Medications for treating pemphigus containing antibodies to fas-ligand

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is the application of a monoclonal antibody against a protein of a human Fas ligand (CD95L, or Apo1L, or FasL) or its antigen-binding fragment for manufacturing a medication for the prevention and/or treatment of skin diseases, associated with the acantholysis of keratinocytes, in particular for the prevention and/or treatment of pemphigus, where the antibody contains amino acid sequences CDR of the antibody NOK-2, F919-7-3, F918-9-4 or F919-9-18 or is produced by the hybridoma ATCC PTA-5045, ATCC PTA-5533, ATCC PTA-5534 or ATCC PTA-5535.

EFFECT: application of the antibodies by the invention or their antigen-binding fragments provides the effective blocking of mechanisms, resulting in pemphigus lesions.

7 cl, 14 dwg

 

The technical field to which the invention relates

The present invention relates to the use of antagonists of Fas-ligand (also known as CD95L or Apo1L and identified in this document as FasL), in particular, to the use of the humanized antibodies to FasL for the prevention and/or treatment of skin diseases associated with acantholysis of keratinocytes, in particular, for the prevention and/or treatment of pemphigus.

The level of technology

Pemphigus is an autoimmune bullous skin condition that is characterized by loss of adhesion between keratinocytes, are caused by autoantibodies directed against desmosomal proteins (desmogleins, dsg). Pemphigus is widely distributed in the world with a frequency of occurrence of a disease constituting 1-5 cases per 100,000 per year, and mainly affects women.

Pemphigus is characterized by loss of adhesion between suprabasally the keratinocytes, which become rounded in the process known as acantholysis. The pathogenesis of pemphigus is subject to major revision, mainly due to the fact that in addition to antibodies to desmoglein acantholysis can cause a new group of antibodies to cholinergic receptors (Kalish, 2000). In addition, it was shown that steric hindrances arising from the binding of antigen-antibody, themselves cannot explain clicks�the use of bubbles (Kitajima, 2002). In addition, it was shown that the formation of desmosome not inhibited by the binding of pemphigus autoantibodies (PVIgG) disease pemphigus, despite the fact that desmosomal connection off within 24-36 hours after treatment autoantibodies PvIgG. At this time there is a consecutive transmission of signals that run PVIgG.

These observations suggest the role of apoptosis in the pemphigus. Indeed, pemphigus is a disease that occurs as a result of loss of cell adhesion (Payne AS et al., 1978), and apoptosis may occur in connection with the separation cells (Marconi et al, 2004). These ideas are strongly supported by the detection of apoptosis in skin damaged by pemphigus. In particular, acantholytic cells Express key markers of apoptosis (Wang X. et al., 2004). More interestingly, it was found that the nuclei labeled with TUNEL, also associated with the arch of the bubble, indicating the presence of apoptotic cells in the skin, environmental damage, before separation. In addition, apoptotic cells expressing Ig and activated caspase-8, detected at the edges of the lesion, on surfaces where no one can see the destruction of intercellular contacts (Wang X et I, 2004). These results show that with pemphigus in keratinocytes before acantholysis occurs apoptosis.

Apoptosis plays a fundamental role in regulating cellular�internal homeostasis and is involved in many pathophysiological processes. Apoptosis may be accompanied by the separation of cells from each other (Rezgui et al., 2000; Bergin et al., 2000), and the loss of interaction between cells and matrix (Giancotti and Ruoslahti, 1999).

Fas (or FasR) is a member of the superfamily of TNF receptors, after binding of Fas-ligand (FasL) triggers apoptosis in many cell systems (Sharma et al., 2000). Intracellular signal for apoptosis caused by Fas-FasL, is carried out through the involvement of a number of adaptor molecules such as FADD (Fas-associated death domain) and FLICE (FADD-like ICE, caspase 8), which in turn inhibited FLIP (protein, FLICE inhibitory) (Juo et al, 1998). The interaction of Fas-FasL involved in the pathological mechanisms of some immunovospalitelnykh and infectious conditions such as AIDS (Bahr et al., 1997) and systemic lupus erythematosus (Kovacs et al., 1997). Skin disease characterized by a metabolic pathway involving Fas-FasL include acute graft-versus-host, toxic epidermal necrolysis and melanoma (Wehrii et al., 2000). In addition, it was reported that lesions similar to acantholytic, detected in cultured keratinocytes treated and PVIgG, and FasR antibodies, despite the fact that PvIgG induces clustering of FasR, FasL and caspase-8 to the cell membrane for several hours before the appearance of lesions (Wang X. et al., 2004). In addition, it was reported that an inhibitor of caspase-1 is similar�th protease substantially inhibits the formation of bubbles in the model of pemphigus in mice (Li et al., 2006). Together, these results show that FasL and external apoptotic pathway play a crucial role in the mechanisms underlying the acantholysis.

Regardless of the nature pemphigus autoantibodies, the impact PVIgG activates the expression of several proapoptotic genes, including FasL, many hours to acantholysis. In addition, intravenous injections of IgG (IVIgG) prevents induced PvIgG activation of FasL and apoptosis in keratinocytes. IVIgG also prevents the acantholysis and apoptosis in vivo (J. Arredondo et al., 2005).

Without treatment the mortality from vulgar pemphigus reaches 100%. Early systemic therapy is necessary for the containment of pemphigus, but the side effects of systemic therapy are the main complication. Treatment includes corticosteroids, medicines containing gold, anti-inflammatory drugs Dapsone or drugs that suppress the immune system (such as azathioprine, methotrexate, cyclosporine, cyclophosphamide, or mycophenolate mofetil). The most traditional is currently the treatment of pemphigus is the use of steroids, which you must enter in throughout your life and may cause severe side effects. The majority of patients suffering from pemphigus die from these side effects. Also, some antibiotics effective, in particular, mini�wedge and doxycycline. Occasionally used intravenous immunoglobulin (IVIg). Plasmapheresis is a process by which plasma containing antibodies is removed from the blood and replace it with liquids, injectable, or plasma donor. Plasmapheresis can be used in addition to systemic drugs to reduce the amount of antibodies in the bloodstream. Also in development are several new molecules: mycophenolate mofetil, PI-0824, PRTX-100 antibodies to CD20 are drugs suppressing the immune system that affect T - and b-cells.

Currently the mortality rate still ranges from 5 to 25%; the most frequently death caused by infection and long-term therapy that inhibits the immune system (primarily corticosteroids), is one of the most important factors still causing a high mortality rate. The immunoglobulin may cause some short term improvement, but, as it turns out, does not lead to long-term remissions, and its price makes it a long-term use impractical.

Also there are several objections against the use of plasmapheresis. Patients weakened by the use of prednisone or other means, suppress the immune system, and undergoing plasma exchange, are at increased risk of sudden death from sepsis. �besides the treatment is very expensive, and to get therapy needed 2-week period of hospitalization. Thus, because of the risk of sudden death from sepsis and the high cost of Plasmapheresis is indicated only in the most difficult, not treatable cases where the patient has an undoubted risk of dying from the disease.

Disclosure of the invention

The aim of the present invention is to provide a therapeutic agent for the treatment of pemphigus. The improvement of new drugs, which blocks FasL, will completely prevent the separation of cells and appearance of skin lesions.

Basically, the present invention relates to the treatment of skin diseases associated with acantholysis of keratinocytes, through the introduction of FasL antagonist. The FasL antagonists can be selected from antibodies to FasL, in particular humanized or human antibodies to FasL, effector molecules, which are nucleic acids, which affect the expression of Fas, such as antisense molecules or molecules capable of RNA interference, such as siRNA molecules; molecules soluble Fas receptor, Malinov, FasL antagonists and low-molecular chemical compounds that inhibit the interaction of Fas-FasL. Antagonists prevent FasL apoptosis of keratinocytes and the subsequent miclet�: the separation (acantholysis). Thus, FasL antagonists, in particular, suitable for the prevention and/or treatment of pemphigus, for example, for the prevention and/or treatment of mucocutaneous of pemphigus.

The present invention relates to a drug containing at least one connection, the vast biological effects of FasL. The expression "the Union, the vast biological effects of FasL" as used in this document refers to all compounds that are capable of completely or at least essentially to block or neutralize the biological effects of FasL. For example, inhibitory or neutralizing effect may be based on inhibition of FasL binding to its natural receptor and, consequently, the suppressed thus signal transmission. This can be achieved, for example, by the use of antibody binding with FasL on their own or with soluble receptors, simulating Fas, or with mottainai, FasL antagonists, blocking, thus, the binding of FasL to the cell receptors. siRNA, inhibiting the expression of Fas or FasL, will lock the system Fas/FasL.

Therapy with antagonists FasL is or monotherapy, or combination therapy with the use of other drugs suitable for the treatment of pemphigus or other skin diseases, in particular, ill�tions, described above. For example, combination therapy FasL antagonists and steroids may provide significant reduction of doses of steroids.

In a preferred embodiment of the present invention provide a therapeutic agent for the treatment of pemphigus, including antibody to Fas-ligand, or the active fragment as an active ingredient. The antibody is preferably a chimeric, humanized or human antibody to FasL or antigen-binding fragment, or derivative, for example, recombinant single-chain antibody. If necessary, the antibody may be anywhereman with effector molecules, for example, cytostatic, cytotoxic and/or radioactive compounds.

Preferred humanized antibodies suitable for treating skin diseases associated with acantholysis of keratinocytes, in particular, pemphigus, in accordance with the present invention described in the patent WO 1997/A ("Anti-Fas ligand antibodies and assay method using the same antibody) or in the patent WO1998/010070 A1 ("Humanized immunoglobulin reacting specifically with Fas Ligand or active fragments thereof and region inducing apoptosis originating in Fas Ligand humanized antibodies"), the contents of which are incorporated in this document by reference. Additional preferred humanized antibodies suitable for treating skin diseases associated with acantholysis ke�of atenolol, in particular, pemphigus, in accordance with the present invention described in the patent US 7,262,277 ("Antagonistic Anti-hFas ligand human antibodies and fragments thereof), the contents of which are also included in this document by reference.

Human or humanized antibodies have at least three potential advantages for use in the treatment of humans compared to mice, and in some cases chimeric antibodies: 1) because the effector portion is from a human antibody, it may be better to interact with other parts of the system; 2) the human immune system is not able to recognize a wireframe plot or the plot of a humanized antibody as foreign, and, therefore, the formation of antibodies in response to such injected antibody should be weaker than in response to totally foreign mouse antibody or a partially foreign chimeric antibody; 3) injected humanized antibodies will presumably have a big half-life compared to natural human antibodies, allowing you to use smaller and less frequent doses.

Other preferred antagonists FasL molecules are soluble FasR, including soluble extracellular portion of Fas-receptor or a modified molecule which is an antagonist of FasL, which has a competitor�Oh or noncompetitive antagonistic activity. Such molecules inhibit the interaction of FasL/FasR, in which FasL binds to a soluble receptor analogue or molecule antagonist FasL binds to its natural receptor, weakening, thus, or completely eliminating the binding of bioactive FasL with its natural receptor. In the treatment using siRNA to determine treatment type and the course of therapy of a subject can be used to analyze the levels of Fas and/or FasL protein, or RNA. Monitoring of levels of Fas and/or FasL protein or RNA can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain proteins Fas and/or FasL-associated symptom, condition or disease. In a particularly preferred aspect the present invention relates to the use of

(i) a monoclonal antibody or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody comprises at least one variable plot heavy chain and at least one variable area light chain, where the amino acid sequence of the region that defines complementarity (CDRs) of the heavy chain are:

(a1) CDR H1: Asn Tyr Trp Ile Glee (SEQ ID NO:1),

(b1) CDR H2: Tyr Leo Tyr Pro Glee Gly Leu Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly (SEQ ID NO:2),

(c1 ) CDR H3: Tyr Arg Asp Tyr Asp Tyr Ala Met Asp Tyr (SEQ ID NO:3) or

(d1) a sequence derived by substituting 1, 2 or 3 amino acids in SEQ ID NOs: 1,2 and/or 3

and/or the amino acid sequence of sections that define complementarity (CDRs), light chain are

(a2) CDR L1: Lys Ser Thr Lys Ser Leu Leu Asn Ser Asp Gly Phe Thr Thy Leu Gly (SEQ ID NO:4),

(b2) CDR L2: Leu Val Ser Asn Arg Phe Ser (SEQ ID NO:5),

(c2) CDR L3: Phe Gln Ser Asn Tyr Leu Pro Leu Thr (SEQ ID NO:6)

or

(d2) a sequence derived by substituting 1, 2 or 3 amino acids in SEQ ID NOs: 4, 5 and/or 6

or

(ii) the antibody or antigen-binding fragment that recognizes the same epitope of human FasL as the antibody (i), for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

In the second particularly preferred aspect the present invention relates to the use of

(i) a monoclonal antibody or its fragment binds an antibody specific for the protein Fas ligand (FasL), where the specified monoclonal antibody produced by using a hybrid cell with No. access FERM BP-5045, or antibody or antibody fragment derived from it, or

(ii) a monoclonal antibody or antigen-binding fragment that recognizes the same samayapuram human FasL, as the antibody (i), for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

In a third particularly preferred aspect the present invention relates to the use of

(i) a monoclonal antibody or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody comprises at least one variable plot heavy chain and at least one variable area light chain, where the amino acid sequence of the region that defines complementarity (CDRs) of the heavy chain are:

(a1) CDR H1: Glu Tyr Pro Met His (SEQ ID NO:7),

(b1) CDR H2: Met Ile Tyr Thr Asp Thr Gly Glu Pro Ser Tyr Ala Glu Glu Phe Lys Gly (SEQ ID NO:8),

(c1) CDR H3: Phe Tyr Trp Asp Tyr Phe Asp Tyr (SEQ ID NO:9) or

(d1) a sequence derived by substituting 1, 2 or 3 amino acids in SEQ ID NOs: 7, 8 and/or 9,

and/or the amino acid sequence of sections that define complementarity (CDRs), light chain are

(a2) CDR L1: Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn (SEQ ID NO:10),

(b2) CDR L2: Tyr Thr Ser Arg Leu His Ser (SEQ ID NO:11),

(c2) CDR L3: Gln Gln Gly Ser Thr Leu Pro Trp Thr (SEQ ID NO:12)

or

(d2) a sequence derived by substituting 1, 2 or 3 amino acids in SEQ ID NOs: 10, 11 and/or 12 or

<> (ii) the antibody or antigen-binding fragment that recognizes the same epitope of human FasL as the antibody (i), for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

In a fourth preferred aspect the present invention relates to the use of

(i) a monoclonal antibody or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody produced by using a hybrid cell with No. access FERM BP-5533, FERM BP-5534 and/or FERM BP-5535, or antibody or antibody fragment derived from it, or

(ii) a monoclonal antibody or antigen-binding fragment that recognizes the same epitope of human FasL as the antibody (i), for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

In a particularly preferred embodiment of the present invention is directed to the use of human antibodies to FasL, or antigen-binding parts, including a variable area light chain and/or variable plot heavy chain, as described in the patent US 7,262,277, the content of which is included in this document way� reference. Particularly preferred human antibodies to FasL suitable for treating skin diseases associated with acantholysis of keratinocytes, in accordance with the present invention include a variable area light chain comprising a polypeptide with the sequence shown in SEQ ID NO. 2 of the patent US 7,262,277 (included in this document by reference), and additionally includes variable plot heavy chain comprising a polypeptide with the sequence shown in SEQ ID NO. 10 or 18 of US patent 7,262,277 (included in this document by reference). In particular, the invention relates to the use of human antibody 3E1 and/or 4G11 to hFas, as described in the patent US 7,262,277 (included in this document by reference) for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

In another preferred aspect, the present invention relates to the use of

(i) human monoclonal antibody or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody comprises at least one variable plot heavy chain and at least one variable area light chain, where the amino acid sequence of plots Oprah�alausa complementarity (CDRs), the heavy chain are:

(a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO:13) or

(a2) CDR H1: Ser His Gly Ile Ser (SEQ ID NO:14),

(b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val Gln Gly (SEQ ID NO:15) or

(b2) CDR H2: Trp Ile Asn Ala Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln Gly (SEQ ID NO:16),

(c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO:17) or

(c2) CDR H3: Glu Thr Met Val Arg Gly Val Pro Cys Asp Tyr (SEQ ID NO:18), or

(d1) a sequence derived by substituting 1, 2 or 3 amino acids in SEQ ID NOs 13, 14, 15, 16, 17 and/or 18,

and/or the amino acid sequence of sections that define complementarity (CDRs), light chain are

(a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ ID NO:19),

(b3) CDR L2: Gly Ala Ser Ser Arg Ala Thr (SEQ ID NO:20),

(C3) CDR L3: Gln Gln Tyr Gly Ser Ser Pro Trp Thr (SEQ ID NO:21)

or

(d3) a sequence derived by substituting 1, 2 or 3 amino acids in SEQ ID NOs: 19, 20 and/or 21

or

(ii) the antibody or antigen-binding fragment that recognizes the same epitope of human FasL as the antibody (i), for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

In a final preferred aspect the present invention relates to the use of

(i) a monoclonal antibody or antigen-binding fragment, spec�ment to protein Fas-ligand (FasL), where the specified monoclonal antibody produced by using a hybrid cell, with no access of ATSS MOUTH-4017 and/or ATSS MOUTH-4018, or antibody or antibody fragment derived from it, or

(ii) a monoclonal antibody or antigen-binding fragment that recognizes the same epitope of human FasL as the antibody (i), for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, particularly of pemphigus.

A medicament of the present invention may be provided in the form of pharmaceutical compositions together with pharmaceutically acceptable carrier. Preferably the pharmaceutical composition is administered by injection or infusion, for example, intravenously, intra-arterially, subcutaneously, intraperitoneally, or by using other suitable methods. The composition can be administered topically or systemically. Preferably the composition is administered systemically.

Pharmaceutical compositions suitable for use in the invention include an active agent in amounts effective to achieve the intended purpose. Effective dose of the drug of the present invention may be in the range of from 0.1 μg to 100 mg, up to a total dose of approximately component of 1 g depending on the method of administration. F�rmaceuticals compositions can be administered daily, for example, once or several times, or every 2-4 days, every week or once in two weeks. The drug can be administered in a single cycle of treatment consisting of injections of one or more medicines, or in several cycles of treatment, each of which consists of injections of one or more drugs. Each treatment cycle may have a duration from one day to several days, months or even years.

Therefore, in accordance with the present invention, the drug can also be used in combination therapy, at least with one additional therapy is effective against skin diseases associated with acantholysis of keratinocytes and, in particular, against pemphigus. The drug will be used or by itself, or in combination with other drugs suppressing the immune system, in particular, with steroids to reduce the dose and/or to minimize their chronic side effects. When used in combination drug will be preferably administered on a monthly basis. When using the drug by itself, it preferably will be administered continuously for a time period depending on each individual case.

In addition�about, the present invention will be explained in more detail using the examples below.

The implementation of the invention

Examples

First, using TUNEL staining, we evaluated the presence of apoptosis in the epidermis of skin surrounding the lesions, in frozen sections of samples from patients with pemphigus and not taking treatment. Fluorescent samples were analyzed using confocal scanning laser microscopy. In suprabasal layers of the epidermis surrounding the lesions, the majority of the keratinocytes had signs of apoptosis compared with normal skin (Fig.1).

To confirm the presence of apoptosis in pemphigus lesions, we used fixed with formalin and placed in paraffin biopsy and discovered the active form of caspase-3. The staining Protocol was carried out using a system UltraVision LP Detection System AP Polymer & Fast Red Chromogen (Lab Vision Corporation, CA, USA) in accordance with the instructions of the manufacturer. Visualization was performed using tablets Fast Red naphthol phosphate substrate. In pemphigus samples we found that a fragment of caspase-3 is localized in the arch, and at the base of the bladder, and some cells were positive in the epidermis surrounding the lesions (Fig.2). Apparently, this result indicates that cell death of keratinocytes occurs to resedimentation, leading to acantholysis.

Since apoptotic keratinocytes are widely represented in pemphigus, we decided to investigate whether the serum of patients with pemphigus cause induction of apoptosis in normal human keratinocytes. To this end, the keratinocytes were seeded on glass slides in the chamber and were grown in medium not containing serum (KGM), to the state of prisonplanet. Cells then were grown in minimal medium for keratinocytes and processed within 48 hours, adding 25% serum from patients who were not taking treatment, and patients who were treated with systemic corticosteroids. As controls used the serum of healthy subjects. Apoptosis was assessed using TUNEL-staining in situ. Evaluated approximately 100 cells, in the areas chosen at random and counted the percentage of TUNEL-positive cells. The serum of patients with pemphigus, but not the serum of healthy subjects or patients under steroid treatment caused apoptosis in human keratinocytes (Fig.3 A-B).

Because the system Fas/FasL involved in many apoptotic processes at the level of the skin (Wehrli et al, 2000), we measured the levels of FasL in the serum of patients with pemphigus using dvuhsostavnogo enzyme immunoassay (ELISA). Serum concentration opredelyalis using the absorption at 450 nm against a standard recombinant protein of human FasL. The FasL levels were very high in the serum of patients who were not taking treatment, and were below the limit of detection in the serum of patients treated with corticosteroids or in the serum of healthy subjects. As a positive control was used the serum of patients with HBV (Fig.4A). One patient FasL levels progressively decreased during systemic therapy with steroids (Fig.4B).

Since FasL contained in high amounts in the serum of patients with pemphigus, we monitored the expression of its cognate receptor FasR. To this end, we applied fixed with formalin and placed in paraffin biopsy and discovered the active form of caspase-3. The staining Protocol was carried out using a system UltraVision LP Detection System AP Polymer & Fast Red Chromogen (Lab Vision Corporation, CA, USA) in accordance with the instructions of the manufacturer. Visualization was performed using tablets Fast Red naphthol phosphate substrate. While in the normal skin FasR expressively only in the basal keratinocytes, in lesions with active pemphigus FasR were detected both in basal and in suprabasal cells. Of greater interest is the fact that when mucocutaneous the pemphigus (PMC) FasR, apparently, is expressed over the entire thickness of the layers of the epidermis and even before the formation of bubbles (Fig.5).

FasL is one of the triggers externally�of activated caspase-8 apoptotic way. We therefore decided to assess whether this path is the value in apoptosis when pemphigus. With this purpose the serum of patients pre-treated with neutralizing antibody to FasL or inhibitor of caspase-8, and added to cultures of keratinocytes. Keratinocytes were grown in KGM and treated with serum of patients with pemphigus, or the serum of patients who were not taking treatment. The serum is pre-treated with neutralizing antibody to FasL (2.5 mg per ml for 30 minutes) or an inhibitor of caspase-8, Z-IETD-FMK (100 μm for 30 minutes). Apoptosis was assessed by TUNEL-staining. The addition of neutralizing antibodies to FasL or inhibitor of caspase-8 partially prevented apoptosis of keratinocytes induced by serum of patients with pemphigus (Fig.6).

In addition, keratinocytes were treated as in Fig.6 and added or antibody to FasL, or immunoglobulins, are not related to FasL. Cells were then homogenized in RIPA buffer for Western blotting. Membranes were incubated with human antibodies to caspase-8 or β-actin. The relative intensity of the bands on autoradiogram quantitatively analyzed using a scanning laser densitometry. The results showed that the caspase-8 was significantly activated in keratinocytes treated with the serum of patients with pemphigus, compared�Yu with untreated cells, whereas the hydrolysis of caspase partially ingibirovala as a result of the preliminary processing of the antibody to FasL (Fig. 7).

Together these results indicate that the serum of patients with pemphigus induced apoptosis of keratinocytes using an external apoptotic path-triggered system Fas/FasL.

Recent studies have shown that components of the adhesive complex cadherin-catenin in epithelial adhesion contacts are targets for caspases during apoptosis (Weiske et al., 2001). To assess whether the apoptotic pathway induced by Fas/FasL, call the division of desmosome, we processed within 72 hours the confluent keratinocytes, cultivated in KGM in the presence of 1.8 mm CaCl2the serum of patients with pemphigus who were exposed or not exposed to therapy. The protein extracts from cultures was analyzed by Western blotting using antibodies to Dsg1 and Dsg3. As an internal control was used for β-actin. We found that the serum of patients with pemphigus able to cleave Dsg1 and Dsg3. In particular, the serum of patients who were not taking treatment, but no patients after therapy with steroids, are largely uncoupled Dsg1 and Dsg3. (Fig.8).

Most importantly, treatment of keratinocytes increasing amounts of FasL (0,1, 10, 100 ng/ml) for 72 hours, led� to splitting dsgs dose-dependent way. Extracts of proteins from the culture was analyzed by Western blotting using antibodies to Dsg1 and Dsg3, as an internal control was used for β-actin. Such doses correspond to the doses found in the serum of patients with pemphigus (Fig.9)

Taking into account that FasL plays an important role in the pathogenesis of pemphigus, we analyzed the antibody to FasL (NOK2, the antibody produced by cell line hybridomas NOK2, access code No. FERM BP-5045). Confluent keratinocytes, cultivated in KGM with 1.8 mm CaCl2were treated within 72 hours: 1. only KGM; 2. the antibody to FasL (NOK2, 15 µg/ml), 3. FasL (500 ng/ml); 4. FasL + antibody to FasL. We provide evidence that antibody to FasL (NOK2) prevents cleavage of dsg, called FasL. We also show that antibody to FasL (NOK2) inhibits apoptosis induced by caspase-8 (Fig.10A). Fig.10B shows that the antibody to FasL (NOK2) prevents intercellular separation caused by FasL, i.e. acantholysis.

To further confirm the Central role of FasL, we used another antibody to FasL (F918-7-3, the antibody produced by cell line hybridomas with access code No. FERM BP-5533; F918-7-4, the antibody produced by cell line hybridomas with access code No. FERM BP-5534; F919-9-18, the antibody produced by cell line hybridomas with access code No. PERM BP-5535). Confluent keratinocytes, cultivated in KGM with 18 mm CaCl 2were treated within 72 hours: only KGM; recombinant FasL (50 ng/ml); a hybrid medium, diluted 1:1 in KGM; FasL + hybrid environment in various dilutions in KGM. We provide evidence that antibodies to FasL prevent splitting dsg3 induced FasL, dose-dependent way (Fig.11A and Fig.11B), the inhibition of activation of apoptosis caused by caspase-8 (Fig.11A). Fig.11C shows that the antibody to FasL contained in the medium from cell lines of hybridomas FERM BP-5535, prevents intercellular separation caused by FasL, i.e. acantholysis.

To explore whether the external apoptotic pathway cleavage of dsg, we pre-treated confluent keratinocytes inhibitor of caspase-8, Z-IETD-FMK (100 μm for 30 minutes) or an antibody to FasL (NOK2, 15 µg/ml). Then the cells were incubated for 72 hours with serum of healthy subjects or patients with pemphigus and not taking treatment. The protein extracts from cultures was analyzed by Western blotting using antibodies to Dsg3 and antibody to caspase-8. Vinculin was used as internal control (Fig.12A). Fig.12B shows that the separation of the cells (i.e. acantholysis) prevented the antibody to FasL or inhibitor of caspase-8. These results indicate that inhibition of apoptotic path-activated FasL or caspase-8, PR�dataset as the activation of caspase-8, and the cleavage of dsg, and blocks, thus, the acantholysis.

In conclusion, we have shown that FasL exhibits dual activity, activating both the external apoptotic pathway mediated by caspase-8 and cleavage of Dsg. In accordance with our work of Wang and colleagues (Wang et al., 2004) have suggested that apoptosis may be the cause of the phenomenon of acantholysis. They showed that PV-IgO antibody to Fas-receptor (anti-FasR) produce lesions in vitro in a similar fashion, leading to: (1) secretion of soluble FasL; (2) increased amounts of FasR, FasL (soluble and membrane), proteins Bax and p53; (3) reducing the level of cell BC1-2; (4) enrichment by caspase-8 and activation of caspases 1 and 3; (5) joint aggregation FasL and FasR with caspase-8 in a membrane signaling complex inducing cell death (DISC). Therefore, the signaling pathway of cell death mediated by Fas, apparently, involved in the formation of foci of the lesions.

To study the long pemphigus and was widely used well-founded the animal model. Passive delivery PVIgG in neonatal mice caused the separation of cells and the formation of the bubble. This model was used to evaluate the involvement of apoptosis and FasL in the pathogenesis of pemphigus. We subcutaneously injected with PVIgG (5 mg/g/every two weeks), purified from serum of patients, newborn C57BL/6N CrI. As controls used normal new�born mice treated IgG purified from serum of healthy subjects (NIgG). Animals were sacrificed 20 hours after the injection.

Staining with hematoxylin and eosin shows that the bubble develops only in mice treated PVIgG, but not in mice treated with normal human IgG (Fig.13A). Apoptosis was detected or by using TUNEL or activated caspase-3 only in mice treated PVIgG (Fig.13V).

To evaluate the role of FasL in vivo, mice were treated with PVIgG or PVIgG together with the antibody to FasL (clone MFL3, the antibody is specific for mouse). Antibody to FasL (40 µg/mouse) was administered 3 hours prior to injection PVIgG, and it prevented the formation of a bubble in mice, as shown by staining H &E. in addition, the length of cracks in mice treated with antibody to FasL was significantly reduced (Fig.14).

In conclusion, we have shown that FasL exhibits dual activity, activating both the external apoptotic pathway mediated by caspase-8 and cleavage of Dsg. Most importantly, the blocking of FasL protects against acantholysis in vitro and in vivo.

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1. The use of monoclonal antibodies or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody comprises at least one variable plot heavy chain and at least one variable area light chain, where the amino acid sequence of the region, complementarity determining (CDR) of the heavy chain are:
(a1) CDR H1: Asn Tyr Trp Ile Gly (SEQ ID NO:1),
(b1) CDR H2: Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn Tyr Asn Glu Lys Phe Lys Gly (SEQ ID NO:2),
(c1) CDR H3: Tyr Arg Asp Tyr Asp Tyr Ala Met Asp Tyr (SEQ ID NO:3)
and the amino acid sequence of sections that define complementarity (CDRs), light chain are
(a2) CDR L1: Lys Ser Thr Lys Ser Leu Leu Asn Ser Asp Gly Phe Thr Thy Leu Gly (SEQ ID NO:4),
(b2) CDR L2: Leu Val Ser Asn Arg Phe Ser (SEQ ID NO:5),
(C2) CDR L3: Phe Gln Ser Asn Tyr Leu Pro Leu Thr (SEQ ID NO:6)
for the prevention and/or treatment of skin diseases associated with acantholysis of keratinocytes, where therapy anti-FasL antibody is a monotherapy.

2. The use of monoclonal antibodies or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody produced by using a hybrid cell, and�eUSA the access number FERM BP-5045, for the prevention and/or treatment of skin diseases associated with acantholysis of keratinocytes, where therapy anti-FasL antibody is a monotherapy.

3. The use of monoclonal antibodies or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody comprises at least one variable plot heavy chain and at least one variable area light chain, where the amino acid sequence of the region, complementarity determining (CDR) of the heavy chain are:
(a1) CDR H1: Glu Tyr Pro Met His (SEQ ID NO:7),
(b1) CDR H2: Met Ile Tyr Thr Asp Thr Gly Glu Pro Ser Tyr Ala Glu Glu Phe Lys Gly (SEQ ID NO:8),
(c1) CDR H3: Phe Tyr Trp Asp Tyr Phe Asp Tyr (SEQ ID NO:9)
and the amino acid sequence of plots, complementarity determining (CDR), light chain are
(a2) CDR L1: Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn (SEQ ID NO:10),
(b2) CDR L2: Tyr Thr Ser Arg Leu His Ser (SEQ ID NO:11),
(C2) CDR L3: Gln Gln Gly Ser Thr Leu Pro Trp Thr (SEQ ID NO:12)
for the prevention and/or treatment of skin diseases associated with acantholysis of keratinocytes, where therapy anti-FasL antibody is a monotherapy.

4. The use of monoclonal antibodies or antigen-binding fragment specific for the protein Fas ligand (FasL), where the specified monoclonal antibody produced � via a hybrid cell, having the access number FERM BP-5533, FERM BP-5534 and/or FERM BP-5535,
for the prevention and/or treatment of skin diseases associated with acantholysis of keratinocytes, where therapy anti-FasL antibody is a monotherapy.

5. The use according to any one of claims.1-4, where the antibody or antigen-binding fragment is selected from partially or fully humanized antibody, a partially or fully humanized single-chain antibody or fragment.

6. The use according to any one of claims.1-4, where the skin disease associated with activation of apoptotic path and/or splitting desmoglein.

7. The use according to any one of claims.1-4, where the skin disorder is pemphigus.



 

Same patents:

FIELD: medicine.

SUBSTANCE: claimed invention relates to the field of biotechnology. Claimed are versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a determined amino acid sequence. An epitope of the antibody from 11 amino acids is determined by the Biacore method. Disclosed are: an immunoconjugate of the antibody with a medication or means for inhibiting cell growth, where the antibody is bound with means covalently, and versions of the composition, based on an effective quantity of the immunoconjugate or the antibody, used for inhibiting B-cell proliferation; as well as a method of determining CD79b in a sample with the application of the antibody. Described are: an antibody-coding polynucleotide, as well as an expression vector and an isolated cell, containing the vector for obtaining the antibody. Disclosed are versions of applying the antibody or immunoconjugate for obtaining the medication for inhibiting the growth of CD79b-expressing cells for the treatment of an individual, affected with cancer, for the treatment of proliferative disease or for inhibiting B-cell proliferation.

EFFECT: invention provides novel antibodies, which can find further application in the therapy of proliferative CD79b-associated diseases.

91 cl, 8 tbl, 9 ex, 20 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology. Claimed is application of monoclonal antibody against human Fas ligand protein (CD95L, or Apo1L, or FasL) or its antigen-binding fragment for manufacturing medication for prevention and/or treatment of skin diseases, associated with acantholysis of keratinocytes, in particular for prevention and/or treatment of pemphigus, where antibody contains amino acid sequences CDR of 3E1 antibody or is produced by hybridoma ATCC PTA-4017.

EFFECT: application of antibody in accordance with invention or its antigen-binding fragment provides more effective prevention of desmoglein (dsg3) cleavage in comparison with application of other antibodies.

11 cl, 16 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. What is presented is a monoclonal anti-IFNAR1 antibodies with L234F, L235E and P331S Fc mutations of human IgG1 possessing a lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors as compared to a non-modified antibody. There are described the recovered nucleic acid providing expression of the above antibody containing a nucleotide sequence coding the antibody, and a pharmaceutical composition based on the above antibody.

EFFECT: using the invention provides the antibody possessing the lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors that provides reducing the undesired effector functions in treating chronic inflammation and autoimmune conditions.

9 cl, 34 dwg, 7 tbl, 36 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology. There are presented versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a specified amino acid sequence and at least one free cysteine amino acid residue specified in A118C (according to the European Numeration) in the heavy chain and V205C (according to the Kabat numeration) in the light chain. There are disclosed: versions of a conjugate compound of the antibody and a drug preparation, wherein the antibody is bond to the drug preparation through free cysteine; an antibody-based pharmaceutical compound for treating cancer; method for detecting CD79b or cancer cells, as well as a method for inhibiting cell proliferation using the conjugate compound. What is described is a method for producing the conjugate compound.

EFFECT: invention can find further application in the therapy of CD79b-associated cancer diseases, including treating haemopoietic tumours in mammals.

70 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to monoclonal antibodies binding to c-Met and able to inhibit both ligand-dependent, and ligand-independent c-Met activation. The above antibodies, as well as a composition containing them can be used for producing a therapeutic agent for treating cancer.

EFFECT: invention enables specifically inhibiting both ligand-dependent, and ligand-independent c-Met activation, particularly inhibiting c-Met dimerisation.

34 cl, 111 dwg, 4 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: presented invention refers to immunology. There are described versions of antibodies or antigen-binding fragments binding to human 4-1BB. One of the versions is characterised by the presence of respective 3 CDR light chain sites and 3 CDR of heavy chain sites. The other version is characterised by the presence of the heavy and light chain with respective amino acid sequences. There are described versions of a pharmaceutical composition for reducing tumour growth or for treating cancer in an individual, as well as methods for reducing the tumour growth or treating cancer in the individual using the versions of antibodies or antigen-binding fragments in a therapeutically effective amount. What is described is a method of treating cancer with using a combination of the antibody and an immunotherapeutic agent. There are disclosed: versions of coding nucleic acids, an expression vector and a host cell containing the antibody expression vector. What is disclosed is a method for producing the antibody with using the cell.

EFFECT: invention provides the new agonist anti-human 4-1BB (also called CD137 or TNFRSF9) antibodies, which recognise an epitope within the amino acid residues K115, C121, R134, R154, V156 of the antigen, have Kd affinity measured by the BIACORE method and approximated to nM, eg 0,4 nM or 8 nM (for the anti-IgG1 antibody format) that can find application in the therapy of cancer and cancerous diseases.

19 cl, 8 dwg, 11 tbl, 9 ex

FIELD: biotechnology.

SUBSTANCE: humanised monoclonal antibody (MAb) specific to syndecan-1 (CD138) is proposed, which is characterised by amino acid and nucleotide sequences. According to the present invention, the antibody belongs to isotype IgG4 kappa, at that the constant fragments are fully human and modified by administration of sites IgG3 imparting to antibody the independent antibody-dependent and complement-dependent cytotoxicity, and amino acid substitutions that enable to increase the antibody gun. The variable sites are presented by murine hypervariable and human framework sites, providing an increase in binding affinity with a ligand. Each chain additionally comprises a signal secretory sequence at the N-terminus cleaved upon secretion from the producing cells. The nucleotide sequences are optimised to enhance antibody production in mammalian cells. The present invention may find additional application in the treatment of diseases associated with CD138, including malignant tumours (cancer).

EFFECT: increased efficiency of use of the compounds.

2 cl, 2 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunology. What is disclosed is using a peptide with an amino acid sequence GLAGGSAQSQRAPDRVL for selecting CεmX-specific antibodies and antigen-binding fragments of these antibodies. What is presented is the CεmX-specific antibody, as well as a method providing selecting the antibodies specifically binding the peptide according to the invention, and a method of treating IgE-mediated diseases involving administering the antibody into an individual according to the invention.

EFFECT: it has been disclosed that the monoclonal antibodies specifically binding the CεmX segment GLAGGSAQSQRAPDRVL can effectively bind to mIgE in human B-cells and are applicable for targeting to these B cells for treating IgE-mediated diseases.

14 cl, 5 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.

EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.

33 cl, 21 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.

EFFECT: invention can find further application in diagnosing and treating cancer.

43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology. Claimed is application of monoclonal antibody against human Fas ligand protein (CD95L, or Apo1L, or FasL) or its antigen-binding fragment for manufacturing medication for prevention and/or treatment of skin diseases, associated with acantholysis of keratinocytes, in particular for prevention and/or treatment of pemphigus, where antibody contains amino acid sequences CDR of 3E1 antibody or is produced by hybridoma ATCC PTA-4017.

EFFECT: application of antibody in accordance with invention or its antigen-binding fragment provides more effective prevention of desmoglein (dsg3) cleavage in comparison with application of other antibodies.

11 cl, 16 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compound, represented by the following formula

,

or its pharmaceutically acceptable salt. In claimed formula each symbol has values, determined in formula of invention. Versions of formula [I] compound and particular compounds are also objects of invention. In addition, invention relates to pharmaceutical composition, ITK inhibitor and means for treatment or prevention of inflammatory diseases, allergic diseases, autoimmune diseases, transplant rejection and other diseases and methods of treating said diseases.

EFFECT: claimed compounds inhibit induced T-cellular kinase (ITK).

32 cl, 86 tbl, 387 ex

Treatment // 2554801

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, and concerns using a human Annexin-1 antibody (Anx-A1), which has the sequence SEQ ID NO: 23 for treating a disease caused by abnormal T-cell activation. The group of inventions also concerns a method of treating a disease caused by abnormal T-cell activation, involving administering a therapeutic amount of the above antibody into an individual in need thereof; using the above antibody for producing a therapeutic agent for treating a disease caused by abnormal T-cell activation.

EFFECT: group of inventions provides treating the disease caused by abnormal T-cell activation.

9 cl, 11 ex, 22 dwg

FIELD: medicine, pharmaceutics.

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

EFFECT: obtaining novel heteroaryl derivatives.

27 cl, 41 ex

FIELD: medicine.

SUBSTANCE: method involves administering intravenously a suspension of allogenic mesenchymal stem cells at 1.5×106 per 100 g of the animal's body weight in a physiological solution 2 ml. Animals' mortality has made 27% in the primary group, and 94% in the reference group.

EFFECT: method of treating infectious peritonitis experimentally opens up new possibilities for using the cell techniques for reducing the rate of patient's death caused by peritonitis.

11 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a new hydrate of 2-amino-2-(2-(4-octylphenyl)ethyl)propane-1,3-diol hydrochloride salt in the crystalline form with the characteristics below. The hydrate can be used for producing a drug or for treating or preventing a transplanted organ or tissue rejection, or autoimmune diseases in a therapeutically effective amount. The above hydrate is characterised by an X-ray powder diffractogram having peaks at approximately 2.9, 17.2, 30.6, 28.2, 24.4, 8.6 and 25.9 degrees 2-Theta with a limit of error of ±0.2 degrees for each value of 2θ, having a purity of 90% or more, and containing 5.2 to 5.9% of water.

EFFECT: invention also characterises a pharmaceutical composition with using the above hydrate.

4 cl, 4 dwg, 8 tbl, 14 ex

Fingolimod salts // 2543621

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new 2-amino-2-[2-(4-C2-20alkylphenyl)ethyl]propan-1,3-diol salts specified in tartrate, lactate benzoate, succinate, malonate, acetate and propionate in the crystalline form. Each of the above salts is characterised by powder X-ray pattern data. Compounds in the therapeutically effective amount can be used in treating autoimmune diseases.

EFFECT: crystalline salts of the present invention possess higher stability, better solubility, more convenient to store and handle.

11 cl, 7 dwg, 1 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula IV, VIII-A and X, and to their pharmaceutical acceptable salts possessing the inhibitory activity on PI3-kinase (phosphoinositide-3-kinase). In compounds of formula IV and IX and Wd is specified in a group consisting of, , , and each of which can be substituted. In formula VIII-A, the group Wd represents the group or , wherein Ra is hydrogen, R11 is amino; in compound IV, Wa2 represents CR5; Wa3 represents CR6; Wa4 represents N or CR7; in compound IX, Wa1 and Wa2 independently represent CR5, N or NR4, and Wa4 independently represents CR7 or S, wherein no more than two neighbouring atoms in a ring represent atom or sulphur; Wb5 represents N; B represents a grouping of formula II, as well as in case of compound IV, B means C1-C10alkyl, C3-C10cycloalkyl, C3-C10heterocycloalkyl having one to six ring heteroatoms specified in N, O and S; in case of compound IX, B also means C1-C10alkyl, C3-C10cycloalkyl or 6-merous heterocycloalkyl having nitrogen atom; Wc represents C6-C10aryl or 5-18-merous heteroaryl having one or more ring heteroatoms specified in N, O and S, or phenyl or 6-merous heteroaryl respectively is equal to an integer of 0, 1, 2, 3 or 4; X is absent or represents -(CH(R9))z-, respectively; z is equal to 1; Y is absent. The other radical values are specified in the patent claim.

EFFECT: compounds can be used for treating such diseases, as cancer, bone disorders, an inflammatory or immune disease, diseases of the nervous system, metabolic disorders, respiratory diseases, thrombosis or cardiac diseases mediated by PI3-kinase.

68 cl, 11 dwg, 7 tbl, 55 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan, methods of their obtaining, pharmaceutical compositions, containing them, and their application for obtaining pharmaceutical compositions for treatment of different conditions and/or diseases. In particular claimed invention relates to potassium salt of D-isoglutamyl-D-tryptophan (1:1) and magnesium salt of D-isoglutamyl-D-tryptophan (2:1).

EFFECT: obtaining pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan.

22 cl, 15 dwg, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and biotechnology. There are presented variants of antagonist antibodies binding to the interleukin-7 receptor (IL-7R). There are described: variants of nucleic acids coding the antibodies; a host cell recombinant producing the antibody; a pharmaceutical composition inhibiting the human IL-7R function, and methods of treating and/or preventing: an autoimmune disease specified in the group of type 1 diabetes, lupus, multiple sclerosis, rheumatoid arthritis; type 2 diabetes or graft-versus-host disease based on using the antibody.

EFFECT: invention provides the antagonist anti-IL-7R antibodies that can find application in medicine.

17 cl, 15 dwg, 8 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology, namely, to peptide for stimulation of Hsp70 (heat shock protein 70) or for protection of skin against ultraviolet radiation. Peptide can be represented by peptide with general formula R1-AA1-AA2-AA3-AA4-R2, its stereoisomers, their mixture and/or their cosmetically or pharmaceutically acceptable salt. AA1 represents -His-; AA2 is selected from the group, consisting of -His-, -Leu- and -Pro-; AA3 represents -Leu-; AA4 is selected from the group, consisting of -Arg- and -Asn-.

EFFECT: claimed invention makes it possible to obtain novel peptide, which is effective for stimulation of Hsp70 synthesis or for protection of skin against ultraviolet radiation.

17 cl, 1 tbl, 16 ex

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