Preparations, containing antibodies against fas ligand, for treating pemphigus

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

 

The technical field to which the invention relates

The present invention relates to the use of antagonists of Fas ligand (also referred to as CD95L or Apo1L, and further abbreviated as FasL), in particular, to the use gumanitarnyh antibodies against FasL for the prevention and/or treatment of skin diseases associated with acantholysis of keratinocytes, in particular for the prophylaxis and/or treatment of pemphigus (pemphigus).

The level of technology

Pemphigus (pemphigus) is an autoimmune bullous skin disease, which is characterized by loss of adhesion of keratinocytes caused by authenti-bodies directed against desmosome proteins (desmogleins, dsg). Pemphigus is prevalent worldwide with a frequency of approximately 1-5 cases per 100,000 per year and predominantly affects women.

Pemphigus is characterized by loss of adhesion suprabasal keratinocytes, which are 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 may cause a new group of antibodies to cholinergic receptors (Kalish, 2000). In addition, it was shown that bubble formation cannot simply be explained by steric hindrances arising from the binding of antigen-antibody (Kitajima, 2002). It was also showing�but formation of desmosome not inhibited upon binding of autoantibodies of pemphigus (PvIgG) with bubbles, while desmosome connection dissotsiiruyut in 24-36 h after treatment autoantibodies PvIgG. At the same time, the number of stages of signal transmission that runs PvIgG.

These observations suggest that when pemphigus play a role in apoptosis. Indeed, pemphigus is a disease caused by loss of cell adhesion (Payne AS et al., 1978), and apoptosis may occur in connection with the separation of cells (Marconi et al., 2004). These ideas are strongly supported by the detection of apoptosis in the affected pemphigus skin. In particular, acantholytic cells Express key markers of apoptosis (Wang X et al., 2004). Even more interestingly, labeled with the TUNEL method kernel was also associated with a set of bubbles, indicating the presence of apoptotic cells in environmental damage to the skin in front of the office. In addition, apoptotic cells expressing Ig and activated caspase-8, are found at the edges of lesions, in those places where no one can see the destruction of intercellular contacts (Wang X et I, 2004). These results convincingly show that with pemphigus in keratinocytes before acantholysis occurs apoptosis.

Apoptosis plays a fundamental role in the regulation of cell homeostasis and Vallecano 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 representative of the superfamily of TNF receptors, which upon binding to Fas ligand (FasL) triggers apoptosis in many cell systems (Sharma et al., 2000). Intracellular signaling in induced Fas-FasL apoptosis is carried out with the involvement of a number of adaptor molecules 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 is involved in the pathological mechanisms of certain immuno-inflammatory and infectious diseases such as AIDS (Bahr et al., 1997) and systemic lupus erythematosus (Kovacs et al., 1997). A skin disease characterized by a metabolic pathway involving Fas-FasL include acute graft versus host disease, toxic epidermal necrolysis and melanoma (Wehrii et al., 2000). In addition, it was reported that lesions similar to acantholytic, observed in cultured keratinocytes treated and PvIgG, and FasR antibodies, whereas PvIgG causes clustering of FasR, FasL and caspase-8 to the cell membrane for a few hours before the occurrence of damage (Wang X et al., 2004). In addition, it was reported that the caspase-1-like ing�inhibitor substantially inhibits the formation of bubbles in the model of pemphigus in mice (Li et al., 2006). Overall, these results show that FasL and external apoptotic pathway play a crucial role in the mechanisms underlying the acantholysis.

Whatever the nature of the autoantibodies in pemphigus, the impact PvIgG enhances the expression of several apoptotic genes, including FasL, many hours to acantholysis. In addition, intravenous IgG (ivIgG) prevents caused PvIgG increase in FasL and apoptosis in keratinocytes, and prevents acantholysis and apoptosis in vivo (Arredondo J et al., 2005).

Without treatment the mortality from pemphigus vulgaris reaches 100%. Early systemic therapy is necessary to control pemphigus, but the side effects of systemic therapy are the main complication. Treatment includes corticosteroids, medications containing gold, anti-inflammatory drug Dapsone or drugs that suppress the immune system (such as azathioprine, methotrexate, cyclosporine, cyclophosphamide, or mycophenolate mofetil). Currently the most common method of treatment of pemphigus are steroids that must be put into place throughout life and can cause severe side effects. The majority of patients with pemphigus die from these side effects. Also, some antibiotics effective, particularly minocycline and doxycycline. Sometimes applied HV�trevenna introduction immunoglobulin (ivIg). Plasmapheresis is a process by which are removed from the blood plasma containing antibodies and replace it with an injected fluid or donor plasma. Plasmapheresis can be used in addition to systemic drugs to reduce the amount of antibodies in the bloodstream. Are in development and several new molecules: mycophenolate mofetil, PI-0824, PRTX-100 antibodies against CD20 are immunosuppressive drugs that affect T - and b-cells.

Current mortality still ranges from 5 to 25%; the most frequent cause of death are infections, and one of the most important factors that still cause a high mortality rate, is a long-term immunosuppressive therapy (primarily corticosteroids). The immunoglobulin may cause some short term improvement, but it seems like does not give long-term remission, and its cost makes long-term use impractical.

Also there are some objections against the use of plasmapheresis. Patients weakened by the use of prednisone or other immunosuppressive funds, and subjected to plasmapheresis, are at increased risk of sudden death from sepsis. In addition, it is a very expensive treatment, but its application required a 2-week period of hospitalization. Thus, because R�ska sudden death from sepsis and the high cost of plasmapheresis is indicated only in the most difficult untreatable cases where the patient is certainly at risk of dying from the disease.

Summary of the invention

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

In General, 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 against FasL, in particular humanized or human antibodies against FasL, nucleic acid molecules that affect the expression of Fas, such as antisense molecules or molecules capable of RNA interference, for example, molecules siPHK; of soluble receptor molecules Fas, FasL neutralizing maleinos, and low molecular weight chemical compounds that inhibit the interaction of Fas-FasL. Antagonists prevent FasL apoptosis of keratinocytes and subsequent intercellular separation (acantholysis). Thus, FasL antagonists are particularly suitable for the prophylaxis and/or treatment of pemphigus, e.g., for the prevention and/or treatment of skin and mucosal pemphigus.

The present invention relates to pharmaceutical preparations containing at least one soybean�the errors, the vast biological effects of FasL. The expression "the Union, the vast biological effects of FasL", the present invention relates to all compounds that are capable of completely or at least substantially inhibit, or neutralize the biological effects of FasL. For example, inhibiting or neutralizing the effect may be based on inhibition of FasL binding to its natural receptor, and thereby caused them to transmit signals. This is achieved, for example, by using antibody that binds to FasL by itself or with soluble receptors, simulating Fas, or with FasL neutralizing Malinov, blocked the binding of FasL to the cell receptors. Interfering with the expression of Fas or FasL, siRNA will block the system Fas/FasL.

Therapy using FasL antagonists is either monotherapy or combination therapy along with other drugs suitable for the treatment of pemphigus or other skin diseases, in particular, as described above. For example, combination therapy FasL antagonists and steroids can provide a significant reduction in steroid dose.

In a preferred embodiment of the present invention provides a therapeutic agent for the treatment of pemphigus, including antibody against Fas ligand person or its active fragments� as the active ingredient. Preferably, the antibody is a chimeric, a humanized or human antibody against FasL or its antigen-binding fragment or derivative, e.g., recombinant single-chain antibody. Optionally, antibodies can be anywhereman with effector molecules, e.g., cytostatic, cytotoxic and/or radioactive compounds.

Preferred humanized antibodies suitable for the treatment of skin diseases associated with acantholysis of keratinocytes, in particular pemphigus, in accordance with the present invention, is described in WO 1997/002290A1 ("Anti-Fas ligand antibodies and assay method using the same antibody) or in WO 1998/010070 Al ("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 by reference. Other preferred humanized antibodies suitable for the treatment of skin diseases associated with acantholysis of keratinocytes, in particular pemphigus, in accordance with the present invention, is described in US 7,262,277 ("Antagonistic anti-hFas ligand human antibodies and fragments thereof), the content of which is also incorporated by reference.

Human or humanized antibodies have at least three potential advantages over mouse and in some cases chimeric antibodies for use in the treatment of a person: 1) because the effect of the�RNA part comes from the person, it may be better to interact with other parts of the system; 2) the human immune system will not recognize a wireframe plot or the plot of a humanized antibody as foreign, and therefore the antibody response to this typed antibody should be weaker than a totally foreign mouse antibody or a partially foreign chimeric antibody; 3) introduced humanized antibodies will presumably have a half-life, closer to natural human antibodies that will allow you to enter smaller and less frequent doses.

Other preferred antagonists are soluble FasL molecules FasR, including soluble extracellular portion of Fas-receptor, or molecules modified by FasL antagonists with activity competitive or non-competitive antagonists. Such molecules inhibit the interaction of FasL/FasR that FasL binds to a soluble receptor analogue or molecule antagonist FasL binds to its natural receptor, thereby reducing 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 for the treatment of the subject, you can use the analysis of the level of protein or RNA of the Fas and/or FasL. Monitoring the level of protein or RNA of the Fas and/or FasL can be used for performance�Azania of treatment outcome and to determine the effectiveness of the compounds and compositions, modulating the level and/or activity of certain proteins Fas and/or FasL associated with any symptom, condition or disease.

In one particularly preferred aspect the present invention relates to the use of:

(i) a monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), and the monoclonal antibody includes at least one variable region of the heavy chain and at least one variable region light chain, wherein the amino acid sequence of sections that define complementarity (CDRs) 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) 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) of the 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 FR�ment, recognizing 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, in particular pemphigus.

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

(i) a monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), and the monoclonal antibody produced by the hybrid cells ohms with an access number FERM BP-5045, or antibody or antibody fragment, derived from it, or

(ii) a monoclonal antibody or its 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, in particular pemphigus.

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

(i) a monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), and the monoclonal antibody includes at least one variable region of the heavy chain and at least one variable region light chain, wherein the amino acid consisten�Telenesti plots define 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) of the 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, in particular pemphigus.

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

(i) a monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), and this monoclonal antibody is produced by cells of hybridomas with an access number PERM BP-5533, FERM BP-5534 and/or FERM BP-5535 or �ntitle or antibody fragment, derived from it, or

(ii) a monoclonal antibody or its 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, in particular pemphigus.

In a particularly preferred embodiment of the present invention is directed to the use of human antibodies against FasL or antigen-binding parts, comprising a variable region light chain and/or variable region of the heavy chain, as described in US 7262277, the content of which is incorporated by reference. Particularly preferred human antibodies against FasL, suitable for the treatment of skin diseases associated with acantholysis of keratinocytes in accordance with the present invention, includes a variable region light chain comprising a polypeptide with the sequence shown in SEQ ID NO 2 from US 7262277 (incorporated by reference) and include the variable region of the heavy chain comprising a polypeptide with the sequence shown in SEQ ID NO. 10 or 18 of US 7262277 (incorporated by reference). In particular, the invention relates to the use of human antibody 3E1 (produced by the cells of hybridomas with a number of ATSS MOUTH-4017) and/or 4G11 (produced by the cells Gebr�houses with a number of ATSS MOUTH-4018) against hFas, as described in US 7262277 (incorporated by reference) for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes, in particular pemphigus.

In the following preferred aspect the present invention relates to the use of:

(i) human monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), and the monoclonal antibody includes at least one variable region of the heavy chain and at least one variable region light chain, wherein the amino acid sequence of sections that define complementarity (CDRs) of 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 complemen-arnosti (CDRs) of the light chain, are:

(a3) CDR L1: Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala (SEQ ID N: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, in particular pemphigus.

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

(i) a monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), and this monoclonal antibody is produced by cells of hybridomas with a number of ATSS MOUTH-4017 and/or ATSS MOUTH-4018, or antibody or antibody fragment, derived from it, or

(ii) a monoclonal antibody or its 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, in particular pemphigus.

Cells hybrid with access numbers of ATSS MOUTH-4017 and ATSS MOUTH-4018 were deposited on 29 January 2002 in the American collection tipovi� cultures (ATSS), 10801 University Boulevard, Manassas, Virginia 20110-2209 (USA).

A medicament of the present invention may be presented in the form of pharmaceutical compositions together with pharmaceutically acceptable carrier. Preferably the pharmaceutical composition is introduced by injection or infusion, e.g., intravenously, intraarterially, subcutaneously, intraperitoneally or by other suitable means. The composition may be administered topically or systemically. Preferably the composition is injected systemically.

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

P�this, in accordance with the present invention, the drug can also be used in combination therapy with at least one additional therapy is effective against skin diseases associated with acantholysis of keratinocytes, in particular, against pemphigus. The drug should be used by itself or in combination with other immunosuppressive drugs, in particular, with steroids in order to reduce their dose and/or minimize chronic side effects. The combined use of the drug is preferably administered on a monthly basis. When use of the drug itself, it is preferably administered continuously for a certain period, depending on individual case.

Hereinafter the present invention will be disclosed in more detail in the following examples.

EXAMPLES

First method TUNEL staining was evaluated the presence of apoptosis in the epidermis of the skin around damage foci in frozen sections from patients with pemphigus who did not receive treatment. Fluorescent samples were analyzed by confocal scanning laser microscopy. In suprabasal layers of the epidermis around the foci of damage most of the keratinocytes was apoptosis compared with normal skin (Fig.1).

For tog� to confirm the presence of apoptosis in bubble damage we used formalin fixed and filled with paraffin biopsy and detected the active form of caspase-3. The method of staining was performed using system Ultra Vision LP Detection System AP Polymer & Fast Red Chromogen (Lab Vision Corporation, CA, USA) according to the manufacturer's instructions. Visualization was performed using tablets Fast Red naphthol phosphate substrate. In affected with pemphigus samples it was found that the fragment of caspase-3 is localized in the arch, and at the base of bubbles, and some cells were positive in the epidermis around damage foci (Fig.2). Apparently, this result suggests that cell death of keratinocytes occurs before the separation of keratinocytes, leading to acantholysis.

Since apoptotic keratinocytes are widely represented in pemphigus, we decided to investigate whether the serum of patients with pemphigus cause apoptosis in normal human keratinocytes. The keratinocytes were sown on the plates and were cultured in serum-free medium (KGM) to the state of preconferences. Then the cells were cultured in basal medium for keratinocytes and processed within 48 hours by addition of 25% serum from either patients not receiving treatment, or from patients receiving systemic corticosteroids. As a control, the blood serum of healthy subjects. And�optos was assessed using TUNEL staining in situ. Evaluated approximately 100 cells in randomly selected fields and counted the contents of TUNEL-positive cells. Serum of patients with pemphigus, but not the serum of healthy subjects or patients exposed to steroid treatment caused apoptosis in human keratinocytes (Fig.3 A-B).

Because 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 method dvuhsostavnogo enzyme immunoassay (ELISA). The concentration in serum was determined by absorption at 450 nm against a standard is recombinant protein of human FasL. The level of FasL was very high in the serum of patients not receiving treatment, and was below the limit of detection in the serum of patients receiving corticosteroid therapy, or in the serum of healthy subjects. As a positive control, the serum HBV patients (Fig.4A). In one patient the level of FasL progressively decreased with systemic steroid therapy (Fig.4B).

Since FasL is contained in large quantities in the serum of patients with pemphigus, we studied the expression of its own receptor FasR. This used formalin fixed and filled with paraffin biopsy and detected the active form of caspase-3. The method of staining was performed using systems� UltraVision LP Detection System AP Polymer & Fast Red Chromogen (Lab Vision Corporation, CA, USA) according to the manufacturer's instructions. Visualization was performed using tablets Fast Red naphthol phosphate substrate.

At the time, as in normal skin FasR expressively only in the basal keratinocytes in tissue lesions with active pemphigus FasR was detected both in basal and in suprabasal cells. Even more interestingly, cutaneous-mucosal pemphigus (PMC) FasR as if expressed in all layers of the epidermis and even before the formation of bubbles (Fig.5).

FasL is one of the main triggers activated by caspase-8 apoptotic way. We therefore decided to evaluate whether he's playing a role in apoptosis in pemphigus. For this purpose the serum of patients pre-treated with neutralizing antibody against FasL or inhibitor of caspase-8, which was added to culture keratinocytes. Keratinocytes were cultured in a COMMERCIAL environment and treated with serum of patients with pemphigus or serum of patients not receiving treatment. Serum pre-treated with neutralizing antibody against 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 using staining TLTNEL. The addition of neutralizing antibodies against FasL inhibitor or caspase-8 partially prevented apoptosis of keratinocytes induced by serum of patients with pemphigus (Fig.6).

In addition, keratinocytes were treated the same way as in Fig.6, and added either an antibody against FasL or foreign immunoglobulins. Then the cells are homogenized in RIPA buffer for analysis by western blotting. Membranes were incubated with antibodies against caspase-8 or β-actin. The relative intensity of the bands on autoradiographs analyzed quantitatively by scanning laser densitometry. The results showed that the caspase-8 was significantly activated in the keratinocytes treated with the serum of patients with pemphigus, compared with untreated cells, whereas caspana activity partially zingiberales pre-treated with antibody against FasL (Fig.7).

Overall, these results indicate that the serum of patients with pemphigus induced apoptosis of keratinocytes through the external apoptotic pathway triggered by system Fas/FasL.

Recent studies have shown that the components of the adhesion of 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 were treated for 72 hours under confluently keratinocytes cultured in the COMMERCIAL environment in the presence of 1.8 mm CaCl2, zaworotko� patients with pemphigus, exposed or not exposed to therapy. Protein extracts from cultures were analyzed by the method of western blotting using antibodies against Dsg1 and Dsg3. As an internal control, the β-actin. We found that the serum of patients with pemphigus able to cleave Dsg1 and Dsg3. In particular, the serum of patients not receiving treatment, but no patients under therapy with steroids, strikingly uncoupled Dsg1 and Dsg3 (Fig.8).

Most importantly, treatment of keratinocytes with increasing doses of FasL (0,1, 10, 100 ng/ml) for 72 hours caused the breakdown of proteins dsg dose-dependent manner. Protein extracts from cultures were analyzed by the method of western blotting using antibodies against Dsg1 and Dsg3. As an internal control, the β-actin. These doses correspond to doses detected in the serum of patients with pemphigus (Fig.9).

Given that FasL plays an important role in the pathogenesis of pemphigus, we tested the antibody against FasL (NOK2 - antibody produced by the cell line of hybridomas NOK2, the access number FERM BP-5045). Under confluently keratinocytes, cultivated in KGM with 1.8 mm CaCl2were treated within 72 hours: 1) only KGM; 2) an antibody to FasL (NOK2, 15 µg/ml), 3) FasL (500 ng/ml); 4) FasL + antibody to FasL. We received evidence that the antibody against FasL (NOK2) prevents induced FasL cleavage of dsg. We also showed �the antibody against FasL (NOK2) inhibits apoptosis, caused by caspase-8 (Fig.10A). From Fig.10B shows that the antibody against FasL (NOK2) prevents FasL caused intercellular separation, i.e. acantholysis.

In order to further confirm the Central role of FasL, we used another antibody against FasL (F918-7-3 - the antibody produced by the cell line of hybridomas with an access number FERM BP-5533; F918-7-4 - the antibody produced by the cell line of hybridomas with an access number FERM BP-5534; F919-9-18 - the antibody produced by the cell line of hybridomas with an access number FERM BP-5535). Under confluently keratinocytes, cultivated in KGM with 1.8 mm CaCl2were 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 received evidence that antibodies to prevent FasL FasL caused the splitting of dsg3 dose-dependent manner (Fig.11A and Fig.11B), caused by inhibiting caspase-8 activation in apoptosis (Fig.11A). From Fig.11C shows that the antibody to FasL contained in the medium from the cell line of hybridomas FERM BP-5535, prevents FasL caused intercellular separation, i.e., the acantholysis.

To set whether the external apoptotic pathway cleavage of dsg, we pre-processed under confluently keratinocytes inhibitor of caspase-8, Z-IETD-FMK (100 μm for 30 minutes) or an antibody to FasL (NOK2, 15 �kg/ml). Then the cells were incubated for 72 hours with serum of healthy subjects or in some untreated patients with pemphigus. Protein extracts from cultures were analyzed by the method of western blotting using antibodies against Dsg3 and anti-caspase-8. As an internal control was used vinculin (Fig.12A). From 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 FasL or apoptotic path-activated caspase-8, prevents the activation of caspase-8 and cleavage of dsg, thereby blocking the acantholysis.

In order to evaluate the role of human antibodies against FasL (human antibody 3E1 generated by the cell line of hybridomas with a number of ATSS MOUTH-4017 (antibody MOUTH-4017), and human antibody 4G11 produced by the cell line of hybridomas with a number of ATSS MOUTH-4018 (antibody MOUTH-4018)), keratinocytes were cultured in OHMS (1.8 mm CaCl2and was treated with recombinant FasL (50 ng/ml) by one or in combination with antibodies of the MOUTH-4017 and MOUTH-4018 at different dilutions. As a control, the humanized antibody BP-5035 and BP-5045 against FasL, already tested in Fig.10-12. From Fig.13A shows that while FasL itself cleaves Dsg3 and activates caspase-8, human antibodies� MOUTH-4017 and MOUTH-4018 and humanized antibodies BP-5035 and BP-5045 prevent this effect (western-blotting). From Fig.13C shows that while FasL itself that causes separation of cells and acantholysis, human antibodies of the MOUTH-4017 and MOUTH-4018 against FasL at different concentrations prevent this effect.

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 have shown that PV-IgG and antibodies against Fas-receptor (anti-FasR) cause damage in vitro in a similar manner, by causing: (1) secretion of soluble FasL; (2) increasing the cellular content of FasR, FasL (soluble and membrane), proteins Bax and p53; (3) a decrease in the level of cellular Bcl-2; (4) increase in 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), So the signaling pathway of cell death mediated by Fas, apparently, involved in the formation of foci of damage.

For the study of pemphigus for a long time was widely used well-founded the animal model. Passive transfer of PvIgG in newborn mice causes separation of cells and the formation of bubbles. The model was applied to evaluate the involvement of apoptosis and FasL in the pathogenesis of pemphigus. We enter�and subcutaneously PvIgG (5 mg/g body weight), purified from serum of patients, newborn C57BL/6N CrI. As a control, the normal newborn mice treated with IgG purified from serum of healthy subjects (NIgG). The animals were sacrificed 20 hours after the injection.

Staining with hematoxylin and eosin showed that the bubbles appear only in mice treated PvIgG, but not in mice treated with normal human IgG (Fig.14A). Apoptosis was detected either using TUNEL or activated caspase-3 only in mice treated PvIgG (Fig.14V).

In order to evaluate the role of FasL in vivo, mice were treated with PvIgG or PvIgG plus antibody against FasL (clone MFL3 specific for mouse). Antibody against FasL (40 µg/mouse) was administered 3 hours after injection PvIgG, and it prevented the formation of bubbles in mice, as seen by staining H&E. in addition, the length of cracks in mice treated with antibody to FasL were significantly decreased (Fig.15).

To compare the effectiveness of various anti-FasL antibodies (Fig.16) human keratinocytes were cultured in KGM with 1.8 mm l2and was treated with PVIgG (1.5 mg/ml) purified from the serum of patients containing antibodies against Dsgl and Dsg3, either alone or in combination with anti-FasL antibody at a concentration of 10 μg/ml Were used the following antibodies: human antibodies against human FasL produced hibrido�Neu cell line of ATSS MOUTH-4017, human antibodies against human FasL produced by hybrid cell line of ATSS MOUTH - 4018, and mouse antibodies against human FasL prior art (Puviani et al., 2003). Western blotting demonstrates that although PVIgG induces cleavage of Dsg3, human anti-FasL MOUTH-4017 are more effective in the prevention of spina desmoglein (acantholysis) in comparison with human anti-FasL MOUTH-4018 and mouse anti-FasL prior art (Puviani et al., 2003). The right panel shows densitometric analysis of Western blotting.

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 human monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), which
the amino acid sequence of sections that define complementarity (CDRs) of the heavy�Loy chain are:
(a1) CDR H1: Arg His Gly Ile Thr (SEQ ID NO:13)
(b1) CDR H2: Trp Ile Asn Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val Gln Gly (SEQ ID NO:15)
(c1) CDR H3: Glu Thr Met Val Arg Gly Val Pro Leu Asp Tyr (SEQ ID NO:17)
and the amino acid sequence of sections that define complementarity (CDRs) of the 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)
for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes.

2. The use according to claim 1, wherein the antibody or antigen-binding fragment selected from partially or fully humanized antibody, a partially or fully humanized single-chain antibodies or fragments thereof.

3. The use according to claim 1, wherein the skin disease is associated with activation of apoptotic path and/or splitting desmoglein.

4. The use according to claim 1, wherein the skin disorder is pemphigus (pemphigus).

5. The use according to any one of claims.1-4 in combination therapy with at least one additional therapy is effective against this skin disease.

6. The use according to claim 5 in combination with at least one additional immunosuppressive drug, in particular with at least trimduplicates steroid.

7. The use of monoclonal antibody or its antigen-binding fragment specific for the protein Fas ligand (FasL), produced by cells of hybridomas with a number of ATSS MOUTH-4017, for the manufacture of a medicament for the prophylaxis and/or treatment of skin diseases associated with acantholysis of keratinocytes.

8. The use according to claim 7, in which a skin disease associated with activation of apoptotic path and/or splitting desmoglein.

9. The use according to claim 7, in which skin disease is pemphigus (pemphigus).

10. The use according to any one of claims.7-9 in combination therapy with at least one additional therapy is effective against this skin disease.

11. The use according to claim 10 in combination with at least one additional immunosuppressive drug, in particular with at least one additional steroid.



 

Same patents:

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.

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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.

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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.

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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.

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43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. What is described is an immunoconjugates targeting CD138-expressing cells containing: IgG4 anti-CD138 antibody containing a heavy chain having CDRs from SEQ ID NO: 1 presented in the description and at least 70% identical to SEQ ID NO: 1, and a light chain having CDRs from SEQ ID NO: 2 presented in the description and at least 70% identical to SEQ ID NO: 2; and an effector molecule presenting a tubulin polymerisation inhibitor, and wherein the effector molecule is attached to the above engineered target antibody through a cleaved linker containing a disulphide bond. Besides, disclosed are a pharmaceutical composition and a kit for inhibition, delaying and/or prevention of tumour growth and/or CD138-expressing tumour cell propagation, wherein the composition contains the above immunoconjugate in an effective amount, and one or more pharmaceutically acceptable excipients; the kit comprises pharmaceutical compositions in independent containers, wherein one container comprises an effective amount of the pharmaceutical composition according to the present invention, while the other container contains a second pharmaceutical composition containing an effective amount of a co-drug, preferentially a cytotoxic agent, and one or more pharmaceutically acceptable excipients. There are presented the following methods implying administering the above immunoconjugate: a) a method of treating multiple myeloma in an individual; b) a method for immunoconjugate-mediated drug delivery; c) a method for inhibition, delaying and/or prevention of CD138-expressing tumour cell growth in a cell culture; d) a method for inhibition, delaying and/or prevention of the growth of a tumour containing CD138 tumour cells, and/or propagation of the tumour cells of this tumour in the patient; e) a method for inhibition, delaying and/or prevention of the tumour growth and/or propagation of CD138-expressing tumour cells in the patient; f) a method of treating the individual suffering a condition expected to be improved by suppressing myeloma cell survival; g) a method for inhibition, delaying and/or prevention of the growth of the tumour containing CD138 tumour cells, and/or propagation of the tumour cells in the individual; h) a method for reducing a cell count in a direct or mediated contact to the CD138-expressing tumour cells in the individual; i) a method for reducing a cell count in a direct or mediated contact to the CD138-expressing tumour cells.

EFFECT: invention enables preparing a homogenously targeted agent for treating the CD138-related diseases.

45 cl, 13 dwg, 5 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 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: claimed are: application of (R)-5-[3-chloro-4-(2,3-dihydroxypropoxy)benz[2]ylidene]-2-([2]-propylimino)-3-ortho-tolylthiazolidin-4-one (compound 1) or its salt for obtaining a medication for prevention and/or treatment of a disease or disorder, associated with the immune system activation, where the medication represents a set of Compound 1 doses; with the dose inducing the heart desensitisation in an initial phase of treatment and being lower than the final dose; with the said initial phase of treatment the dose is introduced with a frequency ensuring support of the heart desensitisation until the next acute reduction of heart rate occurs, after which the dose is titrated with increase to the final dose of Compound 1; a corresponding method of treatment and the set of doses.

EFFECT: invention ensures reduction and minimisation of undesirable side effects of Compound 1 (acute reduction of heart rate, atrioventricular conduction, or fatigue and dizziness) aimed at increase of Compound 1 tolerance and safety, as well as minimises problems, associated with monitoring at the initial phase of treatment or after interruption of treatment in the period of repeated treatment.

22 cl, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and represents a suspension for treating psoriasis, containing calcipotriol monohydrate in the form of nanocrystals having the particle size distribution within the range of 200-600 nm; the particles are dispersed in an aqueous phase containing a non-ionic polymer surfactant specified in a group consisting of a surfactant in the form of poloxamers or polysorbates, in the amount of 0.01-5 wt % calculated using a suspension for preventing development of aggregation and/or calcipotriol monohydrate nanocrystal growth; the calcipotriol monohydrate nanocrystals are produced in the suspension by processing the suspension by a method involving the stages of reduction in crystalline calcipotriol monohydrate particle size in an aqueous phase to form microparticles having the particle size distribution within the approximate range of 5-20 mcm and the average approximate particle size of 10 mcm; the suspension is exposed to three high-pressure homogenisation cycles for 7-15 minutes each; in the first, second and third cycles, the pressure makes 300-800 bars, 800-1,200 bars and 1,200-1,700 bars respectively.

EFFECT: invention provides creating the local composition containing calcipotriol as an active agent, however being free from propylene glycol as a solvent.

34 cl, 8 ex, 5 tbl, 9 dwg

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