R1a R1b R1c are given in the invention formula.

EFFECT: increased efficiency of application of compounds.

12 cl, 8 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new crystalline forms of acid addition salts of (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine, wherein the acid is specified in methanesulphonic, maleic, fumaric, citric, orotic, 10-camphor sulphonic acids and fencifose. The salts possess the agonist properties of neuronal nicotine receptor (NNR) and can be used for managing or preventing pain, an inflammation or a CNS disorder. Each of the crystalline salts is characterised by an X-ray powder diffraction diagram. The invention also involves an amorphous form of (R)-5-((E)-2-pyrrolidin-3-ylvinyl)pyrimidine monocitrate and polymorphic forms of the above crystalline salts.

EFFECT: invention refers to a pharmaceutical composition containing an effective amount of the presented salts.

19 cl, 8 dwg, 33 ex

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