Antibody preparation

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to formulations containing a human anti-CD20 monoclonal antibodies, a surfactant: polysorbate or poloxamer, trehalose, histidine buffer, to methods for preparing and using it.

EFFECT: group of inventions provides the formulations for enteral introduction of good storage stability at temperature 2-8° and 25°C and good stability at such physical factors, as aggregation, and chemical factors, as fragmentation.

5 cl, 2 ex, 2 tbl

 

This invention relates to a composition with anti-CD20 monoclonal antibody to the method of manufacture of the composition and applications of this structure.

The level of technology

The CD20 molecule (also called human B-lymphocyte differentiation antigen or VR) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 KD located on pre-b and Mature b lymphocytes (Valentine et al. (1989) J. Biol. Chem. 264(19):11282-11287; and Einfield et al. (1988) EMBO J. 7(3):711-717). CD20 is found on the surface of more than 90% of b-cells in peripheral blood or lymphoid organs, is expressed during early pre-b-cell development and is maintained to the stage of differentiation of plasma cells. CD20 is present on normal b cells and malignant b cells. In particular, CD20 is expressed on more than 90% of b-cell non-Hodgkin's lymphomas (non-Hodgkin's lymphomas, NHL) (Anderson et al. (1984) Blood 63(6): 1424-1433)), but it is not found on hematopoietic stem cells, Pro-b cells, normal plasma cells or other normal tissues (Tedder et al. (1985) J Immunol. 135(2):973-979).

85-amino acid stretch of carboxyl end of the protein CD20 is located in the cytoplasm. The length of this section differs from the length of other specific b-cell surface structures, such as IgM, IgD and heavy chains of IgG or antigens of the major histocompatibility class I1 α - or β-chains, to the that have a relatively short intracytoplasmic parcels 3, 3, 28, 15, and 16 amino acids, respectively (Komaromy et al. (1983) NAR 11:6775-6785). From the last 61 amino acids carboxyl end 21 is acid residue, whereas only 2 are basic, indicating that this area has a strong negative charge. The number in GenBank (GenBank Accession No NP-690605. It is believed that CD20 may participate in the regulation of the first stage(s) of the processes of activation and differentiation of b-cells (Tedder et al. (1986) Eur. J. Immunol. 25 16:881-887) and can function as channels for calcium ions (Tedder et al. (1990) J. Cell. Biochem. 14D: 195).

There are two different types of anti-CD20 antibodies, significantly differing in the nature of a CD20 binding and biological activities (Cragg, M.S., et al, Blood, 103 (2004) 2738-2743; and Cragg, M.S., et al, Blood 101 (2003) 1045-1051). Antibodies of the first type, such as rituximab, are powerful in relation mediated by complement cytotoxicity, whereas antibodies to type II, such as tositumomab (Bexxar®, B1), V and AT effectively initiate death of the target cells through the caspase-independent apoptosis with concomitant release of phosphatidylserine.

Common features of anti-CD20 antibodies (type I and II are shown in table 1.

Table 1
Properties anti-CD20 antibodies (type I and II
anti-CD20 is ntitle type I anti-CD20 antibody type II
the CD20 epitope type Ithe epitope of CD20 type II
Localization of CD20 into lipid raftsNo localization of CD20 into lipid rafts
Increased CDC (isotype IgG1)Reduced CDC (isotype IgG1)
Antibody-dependent cellAntibody-dependent cell
cytotoxicity (isotype IgG1)cytotoxicity (isotype IgG1)
Full binding capacityReduced binding capacity
Homotypically aggregationStronger homotypically aggregation
Apoptosis when cross stitchingStrong induction of cell death without cross-stitch

The invention

In one aspect the invention relates to a pharmaceutical composition containing:

from 1 to 150 mg/ml anti-CD20 antibody;

from 1 to 100 mm of a buffer;

perhaps from 0.001 to 1% surfactant; and

possibly from 1 to 800 mm tonicheskogo the agent;

at pH in the range from 4.5 to 7.0.

Preferably specified anti-CD20 antibody is an antibody type II. More preferably the specified anti-CD20 antibody is a humanized B-Ly1 antibody.

Detailed description of the invention

The term "antibody" includes various forms of antibodies, including, without limitation, whole antibodies, human antibodies, humanized antibodies and antibodies obtained by genetic engineering, such as monoclonal antibodies, chimeric antibodies or recombinant antibodies as well as fragments of such antibodies, as long as there are their characteristic properties in accordance with the invention.

"Fragments of antibodies include the portion of the antibody is a full length, usually at least the connecting portion of the antigen or its variable region. Examples of fragments of antibodies include double antibody, single-chain molecules, antibodies, immunotoxins and polyspecific antibodies formed from fragments of antibodies. In addition, fragments of antibodies include single-chain polypeptides having the characteristics of a VH chain, namely the ability to unite with the VL chain, or characteristics of the VL chain that binds the CD20 antigen, namely the ability to unite with the VH chain for the formation of functional antigen-binding pocket.

"Antibody fragments" also include these fragments, which themselves are not able to provide effector functions (ADCC/CDC), but provide these functions in accordance with the invention, after combining with a constant domain(s) of the corresponding antibodies.

Used in this document the terms "monoclonal antibody" or "composition with a monoclonal antibody" refers to the production of antibody molecules of single amino acid composition. Thus, the term "human monoclonal antibody" refers to antibodies, demonstrating the same binding specificity which have variable and constant region derived from human germline immunoglobulin sequences. In one embodiment the human monoclonal antibody produced by hybridoma, which includes a b cell obtained from a transgenic non-human animal, e.g. a transgenic mouse, with a genome containing the transgene of human heavy chain and human transgene light chain, merged with immortalizing cell.

The term "chimeric antibody" refers to monoclonal antibody containing the variable region, i.e. binding region, from one source or type, and at least part of a constant region derived from a variety of sources and types, typically obtained using the techniques of recombina is based DNA. Especially preferred are chimeric antibodies containing mouse variable plot and human constant plot. Such murine/human chimeric antibodies are the product of downregulation of immunoglobulin genes containing the DNA segments encoding the variable sections of mouse immunoglobulin, and the DNA segments encoding the constant parts of the human immunoglobulin. Other forms of "chimeric antibodies"covered by this invention are those in which the class or the subclass has been modified or changed compared to the original antibody. Such "chimeric" antibodies, also referred to as "antibodies with switching class. Methods of obtaining chimeric antibodies include conventional techniques of recombinant DNA and gene transfection, now well known in this field. See, for example, Morrison, S.L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. patent 5202238 and 5204244.

The term "humanitariannet antibody" refers to antibodies that have been modified frame areas or areas that define complementarity" (CDR) so that they contain the CDR of an antibody of a different specificity than the parent immunoglobulin. In the preferred embodiment of the murine CDR-grafted frame section of human antibodies to receive the "gumanitarnogo antibodies". See, nab is emer, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. Especially preferred CDRs correspond to those of the presented sequences that recognize these antigens for chimeric and bifunctional antibodies.

The term "human antibody", as used herein, includes antibodies with variable and constant parts derived from human germline immunoglobulin sequences. Human antibodies are well known in this area (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Pharmacol. 5 (2001) 368-374). On the basis of such technology it is possible to obtain human antibodies against a variety of targets. Examples of human antibodies are, for example, described in Kellermann, S. A., et al., Curr Opin Biotechnol. 13 (2002) 593-597.

The term "recombinant human antibody", as used herein, includes all human antibodies that receive, Express, create, or produce by using recombinant means, such as antibodies isolated from a host cell (host cell), such as cell NS0 or Cho, or animal (e.g. a mouse)that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector, transfitsirovannykh in the host cell (the cell-master). Such recomb Nannie human antibodies have variable and constant parts, derived from the germline sequence of a human immunoglobulin, in rearranged form. Recombinant human antibodies in accordance with the invention were subjected to somatic giperbolinovaya in vivo. Thus, the amino acid sequence of plots VH and VL of the recombinant antibodies are sequences that, being derived from and related to human germline sequences of VH and VL, may not naturally exist in the set of human germline antibody in vivo.

Used in this document the terms "specific binding" or "specifically binds to" refers to the antibody specifically binding to the antigen CD20. Preferably has a binding affinity of KD 10-9mol/l or lower (for example, 10-10mol/l), preferably a KD value of 10-10mol/l or lower (for example, 10-12mol/l). The affinity of binding is determined using standard analysis of binding, such as the method of surface plasma resonance (Biacore®).

The term "nukleinovokisly molecule"as used herein includes DNA and RNA. Nukleinovokisly molecule can be single-stranded or double-stranded, but it is preferable to double-stranded DNA.

"Constant domains" h is involved directly in binding the antibody to the antigen, but are involved in effector functions (ADCC, the reaction of complement fixation and CDC).

The term "variable area" (variable area light chain (VL), variable plot heavy chain (VH))used in this document, means a pair of light and heavy chains, which is directly involved in the binding of an antibody to the antigen. The variable domains of human light and heavy chains have the same General structure and each domain contains four frame plot (framework region, FR), the sequence of which is largely conservative, United the three hypervariable sites (or sites that define complementarity, CDR). Frame sections take the conformation of b-list, and the CDR can form loops connecting structure of the b-sheet. CDRs in each chain are held in their three-dimensional structure using the frame sections and form together with the CDRs from the other chain antigen-binding site. CDR3-areas of heavy and light chain antibodies play a particularly important role in the binding specificity/affinity of an antibody in accordance with the invention and, therefore, provide further subject of the invention.

The term "hypervariable area" or "antigennegative part of the antibody", as used herein, refers to amino acid residues of an antibody which are responsible is and binding to the antigen. Hypervariable site contains amino acid residues of the areas that define complementarity"or "CDR". "Frame parts", or "FR"are the portions of the variable domain, which differ from the residues of the hypervariable sites identified here. Thus, the light and heavy chains of the antibodies contain from N-Terminus to the C-end of the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In particular, CDR3 of the heavy chain is the portion which makes the greatest contribution to the binding to the antigen. The field of the CDR and FR are defined in accordance with the standard system definitions of Kabat, Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop".

The term "CD20" and "CD20-antigen" are used herein interchangeably and include any variants, isoforms and species homologs of human CD20, which are naturally expressed by cells or are expressed on cells transfected with CD20 gene. The binding of an antibody of the invention with the CD20 antigen mediates the destruction of cells expressing CD20 (e.g., tumor cells) by inactivation of CD20. The destruction of cells expressing CD20, may be performed by one or more of the following mechanisms.

Synonyms CD20 taken in this area include the B-lymphocyte antigen CD20, which-lymphocyte surface antigen B1, Leu-16, Bp35, BM5 and LF5.

The term "anti-CD20 antibody" in accordance with the invention refers to an antibody that is specific binds the CD20 antigen. Depending on the binding properties and biological activity of anti-CD20 antibodies CD20-antigen can distinguish between two types of anti-CD20 antibodies (type I and type II anti-CD20 antibody) according to Cragg, M.S., et al., Blood 103 (2004) 2738-2743; and Cragg, M.S., et al, Blood 101 (2003) 1045-1051, see table 2.

Table 2
Properties anti-CD20 antibodies (type I and II
anti-CD20 antibodies type Ianti-CD20 antibody type II
the CD20 epitope type Ithe epitope of CD20 type II
Localization of CD20 into lipid raftsNo localization of CD20 into lipid rafts
Increased CDC (isotype IgG1)Reduced CDC (isotype IgG1)
Antibody-dependent cellular cytotoxicity (lgG1 isotype)Antibody-dependent cellular cytotoxicity (isotype IgG1)
Full binding capacityReduced binding capacity
Homotypically aggregationStronger homotypically aggregation
Apoptosis when cross stitchingStrong induction of cell death without cross-stitch

One of the most important properties of anti-CD20 antibodies (type I and II is their way of bonding. Thus, anti-CD20 antibodies (type I and II can be classified according to the binding capacity of the specified anti-CD20 antibody to CD20 on Raji cells (ATSC No. CCL-86) compared with rituximab.

Used in this document, the term "anti-CD20 antibody can be used for antibody I or type II. Preferably this antibody type II.

Anti-CD20 antibodies of the first type are relevant binding capacity of the specified anti-CD20 antibody to CD20 on Raji cells (ATSC No. CCL-86) to the binding ability of rituximab from 0.8 to 1.2, preferably from 0.9 to 1.1. Examples of such anti-CD20 antibodies of the first type include rituximab (WO 94/11026), 1F5 IgG2a (ESAS, hybridoma; Press et al., Blood 69/2:584-591 (1987)), HI47 IgG3 (ESAS, hybridoma), 2S6 IgG1 (as disclosed in WO 2005/103081), 2F2 IgG1 (as disclosed in WO 2004/035607 and WO 2005/103081) and N IgG1 (as disclosed in WO 2004/056312). Preferably specified anti-CD20 antibody type I is a monoclonal antibody that binds to the same epitope as rituximab. AHTH-CD20 antibody type II have compared the binding properties of the specified anti-CD20 antibody to CD20 on Raji cells (ATSC No. CCL-86) to the binding ability of rituximab from 0.3 to 0.6, preferably from 0.35 to 0.55, more preferably from 0.4 to 0.5. Examples of such anti-CD20 antibody type II include tositumomab (B1 IgG2a), humanitariannet B-Ly1 antibody IgG1 (chimeric humanitariannet IgG1 antibody, disclosed in WO 2005/044859), 11 B8 IgG1 (as disclosed in WO 2004/035607) and AT IgG1. Preferably specified anti-CD20 antibody type II is a monoclonal antibody that binds to the same epitope as humanitariannet B-Ly1 antibody (disclosed in WO 2005/044859).

"The ratio of the binding ability of anti-CD20 antibodies to CD20 on Raji cells (ATSC No. CCL-86) to the binding ability with rituximab" determined by direct immunofluorescent analysis (measured by the mean fluorescence intensity (MFI, mean fluorescence intensity)) using said anti-CD20 antibody conjugated with Su, and rituximab conjugated to A in FACS analysis (Becton Dickinson) with Raji cells (ATCC no CCL-86), as described in example No. 2, and calculated as follows.

MFI is the mean intensity of fluorescence. Used herein, the term "degree S-tagging" means the number of molecules S-label on the antibody molecule.

Usually specified anti-CD20 antibody type I is the ratio of the binding capacity of the specified anti-CD20 antibodies (type I) with CD20 on Raji cells (ATSC No. CCL-86) to the binding ability with rituximab at,8 1,2, preferably from 0.9 to 1.1.

Usually specified anti-CD20 antibody type II has binding properties of the specified anti-CD20 antibodies (type I) with CD20 on Raji cells (ATSC No. CCL-86) to the binding ability of rituximab from 0.3 to 0.6, preferably from 0.35 to 0.55, more preferably from 0.4 to 0.5.

In the preferred embodiment of the specified anti-CD20 antibody type II, preferably humanitariannet B-Ly1 antibody has an increased antibody-dependent cellular cytotoxicity (ADCC).

Under used in this document, the term "antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" refers to an antibody with enhanced ADCC, which is determined by any suitable method known to specialists in this field. One of the accepted tests ADCC in vitro is the following:

1) the analysis uses the target cells, known to Express the target antigen recognized by the antigen-binding site antibodies;

2) as effector cells the analysis uses mononuclear cells of peripheral blood (MRPC, RVMS), isolated from the blood of randomly selected healthy donors;

3) the analysis is carried out in accordance with the following Protocol:

I) MRPC distinguish using standard procedures of centrifugation on density and suspended in an amount of 5×10 6cells/ml in cell culture medium RPMI;

II) the target cells are grown using standard methods of cultivation of tissues, harvested in exponential growth phase with a viability of more than 90%, washed in cell culture medium RPMI, mark 100 mccoury "CI, washed twice in cell culture medium and resuspended in cell culture medium with a density of 1 0' cells/ml;

III) 100 ál of the final cell suspension is transferred into each well of 96-well microplates;

IV) the antibody is serially-diluted from 4000 ng/ml to 0.04 ng/ml in cell culture medium and 50 μl of the obtained solutions antibodies are added to the target cells in 96-well-microtiter plate, testing in triplicate various concentrations of antibodies, covering the whole range of concentrations;

V) to control the maximum release (maximum release, MR) in three additional wells in the plate with labeled target cells contribute 50 μl of 2% (VN) aqueous solution of nonionic surfactant (Nonidet, Sigma, St. Louis) instead of the solution of antibodies (p. IV above);

VI) to control spontaneous release (spontaneous release, SR) in three additional wells in the plate with labeled target cells contribute 50 ál of cell culture medium RPMI instead of the solution of antibodies (p. IV above);

VII) then 96-well plate centrifuge is comfort at 50g for 1 minute and incubated for 1 hour at 4°C;

VIII) 50 μl of the suspension RVMS (p. V above) is added to each well for ratio of effector cells: target cells, 25: 1, and put the tablets in an incubator in an atmosphere with 5% CO2at 37°C for 4 hours;

IX) from each well collect cell-free supernatant and using a gamma-counter evaluate experimental release of radioactive substances (experimentally released radioactivity, ER);

X) the percentage of specific lysis calculated for each concentration of antibody in accordance with the formula (ER-MR)/(MR-SR)×100, where ER is the average radioactivity counted (see item IX above) for that the antibody concentration, MR is the average radioactivity counted (see item IX above) for the control of MR (see item V above), a SR is the average radioactivity counted (see item IX above) for the control of the SR (see item VI above);

4) "increased ADCC" is defined or how to increase the maximum percentage of specific lysis observed within the concentration of the antibodies described above and/or as a reduction in the antibody concentration required to achieve half of the maximum percentage of specific lysis observed within the concentration of the antibodies described above. The increase in ADCC correlated with ADCC, measured in the above analysis under the mediation of the same antibody, which was obtained by using t the th same type of host cells (host cells) using the same standard methods of obtaining, cleaning, preparation and storage, which are well known to specialists in this field, but which was not obtained using the host cells created for overexpression GnTIII.

Indicated "increased ADCC" can be obtained by applying glycotechnology to these antibodies, it means improving these natural cell-mediated effector functions of monoclonal antibodies by developing their oligosaccharide component as described in Umana, P., et al., Nature Biotechnol. 17:176-180 (1999) and U.S. patent No. 6602684.

The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells with antibodies in accordance with the invention in the presence of complement. CDC is measured preferably by the treatment of a preparation of cells expressing CD20, antibody anti-CD20 in accordance with the invention in the presence of complement. CDC is considered to be found if the antibody at a concentration of 100 nm for 4 hours induces lysis (cell death) of 20% or more of tumor cells. The analysis is performed preferably with tumor cells, labeled51Cr or Eu, and measured the release of51Cr or Eu. To control spend incubation of tumor target cells with complement, but without antibodies.

Usually anti-CD20 antibodies (type I and II isotype IgG1 demonstrate the characteristic properties CC. Anti-CD20 antibodies of the first type have an increased CDC (isotype IgG1), and anti-CD20 antibodies of type II have reduced CDC (isotype IgG1) in comparison with each other. Preferably the anti-CD20 antibodies and I and type II antibodies are of the IgG1 isotype.

Antibody "rituximab is a genetically engineered chimeric monoclonal antibody containing the human gamma 1 and murine constant domain that is directed against human CD20 antigen. This chimeric antibody contains a human gamma-1 constant domains and is called "SV" in W094/11026 (Anderson et al.). Rituximab is approved for the treatment of patients with relapsed or refractory, low-grade or follicular, CD20-positive b-cell non-Hodgkin lymphoma. Studies of the mechanism of action in vitro showed that rituximab shows the human complement-dependent cytotoxicity (CDC) (Reff et. al, Blood 83(2): 435-445 (1994)). In addition, he has been very active in assays that measure antibody-dependent cellular cytotoxicity (ADCC).

The term "humanitariannet B-Ly1 antibody" refers to humanitarianlaw B-Ly1 antibody, opened in WO 2005/044859, which was derived from murine monoclonal anti-CD20 antibody B-Ly1 (variable plot murine heavy chain (VH): SEQ ID No. 1; variable plot murine light chain (VL): SEQ ID No. 2 - see Poppema, S. and Visser, L, Biotest Bulletin 3: 13-139 (1987)) by chimerization with human constant domain of IgG1 and subsequent humanization (see WO 2005/044859). These "humanized B-Ly1 antibodies are disclosed in detail in WO 2005/044859.

Preferably "humanitariannet B-Ly1 antibody" has variable plot heavy chain (VH)selected from the group SEQ ID No. 3 to SEQ ID No. 20 (with IN-N IN-N and B-HL8, B-HL17 from WO 2005/044859). Especially preferred are SEQ ID№№3, 4, 7, 9, 11, 13 and 15 (IN-N, VN-3-N, N, B-HL8, B-HL11 and B-HL13 from WO 2005/044859). Most preferred of these VH is VN. Preferably "humanitariannet B-Ly1 antibody" has variable area light chain (VL) of SEQ ID No. 20 (B-KV1) from WO 2005/044859. In addition, humanitariannet B-Ly1 antibody is preferably IgG1-antibody. Preferably such humanized B-Ly1 antibodies obtained by modification of carbohydrate associated with the protein molecule (glycoengineered, GE) in the Fc-region in accordance with the procedures described in WO 2005/044859, WO 2004/065540, Umana, P., et al., Nature Biotechnol. 17:176-180 (1999) and WO 99/154342. The most exposed glycosidically humanized B-Ly1 antibodies have modified the structure of glycosylation in the Fc region, preferably having a reduced level of fokusnik residues. Preferably at least 40% or more (in one embodiment between 40% and 60%, in another embodiment at least 50%, and in yet another embodiment at least 70% or more) of the oligosaccharides of the Fc region are nefokusirana. In addition, the oligosaccharides of the Fc-region and preferably have a symmetrical branching. Most preferably "humanitariannet B-Ly1 antibody contains a VH-N and VL B-KV1 from WO 2005/044859. In this document, the specified antibody is also referred to as "HuMab < CD20>". In another most preferred embodiment of the specified antibody has a reduced level of fokusnik residues, as defined above, and/or oligosaccharides of the Fc-region is most preferably have a symmetrical branching. In another most preferred embodiment of the specified antibody shows increased ADCC as defined in this document.

Oligosaccharide component may substantially affect the properties related to the effectiveness of a therapeutic glycoprotein, including physical stability, resistance to degradation by a protease, interaction with the immune system, the pharmacokinetics and specific biological activity. Such properties may depend not only on the presence or absence of oligosaccharides, but also on their specific structures. You can make some generalizations between oligosaccharide structure and function of glycoprotein. For example, some oligosaccharide structures mediate the rapid elimination of the glycoprotein from the bloodstream through the interaction with specific uglevodsvyazyvayushchie proteins, whereas others may be contacted with antibodies and cause unwanted immune Rea is tion (Jenkins et al., Nature Biotechnol. 14:975-81 (1996)).

Mammalian cells are preferred hosts for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in a more suitable for use in human form (Cumming et al., Glycobiology 1:115-30 (1991); Jenkins et al., Nature Biotechnol. 14:975-81 (1996)). Bacteria rarely glycosylase proteins, and other common types of hosts, such as yeast, filamentous fungi, insect cells and plants, which produce patterns of glycosylation associated with rapid elimination from the bloodstream, unwanted immune interactions, and in some specific cases, the reduction of biological activity. Among the mammals in the last two decades, the most frequently used cells of the ovary of the Chinese hamster (Chinese hamster ovary, CHO). In addition to this a suitable structure of the glycosylation of these cells allow the consistent offspring genetically stable, high-performance clonal cell lines. They can be cultivated with high density in a simple bioreactors using serum-free media and allow you to develop a safe and reproducible biological processes. Other commonly used animal cells include cells of the kidneys in baby hamster (baby hamster kidney, BHK), mouse NSO myeloma cells and SP2/0. Just n of the a long time has also been tested products using transgenic animals (Jenkins et al., Nature Biotechnol. 14: 975-981 (1996)).

All antibodies contain carbohydrate structures in the conservative positions in the constant parts of the heavy chains, each isotype with different network N-linked carbohydrate structures with different power to affect the protein structure, secretion or functional activity (Wright, A., and Morrison, S.L., Trends Biotech. 15: 26-32 (1997)). The structure of the attached N-linked carbohydrates varies considerably depending on the degree of processing, and may also include vysokonadezhnye complex oligosaccharides, widely branched and with two branches (Wright, A., and Morrison, S.L., Trends Biotech. 15: 26-32 (1997)). There are typically heterogeneous processing core oligosaccharide structures attached to a specific glycosylation site, so even monoclonal antibodies exist in several glycoform. In addition, it was shown that between cell lines there are significant differences in the glycosylation of antibodies, and even when culturing the cell line under different conditions observed minor differences (Lifely, M.R. et al., Glycobiology 5(8):813-22 (1995)).

One way to achieve a significant increase in activity, while maintaining a simple process of production and, possibly, avoiding significant adverse effects, is to strengthen the natural cell-mediated the effector functions of monoclonal antibodies by engineering their oligosaccharide component, as described in Umana, P., et al., Nature Biotechnol. 17:176-180 (1999) and U.S. patent No. 6602684. Antibody type IgG1, most frequently used in cancer immunotherapy, are glycoproteins that have a conservative N-linked glycosylation site Asn297 in each CH2 domain. Two complex oligosaccharide with two branches attached to Asn297, hidden between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for mediating effector functions of antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al., Glycobiology 5: 813-822 (1995); Jefferis, R., et al., Immunol. Rev. 163: 59-76 (1998); Wright, A. and Morrison, S.L., Trends Biotechnol. 15: 26-32 (1997)).

Previously it was shown that overexpression in the cells of the Chinese hamster ovary (Cho) β(1,4)-N-acetylglucosaminyltransferase I11 ("GnTII17y), glycosyltransferases that catalyze the formation of oligosaccharides with two branches, significantly increases the in vitro ADCC activity antiaromatase chimeric monoclonal antibody (chCE7)produced by projecting cells Cho (see Umana, P., et al., Nature Biotechnol. 17: 176-180 (1999); and WO 99/154342, the content of which is fully incorporated by this reference). Antibody chCE7 belongs to a wide class of unconjugated monoclonal antibodies that have a high tumor affinity and specificity, but too low activity to be clinically for the serious, when produced in standard industrial cell lines, which lack the enzyme GnTIII (see Umana, P., et al., Nature Biotechnol. 17: 176-180 (1999)). This study first showed that a significant increase in ADCC activity can be achieved by creating cells that produce antibodies that they expressed GnTIII, which also leads to an increase in the share of constant area (Fc)-associated oligosaccharides with two branches, including nefokusirana oligosaccharides with two branches, above the levels found in natural antibodies.

The term "expression of CD20 antigen" is meant to emphasize a significant level of expression of CD20 antigen in the cell, preferably on the cell surface of T - and b-cells, more preferably cells from the tumor or cancer, respectively, preferably solid tumors. Patients with CD20-expressing cancer" can be identified using standard assays known in the field. "Expression of CD20 antigen" also preferably used to indicate a significant level of expression of CD20 antigen in the cell, preferably on the cell surface of T - and b-cells, more preferably the b cells in autoimmune diseases. For example, the expression of CD20 antigen is measured by immunohistochemistry (IHC, immunohistochemical detection), FACS analysis (flow, cytometry the definition of analysis) or by PCR-based detection of the corresponding mRNA.

The term "CD20-expressing cancer"as used herein refers preferably to lymphoma (preferably b-cell nehodgkinski lymphomas (NHL)and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, (a) follicular lymphoma, b) lymphoma of small cells with unsplit cores/Burkitt's lymphoma (including the endemic Burkitt's lymphoma, sporadic lymphoma Burkitt and nahodkinskuju lymphoma Burkitt) lymphoma marginal zone (including extranodal b-cell lymphoma marginal zone lymphoma associated with lymphatic tissue mucosa, MALT, nodal b-cell lymphoma marginal zone lymphoma marginal zone of the spleen), (d) lymphoma of the cells of the mantle zone (MCL), e) both lymphoma including diffuse In-both lymphoma (DLCL), diffuse lymphoma, mixed cell type, immunoblastic lymphoma, primary b-cell lymphoma of the mediastinum, angiocentric lymphoma/pulmonary b-cell lymphoma), f), hairy cell leukemia, g) lymphocytic lymphoma, macroglobulinemia waldenstrom, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/lymphoma of the small lymphocytes (SLL), b-cell prolymphocytic leukemia, i) neoplasm of plasma cells, myeloma of plasma cells, multiple myeloma, plasma is itomo, j) Hodgkin's disease.

Preferably CD20-expressing cancer is a b-cell non-Hodgkin lymphoma (NHL). Especially the CD20-expressing cancer is lymphoma mantle cell (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), b-cell diffuse both lymphoma (DLCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, lymphoma marginal zone, post-transplantation lymphoproliferative disorder (PTLD), HIV-associated lymphoma, macroglobulinemia waldenstrom or primary lymphoma of the Central nervous system.

Used in this document, the term "autoimmune disease" refers to a disease or disorder associated with and directed against their own body tissues. Examples of autoimmune diseases or disorders include arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with the 1 5 inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, respiratory distress syndrome in adults (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions, soprovojdayuschiesya T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, deficiency of leukocyte adhesion, systemic lupus erythematosus (SLE), juvenile diabetes, multiple sclerosis, allergic encephalomyelitis, immune reactions associated with immediate hypersensitivity and delayed type, mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, Wegener, including Wegener's granulomatosis, agranulocytosis, vasculitis (including N-associated), aplastic anemia, anemia diamond-Blackfan, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), malignant anemia, partial krasnocletocnaya aplasia (PRCA), a deficiency of factor VIII, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, inflammatory diseases of the Central nervous system (CNS)syndrome of multiple organ lesions, myasthenia gravis, a disease mediated by a complex of antigen-antibody disease autoantibodies to the basal membrane, antiphospholipid syndrome, allergic neuritis, Behcet's disease, syndrome Castleman syndrome?, myasthenic syndrome Lambert-Eaton syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson, bullous pemphigoid, disease, autoimmune polyendocrinopathy, nephropathy, IgM polyneuropathies or IgM-op is radovanu neuropathy, idiopathic thrombocytopenic purple (ITP), thrombotic thrombocytopenic purple (TTP), autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison disease, graves ' disease, autoimmune polyglandular syndromes (or polyglandular syndromes type I), type I diabetes, also called insulin-dependent diabetes mellitus type I (IDDM) and Sheehan syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV-associated), obliterative bronchiolitis (without transplantation) with NSIP (non-specific interstitial pneumonia, nonspecific interstitial pneumonia), Guillain-Barre syndrome, vasculitis of large vessels (including rheumatic rheumatica and giant cell arteritis diagnostics ('s arteritis)), vasculitis of medium vessels (including Kawasaki disease and nodular arthritis), ankylosing spondylitis, Berger disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, trophic atty (gluten enteropathy), cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary heart disease, but are not limited to.

therapeutic formulations of the antibodies, used in accordance with this invention are prepared for storage by mixing the antibody having the desired degree of purity, with the possible pharmaceutically acceptable carriers, excipients and stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients working in the doses and concentrations.

The term "surfactant", as used herein, refers to pharmaceutically acceptable surface-active agent. In the composition of the invention, the amount of surfactant is described by the percentage, expressed as weight/volume. The most commonly used units of weight/volume are mg/ml Suitable pharmaceutically acceptable surfactants include nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG, PEG), but are not limited to. In addition, they include esters of fatty acids and polyethylenimine, polyethylene-polypropyleneglycol, polyoxyethylenated and dodecylsulfate sodium, but are not limited to. The preferred polyethylenimine are esters of polyethylene(20)sorbitan (synonym Polysorbate 20 sold under the trademark Tween 20™) and poly is xiatian(20)-orbitonasal (synonym Polysorbate 80, sold under the trademark Tween 80™). The preferred polyethylene-polypropyleneglycol are those sold under the names Pluronic® F68 or Poloxamer 188™. Most preferred is Poloxamer 188™. The preferred polyoxyethylenesorbitan are those sold under the trademark Myrj™. Preferred monolaurate esters of polyoxyethylene are those sold under the trademark Brij™. When using complex ether polietilensorbit-polyethylene(20)sorbitan (Tween 20™) and polyoxyethylene(20)sorbitrate (Tween 80™), they are mainly used in amounts of from about 0.001 to about 1%, preferably from about 0.005-about 0.1%, even more preferably from about 0.01% to about 0.04 percent (weight/volume).

The term "buffer"as used herein, refers to pharmaceutically acceptable buffer. Suitable pharmaceutically acceptable buffer includes his-tag buffers, citrate buffers, succinate buffers, acetate buffers and phosphate buffers, but is not limited to them. Preferred buffers include L-histidine, or mixtures of L-histidine hydrochloride, L-histidine with isotonic agents and may undergo pH adjustment using acid or base known in this field. Most preferred is L-histidine. Above gested the new buffers, generally used in a concentration of from about 1 mm to about 100 mm, preferably from about 5 mm to about 50 mm, and more preferably about 20 mm. Regardless of which buffer the pH is adjusted to values of about 4.5 to about 7.0 and preferably from about 5.5 to 6.5, more preferably about 6.0, or using acid or base known in this field, or by using appropriate mixtures of buffer components, or both.

The term "isotonic agents", as used herein, refers to pharmaceutically acceptable isotonic agents. Isotonic agents are used for isotonic composition. Isotonic composition is a liquid or liquid recovered from the solid form, such as lyophilized, and refers to a solution having the same toychest, as well as some other solutions with which it is compared, for example, physiological saline and serum. Suitable isotonic agents include (but are not limited to salts, including sodium chloride (NaCl) or potassium chloride, but not limited to, sugars, including glucose, sucrose, trehalose, but not limited to, glycerin, and any component from the group of amino acids, sugars, salt, and their combination is. Isotonic agents, generally used at a final concentration from about 5 mm to about 350 mm.

The term "liquid"used herein in connection with the composition in accordance with the invention, means a composition that is liquid at a temperature of at least from about 2 to about 8°C.

The term "dried"as used herein in connection with the composition in accordance with the invention, means a composition that is dried by freezing of the composition, and then subjected to sublimation of ice from the frozen contents using any of the methods of freeze drying known in this field, for example using commercially available devices drying freeze.

The term "salt"used herein denotes a salt in an amount of from about 1 mm to about 500 mm. Non-limiting examples of salts are salts of any of the combinations of the cations sodium, potassium, calcium or magnesium chloride, phosphate, citrate, succinate, sulfate anions, or mixtures thereof.

The term "amino acid"as used herein refers to the amino acid in amount from about 1 to about 100 mg/ml, including arginine, glycine, ornithine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, isolate is, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, Proline, but not limited to.

The term "sugar"as used herein, refers to pharmaceutically acceptable sugar is used in an amount of from about 25 mm to about 500 mm. Preferred is a number from 100 to 300 mm. More preferred is a number of from 220 to 260 mm. Most preferred is an amount of 240 mm. Suitable sugars include monosaccharides and disaccharides, but not limited to. Non-limiting examples of sugar in accordance with the invention are trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucamine (so-called "meglumine"), galactosamine and Narimanova acid, and combinations thereof. Most preferred is trehalose.

The term "stabilizer" refers to pharmaceutically acceptable stabilizers, such as, for example, amino acids and sugar, described in the previous sections, as well as a commercially available dextrans of any type and molecular weight, known in this area, but is not limited to them.

The term "antioxidant" means a pharmaceutically acceptable antioxidant. It may include excipients, such as methionine, benzyl alcohol, or any other excipient is, used for information, oxidation to a minimum.

The term "treatment" or its equivalent be used, for example, to cancer, refers to a procedure or course of action intended to reduce or eliminate the number of cancer cells in a patient or alleviate symptoms of cancer. "A method of treating cancer or other proliferative disorders does not necessarily mean that the cancer cells or other disorder can actually be eliminated, the number of cells or the disorder may actually be reduced or that the symptoms of cancer or other disorders may actually be weakened. Often a method of treating cancer will be performed even with a low probability of success, but when is nevertheless considered, taking into account the history of the illness and the estimated duration of survival of the patient, that it will have improving effects.

In one aspect, the invention relates to the composition with the antibody anti-CD20 containing:

from about 1 to about 150 mg/ml antibody anti-CD20,

from about 0.001 to about 1% of at least one surfactant, and

from about 1 to about 100 mm of a buffer

at pH from about 4.5 to about 7,0.

In a more preferred embodiment the composition according to the invention contains:

from about 1 to about 150 mg/ml antibody anti-CD20,

- the t of roughly 0.005 to about 0.05% of at least one surfactant, and

from about 1 to about 100 mm of a buffer

at pH from about 4.5 to about 7,0.

In the preferred embodiment of the composition according to the invention contains:

from about 10 to about 30 mg/ml anti-CD20 antibody type II

- 20 mm L-histidine

- 240 mm trehalose, and

- a 0.02% (weight/volume) Polysorbate 20

at pH about 6.

In another preferred embodiment the composition according to the invention contains:

from about 10 to about 30 mg/ml anti-CD20 antibody type II

- a 0.02% (weight/volume) poloxamer 188™,

- 20 mm L-histidine, and

- 240 mm trehalose

at pH about 6.

Preferably the anti-CD20 antibody is an antibody type 1. More preferably the anti-CD20 antibody is an antibody type II. Even more preferably the anti-CD20 antibody is a "humanized B-Ly1 antibody, as described in detail in WO 2005/044859. Most preferably the antibody is HuMab < CD20>. The composition contains the indicated antibody in an amount of from 1 to about 150 mg/ml, more preferably from about 5 to about 100 mg/ml of the indicated antibodies, more preferably from about 10 to about 30 mg/ml of the indicated antibodies or number selected among approximately 5, 10, 15, 20, 25 or 30 mg/ml of the indicated antibodies, and most preferably 25 mg/ml of the indicated antibodies.

The composition in accordance with the laws the AI with the invention can be in liquid form, in dried form or in liquid form recovered from the freeze-dried form.

In one embodiment, the composition in accordance with the invention is a lyophilized composition. Freeze-dried composition in accordance with the invention has the advantage that the increase in stability due to the formation of particles and aggregates of higher molecular weight, which usually is difficult to achieve with liquid formulations with the same concentration of the described anti-CD20 antibodies.

The composition in accordance with the invention can be administered intravenously (in/in), subcutaneously (s/C) or by any other parenteral route, known in the pharmaceutical field.

In addition, the composition in accordance with the invention may contain preservatives (e.g., chloride octadecylsilane-ammonium; chloride hexadecane, benzalkonium chloride, chloride benzene; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; chelating agents such as EDTA; soleobrazutaya counterions such as sodium; metal complexes (e.g. Zn-protein complexes).

Also active phrases which you can be entered in manufactured microcapsules, for example, by methods koatservatsii or by polymerization on the phase boundary, for example in hydroxymethylcellulose or gelatin-microcapsules and polymetylmetacrylate microcapsules, respectively, in colloidal drug-delivery systems (e.g. liposomes, albumen the microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such techniques are disclosed in Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Can be manufactured drugs slow release. Suitable examples of drugs slow release are semi-permeable matrices of solid hydrophobic polymers containing the antibody, which form in the form of rounded particles, e.g. films, or microcapsules. Examples of matrices for sustained release include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly (vinyl alcohol), polylactide (U.S. patent 3773919), copolymers of L-glutamic acid and gamma-ethyl-1-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic acid and glycolic acid, such as the LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid and glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxybutiric acid.

Compositions that will be used for administration in vivo, the Dol shall have to be sterile. This is easily accomplished by filtration through membrane for sterilizing filter.

Preferably the composition of the invention contains one or more isotonic agent in an amount of from about 5 mm to about 350 mm, as defined herein above.

Preferably the composition of the invention contains a sugar in an amount of from about 25 mm to about 500 mm, as defined above.

Also preferably, the composition of the invention also contains one or more of the following ingredients: antioxidants, ascorbic acid, glutathione, preservatives, such as m-cresol, phenol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, thiomersal, benzalkonium, polyethylene glycol, such as PEG 3000, 3350, 4000, 6000, albumin, human serum albumin (HSA), bovine serum albumin (BSA), a polyhydric alcohol, glycerol, ethanol, mannitol, salts, acetate salts (for example sodium acetate, magnesium chloride, calcium chloride, tromethamine, EDTA (e.g. Na-EDTA).

Also preferably, the composition of the invention also contains one or more stabilizer as defined above, and ingredients, also known in this area as "neoprotestant" (lyoprotectant), such as sugars, sugar alcohols, amino acids and dextrans, known in this field.

In a particular embodiment the composition of the invention contains follow what their compositions in liquid form, freeze-dried form or in liquid form, restored from lyophilised forms:

15 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.01% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

140 mm sodium chloride

at pH 6.0;

or

10 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.01% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

140 mm sodium chloride

at pH 6.0;

or

15 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

perhaps from 0.001 to 1% (weight/volume) surfactants,

20 mm L-histidine

at pH 6.0;

or

10 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

0.02% Polysorbate 20 (weight/volume),

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

0.02% Polysorbate 20 (weight/volume),

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1, most preferably HuMab < CD20>

of 0.02% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.01% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.01% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

0,1% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.02% (weight/volume) Polysorbate 80,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

0,1% (weight/volume) Polysorbate 80,

20 mm acetate, and

240 mm trehalose

at pH 5.5;

or

25 mg/ml anti-CD20 antitle type, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

0,1% (weight/volume) Polysorbate 80,

20 mm acetate, and

140 mm sodium chloride

at pH 5.5;

or

30 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.01% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

200 mm trehalose

at pH 6.5.

In the preferred embodiment of the composition according to the invention the composition is in lyophilized form and contains after recovery with the appropriate amount of water for injection:

10 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo B-Ly1 antibody, most preferably HuMab < CD20>

of 0.02% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0.

This composition demonstrates good stability when stored at 2-8°C and 25°C with sufficient stability against physical destinations such as aggregation, and chemical end-points, such as fragmentation.

In the preferred embodiment of the composition according to the invention the composition is in liquid form:

25 mg/ml anti-CD20 antibody type II, preferably gumanitarnogo BLy1 antibodies, most preferably HuMab < CD20>

0,02% weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0.

In a preferred embodiment the composition is used to prevent or reduce metastasis or further dissemination of such a patient suffering from a CD20-expressing cancer. The composition used for increasing the life expectancy of such patients, increasing the survival rate of such patients without progression, increasing the duration of response, which leads to statistically significant and clinically noticeable improvement in treated patients, determined by life expectancy, survival without progression, response rate to treatment or duration of response. In a preferred embodiment the composition is used to increase the proportion of responses to treatment in patients.

In the context of this invention in combination with a composition with anti-CD20 antibody in accordance with the invention can be used for additional other cytotoxic, chemotherapeutic or anti-cancer agents, or compounds that enhance the effects of such agents.

Such agents include, for example: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX, for example, cytoxan®), chlorambucil (CHL, for example, leykeran®), cisplatin (CisP, for example, platinol®), busulfan (e.g., mileran®), melphalan, carmustine (BCNU), p is eposition, triethylenemelamine (TEA), mitomycin C, etc.; antimetabolites, such as methotrexate (MTX), etoposide (VP16, for example, Vepesid®), 6-mercaptopurine (6MP), 6-tioguanin (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g., xeloda®), dacarbazine (DTIC) and the like; antibiotics, such as actinomycin D, doxorubicin (DXR, for example, adriamycin®), daunorubicin (daunomycin), bleomycin, and mithramycin the like; alkaloids, such as Vinca alkaloids such as vincristine (VCR), vinblastine, and the like; and other antitumor drugs, such as paclitaxel (e.g., Taxol®) and derivatives of paclitaxel, cytostatic agents, glucocorticoids such as dexamethasone (DEX, for example, decadron®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, enzymes that destroy amino acids, such as asparaginase, leucovorin and other derivatives of folic acid, and the like of various antitumor agents. As an additional means can also be used following agents: amifostine (for example, ethyol®), dactinomycin, mechlorethamine (nitrogen analogue of mustard), streptozocin, cyclophosphamide, lomustin (CCNU), liposomal doxorubicin (e.g., doxil®), gemcitabine (for example, Gemzar®), liposomal daunorubicin (for example, daunoxome®), procarbazine, mitomycin, docetaxel (e.g., Taxol the p®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy-ethyl-7-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubitsin, mesna, beta-interferon, alpha-interferon, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, tioguanin, thiotepa, uracil mustard, vinorelbine, chlorambucil. Preferably used in combination for treatment with anti-CD20 antibody without such additional agents.

The use of cytotoxic and anticancer agents described above, as well as antiproliferative specific targets of anticancer drugs, such as inhibitors of protein kinases in chemotherapeutic regimens, as a rule, are well characterized in the field of cancer therapy, and their use here is based on the same considerations monitoring tolerance and efficiency and to control the routes of administration and doses, with some modifications. For example, the actual doses of cytotoxic agents can vary depending on the response of the cell culture of the patient, identified using the methods of cultivation of tissues. Typically, the dose will be reduced compared to the amount used in the absence of additional other agents./p>

A typical effective dose of the cytotoxic agent can be in the range recommended by the manufacturer, and being identified as responses in vitro or responses in animal models, they can be reduced to about one order of magnitude of the concentration or quantity. Thus, the actual dose will depend on the decision of the physician, the patient, and therapeutic method based on the reaction in vitro primary cultured malignant cells or cultured tissue sample, or the reactions observed in relevant animal models.

In the context of the present invention can be ionizing radiation in an effective amount and/or in addition to composition with an anti-CD20 antibody in accordance with the invention, it is possible to use radioactive drug. The radiation source can be external or internal to the patient receiving treatment. If the source is external to the patient, therapy is called external radiation therapy (EBRT, external beam radiation therapy). If the source is internal to the patient, therapy called brachytherapy (BT, brachytherapy). Radioactive atoms for use in the context of the present invention can be selected from the group including radium, cesium-137, iridium-192, americium-241, gold-198, Koba the before-57, copper-67, technetium-99, iodine-123, iodine-131 and indium-111, but is not limited to them. You can also mark such antibodies with radioactive isotopes. Preferably the composition of the anti-CD20 antibody in accordance with the invention is used without such ionizing radiation.

Radiation therapy is the standard way of control irresectable or inoperable tumors and/or tumor metastases. Improved results were seen when combining radiation therapy with chemotherapy. Radiation therapy is based on the principle that high doses of radiation delivered to the target area, leading to the deaths of reproductive cells into the tumor and normal tissues. The mode of radiation dose, typically defined in the form of absorbed dose of radiation (Gy), time and fractionation and must be carefully determined by the oncologist. The amount of radiation that a patient receives will depend on various factors, but the two most important are the localization of the tumor in relation to other critical structures or organs of the body and the degree of tumor spread. A typical course of treatment for a patient who is undergoing radiation therapy, flows through the schedule from 1 to 6 weeks with a total dose of from 10 to 80 Gy, administered to the patient 5 days a week parts from approximately 1.8 to 2.0 Gy per day. In the preferred embodiment of this the image is placed introduction people with tumor composition in accordance with the invention and the radiation act synergistically. In other words, the inhibition of tumor growth using agents containing a combination of the invention is enhanced in combination with radiation, possibly with an additional chemotherapeutic or antineoplastic agents.

The parameters of adjuvant radiation therapy, see, for example, in WO 99/60023.

The composition of the antibody is administered to the patient in accordance with known methods by intravenous bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, vnutrizonovoy, subcutaneous, intra-articular, intrasynovial or intrathecal route. Intravenous or subcutaneous administration of the antibody is preferred.

The invention also provides a set characterized by the contents of the container, a composition within the container containing the composition antibodies anti-CD20, as well as insert with instructions for using the composition for the introduction of a specified structure with the anti-CD20 antibody to a patient suffering from a CD20-expressing cancer.

The term "liner" refers to the instructions, usually included in commercial packages of therapeutic products, which may contain information about the indications, usage, dosage, introduction, contraindications and/or warnings concerning the use of such therapeutic products.

In site Cetelem embodiment, the product container may also include a pharmaceutically acceptable carrier. The product may also include a sterile solvent, which is preferably stored in a single secondary container.

Used in this document, the term "pharmaceutically acceptable carrier" includes any and all materials that are compatible with the introduction of drugs, including solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic and delaying absorption agents, and other materials and compounds that are compatible with the introduction of drugs. Except when any conventional medium or agent is incompatible with the active compound, its use in the compositions of the invention may be seen. Supplementary active compounds can also be included in the composition.

In yet another embodiment of the invention, the composition in accordance with the invention contains an anti-CD20 antibody type 1, which is administered together with an anti-CD20 antibody type II in accordance with the invention. The compositions in accordance with the invention can be two separate structures for each of the anti-CD20 antibody. An alternative structure in this case may also contain both antibodies in a single composition.

In yet another embodiment of the invention, the composition in accordance with the invention contains an anti-CD20 antibody, and the specified anti-CD20 antibody centuries the field, together with anti-l-2 active agent. The term "l-2"as used herein refers to a protein l-2 (ID Swiss Prot (Swiss database of amino acid sequences) No. R), member of a family of proteins Bcl-2. The term "active agent is an anti-Bcl-2" includes "anti-Bcl-2 antisense nucleotides and Bcl-2 inhibitors". "Anti-Bcl-2 antisense nucleotides" reduce the mRNA level of Bcl-2 and reduced expression of the protein Bcl-2. Examples of such anti-Bcl-2 antisense nucleotides include oblimersen and SPC-2996. The term "ABT-737"as used herein means N-[4-[4-(4'-chlorobiphenyl-2-ylmethyl)piperazine-1-yl]benzoyl]-3-[3-(dimethylamino)-1(R)-(phenylsulfanyl)propylamino]-4-nitrobenzenesulfonamide; 4-[4-(4'-chlorobiphenyl-2-ylmethyl)piperazine-1-yl]-N-[3-[3-(dimethylamino)-1(R)(phenylsulfanyl)propylamino]-4-nitrophenylarsonic]benzamid, Bcl-2 inhibitor, which is described in WO 2006/099667 or Corey, S., et al., Cancer Cell (2005) 5-6. The term "W-263", as used herein, means Bcl-2 inhibitor, is described in U.S. patent 2007027135. Preferably the active agent is an anti-Bcl-2 choose among oblimersen, SPC-2996, TA-402, hossipole, at-101, drug Obatoclax mesylate (obatoclax mesilate), AND-371191, A-385358, AND-438744, ABT-737, at-101, BL-11, BL-193, GX-15-003, 2-methoxyaniline And3ON-14-1, KF-67544, purpurogallin, TP-TW-37, YC-137 and Z-24. Preferably the active agent is an anti-Bcl-2 is a Bcl-2-protein-binding inhibitor with S anti-Bcl-2 inhibitor active is 5 μm or less. This Bcl-2-protein-binding inhibitor, preferably chosen among hossipole, at-101, drug Obatoclax mesylate, ABT-263 and ABT-737, more preferably from ABT-263 or ABT-737.

In yet another embodiment of the invention, the composition in accordance with the invention contains an anti-CD20 antibody, and the specified anti-CD20 antibody is administered together with an inhibitor of the proteasome. The term "proteasome inhibitor", as used herein, refers to agents that inhibit the activity of the 26S proteasome. These proteasome inhibitors include, in particular, derivatives of peptides, such as peptide aldehydes (e.g., MG132, MG115, SØR-1615, PSI or specific inhibitor immunoproteasome IPSI-001 (Cbz-LnL-CHO = N-carbobenzoxy-leucyl-norleucinal, see U.S. patent 20060241056), boronate peptides (e.g., bortezomib (PS-341) or DFLB), epoxyketone peptides (for example, Apoksiomen, dihydrophenazine or derived ephemeria carfilzomib (PR-171)) or vinylsulfonic peptides (for example, NLVS), and ones derived such as salinosporamide A (NPI-0052), derivatives salinosporamide And lactacystin or derivatives lactacystin (for example, glasto-lactacystin-L-lactone (muraled) or PS-519). Describes the different types and patterns of these proteasome inhibitors, such as Kisselev, A.L, et al., Chem Biol (2001) 739-758, WO 2004/004749 and Joazeiro, C., et al., Res 66(16) (2006) 7840-7842), Kanagasabaphy, et al., Curr Opin Investig Drugs 8 (2007) 447-51, Adams, J., Nat Rev Caner 4 (2004) 349-360 and in U.S. patent 20060241056.

Preferably such a proteasome inhibitor choose among peptide aldehydes (preferably N-carbobenzoxy-leucyl-norleucinal (IPSI-001)), peptide boronates (preferably bortezomib (PS-341)), peptide epoxyketone (preferably derived epoxidizing carfilzomib (PR-171)) or salinosporamide A (NPI-0052). More preferably, such an inhibitor of proteasomes choose among bortezomib (PS-341), carfilzomib (PR-171), salinosporamide A (NPI-0052) or N-carbobenzoxy-leucyl-norleucinal (IPSI-001).

In the preferred embodiment, the proteasome inhibitor is a peptide derivative selected among peptide aldehydes (preferably N-carbobenzoxy-leucyl-norleucinal (IPSI-001)), peptide boronates (preferably bortezomib (PS-341)or peptide epoxyketone. In another preferred embodiment, the proteasome inhibitor is a peptide boronate (preferably bortezomib (PS-341), see, for example, Adams, Cur. Opin. Chem Biol. 6 (2002) 493-500 and U.S. patent 5780454)).

Preferably the inhibitor of the proteasome has antiproteases inhibitory activity with IC50 of 5 μm or less, more preferably 1 μm or less. Cellular analysis to identify those proteasome inhibitors and to determine the IC50 antiproteases inhibitory activity (through serial dilutions and calculations using appropriate Nelly the nonlinear curve (XLfit software (ID Business Solution Ltd, Guildford, Surrey, UK)) described in Moravec et al., Cell Notes 15 (2006) 4-7 and uses reagent for cell analysis Proteasome-Glo™ from Promega cells U266 human myeloma plasma). This analysis dimension with the addition of the mix to measure chymotrypsin-like protease activity associated with the proteasome in cultured cells.

In addition to the IPSI-001 (Cbz-LnL-CHO = N-carbobenzoxy-leucyl-norleucinal) preferred proteasome inhibitors also are the following derivatives of the peptides of U.S. patent 20060241056:

N-carbobenzoxy-homophenylalanine-phenylalanyl, N-carbobenzoxy-leucyl-phenylalanyl, N-carbobenzoxy-alanyl-phenylalanyl, N-carbobenzoxy-glycyl-prolyl-alanyl-phenylalanyl, N-carbobenzoxy-glycyl-prolyl-i.e. phenylalanyl-phenylalanyl, N-carbobenzoxy-glycyl-i.e. phenylalanyl-phenylalanyl, N-carbobenzoxy-leucyl-normativnopravovoe acid, N-carbobenzoxy-i.e. phenylalanyl fenilalanina acid, N-carbobenzoxy-homophenylalanine-fenilalanina acid, N-carbobenzoxy-leucyl-fenilalanina acid, N-carbobenzoxy-glycyl-prolyl-alanyl - fenilalanina acid, N-carbobenzoxy-glycyl-prolyl-i.e. phenylalanyl fenilalanina acid, N-carbobenzoxy-leucyl-leucyl-fenilalanina acid, N-carbobenzoxy-glycyl-i.e. phenylalanyl-phenylalanine is Aronova acid, N-carbobenzoxy-leucyl-norleucine-methylphenylsulfonyl, N-carbobenzoxy-i.e. phenylalanyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-homophenylalanine-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-leucyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-alanyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-glycyl-prolyl-alanyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-glycyl-prolyl-i.e. phenylalanyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-leucyl-leucyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-glycyl-i.e. phenylalanyl-phenylalanine-methylphenylsulfonyl, N-carbobenzoxy-leucyl-norleucine-epoxyketone, N-carbobenzoxy-i.e. phenylalanyl-phenylalanine-epoxyketone, N-carbobenzoxy-homophenylalanine-phenylalanine-epoxyketone, N-carbobenzoxy-leucyl-phenylalanine-epoxyketone, N-carbobenzoxy-alanyl-phenylalanine-epoxyketone, N-carbobenzoxy-glycyl-prolyl-alanyl-phenylalanine-epoxyketone, N-carbobenzoxy-glycyl-prolyl-i.e. phenylalanyl-phenylalanine-epoxyketone, N-carbobenzoxy-leucyl-leucyl-phenylalanine-epoxyketone and N-carbobenzoxy-glycyl-i.e. phenylalanyl-phenylalanine-epoxyketone.

The following examples and graphic materials are provided to facilitate understanding of the present invention, the true limits of which are set out in the accompanying claims. You need to understand h is about to set forth the procedures can be made without deviation from the invention.

EXAMPLES

Example 1

Were made following compositions in liquid, lyophilized form or in liquid form, recovered from dried:

15 mg/ml HuMab < CD20>

of 0.01% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

140 mm sodium chloride,

at pH 6.0;

10 mg/ml HuMab < CD20>

of 0.01% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

140 mm sodium chloride,

at pH 6.0;

15 mg/ml HuMab < CD20>

20 mm L-histidine

at pH 6.0;

10 mg/ml HuMab < CD20>

of 0.02% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

240 mm trehalose,

at pH 6.0;

25 mg/ml HuMab < CD20>

of 0.02% (weight/volume) Polysorbate 20,

20 mm L-histidine, and

240 mm trehalose,

at pH 6.0.

Also make freeze-dried form, which contains after recovery with the appropriate amount of water for injection:

10 mg/ml HuMab < CD20>

of 0.02% (weight/volume) Polysorbate 20,

20 mm L-histidine

240 mm trehalose,

at pH 6.0.

This composition demonstrates good stability when stored at 2-8°C and 25°C with sufficient stability against physical destinations such as aggregation, and chemical end-points, such as fragmentation.

Liquid and lyophilized formulations of medicinal products for parenteral administration in accordance with izobreteniem were developed as follows.

The production of liquid compositions.

The compositions of HuMab < CD20> were made by homogenization of solutions of HuMab < CD20> buffer for products (for example, 20 mm his-tag buffer at pH of about 6.0 or 20 mm his-tag buffer at pH about 6.0 and containing 140 mm sodium chloride and 0.01% (weight/volume)Polysorbate 20). The compositions of HuMab < CD20> can also be obtained by adjusting the protein concentration to the desired concentration by dilution with buffer. If necessary, was added excipients to stabilize the protein and to fit toychest, they can be added in dissolved form or alternatively in solid form. Optionally, the compositions were added surfactant in the form of stock solution. All formulations were sterilized by filtration using filters with a pore size of 0.22 μm, was filled Liquefiable in sterile glass vials and closed with rubber stoppers and lids Alucrimp. These formulations were stored at different temperatures for different time intervals and used for analysis in the time specified in separate paragraphs. The compounds were analyzed by 1) using UV spectrophotometry, 2) using exclusion chromatography (size exclusion chromatography, SEC), 3) the presence of visible and invisible particles, 4) using ion exchange chromatography (ion exchange chromatography, IEC) and (5) on the turbidity of the solution.

Manufacturer lio is lizirovania compositions.

The solution HuMab < CD20> or prepared in accordance with the description above for liquid formulations, or produced as a homogenized solution HuMab < CD20> from HuMab < CD20> 20 mm his-tag buffer at pH about 6.0 and containing sugar and surfactant. All formulations were sterilized by filtration using filters with a pore size of 0.22 μm and was filled Liquefiable in sterile glass vials. The vials were partially closed by rubber plugs, suitable for use in lyophilization processes, and transferred into the drying chamber of lyophilizate. In the framework of the present invention is used any method of drying known in this field. For example, the lyophilization process used for this study includes cooling the composition to room temperature to approximately 5°C (pre-cooling), and then freezing at -40°C (freezing I) with changing temperature from about 1°C./min to 5°C/min In the first stage of drying may occur, increasing the temperature from -40°C to -30°C at the rate of 0.3 to 0.5°C/min, and then is dried at a temperature of -30°C for at least 50 hours at a pressure in the chamber from approximately 75 to 80 mtorr. In the second stage of drying may occur, increasing the temperature from -30°C to 25°C with a speed of 0.1 to 0.3°C/min, and then is dried at the is the temperature value of 25°C for at least 50 hours at a chamber pressure from about 50 to 80 mtorr (applicable schedule of drying are shown in table 1). It is recognized that the compositions of HuMab < CD20>that were dried using the described processes lyophilization, convenient for a quick recovery for about 2-3 minutes. All lyophilized mass in this study had a residual water content of from about 0.1 to 1.0%, according to research by the Karl Fischer method. Vials with lyophilized and kept at different temperatures for different time intervals. Lyophilized compositions were restored in an appropriate volume of water for injection (WFI, water for injection), and then 1) was analyzed using UV spectrophotometry, 2) was determined by the recovery time 3) was analyzed using exclusion chromatography (size exclusion chromatography, SEC), 4) using ion exchange chromatography (ion exchange chromatography, IEC), 5) determined the presence of visible and invisible particles, 6) was determined by the turbidity of the solution.

Exclusion chromatography (SEC) was carried out to identify soluble high-molecular groups (aggregates) and low molecular weight hydrolysis products in the compositions. This method was applied with a suitable tool for HPLC equipped with UV detector (wavelength detection 280 nm) and column Bond GF-250 (a 9.4×250 mm, Agilent)as the mobile phase used a 200 mm solution of sodium phosphate with a pH of 7.0.

Ion exchange chromatography (IEC) was carried out to identify the products of chemical degrades and, changing the total charge HuMab < CD20> in the compositions. This method was applied with a suitable tool for HPLC equipped with UV detector (wavelength detection at 220 and 280 nm) and column Dionex Propac WCX-10 (4 mm × 250 mm); mobile phase a and b are used respectively 10 mm sodium phosphate buffer in water with a pH of 6.0 and 10 mm sodium phosphate buffer pH 6.0+0,75 NaCl with a flow rate of 1.0 ml/min

To determine the concentration of protein was performed by UV-spectroscopy on the spectrophotometer Varian Cary Bio at a wavelength of 280 nm.

To determine the turbidity was measured opalescence in FTU (units turbidity) using turbidimeter HACH 2100AN at room temperature.

The samples were analyzed for the presence of invisible particles using a HIAC Royco PharmaSpec (HRLD-150), as well as visible particles with a tool for visual inspection Seidenader V90-T.

Example 2

Were made following compositions either in liquid or in lyophilized form or in liquid form, recovered from dried:

25 mg/ml HuMab < CD20>

of 0.02% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml HuMab < CD20>

of 0.01% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml HuMab < CD20>

0,1% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml HuMab < CD20>

of 0.02% (weight/volume) Polysorbate 80,

20 mm L-histidine, and

240 mm trehalose

at pH 6.0;

or

25 mg/ml HuMab < CD20>

0,1% (weight/volume) Polysorbate 80,

20 mm acetate, and

240 mm trehalose

at pH 5.5;

or

25 mg/ml HuMab < CD20>

0,1% (weight/volume) Polysorbate 80,

20 mm acetate, and

140 mm sodium chloride

at pH 5.5;

or

30 mg/ml HuMab < CD20>

of 0.01% (weight/volume) poloxamer 188™,

20 mm L-histidine, and

200 mm trehalose

at pH 6.5.

Liquid and lyophilized formulations of the drug product for parenteral administration was prepared as follows.

The production of liquid compositions.

The compositions of HuMab < CD20> produced by homogenization of solutions of HuMab < CD20> buffer for products (for example, 20 mm his-tag buffer at pH about 6.0 and containing 240 mm trehalose and 0.02% (weight/volume)) poloxamer 188™). The compositions of HuMab < CD20> also can be obtained by diafiltration 10-40 mg/ml solutions of HuMab < CD20> buffer for products (for example, 20 mm his-tag buffer at about pH 6,0) by tangentially-flow filtration (TFF, tangential flow filtration) to increase the protein concentration above the target concentration of the be the spacecraft and to replace the buffer. The compositions of HuMab < CD20> can also be obtained by adjusting the protein concentration to the desired concentration by dilution with buffer. To stabilize the protein and to fit toychest added excipients in dissolved form or alternatively in solid form. Optionally, the compositions were added surfactant in the form of stock solution. All formulations were sterilized by filtration using filters with a pore size of 0.22 μm, was filled Liquefiable in sterile glass vials and closed with rubber stoppers and lids Alucrimp. These formulations were stored at different temperatures for different time intervals and used for analysis in the time specified in separate paragraphs. The compounds were analyzed by 1) using UV spectrophotometry, 2) using exclusion chromatography (size exclusion chromatography, SEC), 3) the presence of visible and invisible particles,

4) using ion exchange chromatography (ion exchange chromatography, IEC) and

5) on the turbidity of the solution.

Manufacturer of lyophilised compositions.

Solutions HuMab < CD20> have been prepared in accordance with the description above for liquid formulations. All formulations were sterilized by filtration using filters with a pore size of 0.22 μm and was filled Liquefiable in sterile glass vials. The vials were partially closed by rubber plugs, suitable for use in the project in the processes of lyophilization, and transferred into the drying chamber of lyophilizate. In the framework of the present invention is used any method of drying known in this field. For example, the lyophilization process used for this study includes cooling the composition to room temperature to approximately 5°C (pre-cooling), and then freezing at -40°C (freezing I) with changing temperature from about 1°C./min to 5°C/min In the first stage of drying may occur, increasing the temperature from -40°C to -30°C at the rate of 0.3 to 0.5°C/min, and then is dried at a temperature of -30°C for at least 50 hours at a pressure in the chamber approximately 75 to 80 mtorr. In the second stage of drying may occur, increasing the temperature from -30°C to 25°C with a speed of 0.1 to 0.3°C/min, and then is dried at a temperature of 25°C for at least 50 hours at a chamber pressure from about 50 to 80 mtorr (applicable schedule of drying are shown in table 1). In the compositions of HuMab < CD20>that were dried using the described processes lyophilization, was identified residual water content of from about 0.1 to 1.0%, according to research by the Karl Fischer method. Vials with lyophilized and kept at different temperatures for different time intervals. Lyophilized compositions were restored in an appropriate volume of water is La injection (WFI, water for injection), and then 1) was analyzed using UV spectrophotometry, 2) were analyzed using exclusion chromatography (size exclusion chromatography, SEC), 3) using ion exchange chromatography (ion exchange chromatography, IEC), 4) determined the presence of visible and invisible particles, 5) was determined by the turbidity of the solution.

Exclusion chromatography (SEC) was carried out to identify soluble high-molecular groups (aggregates) and low molecular weight hydrolysis products in the compositions. This method was applied with a suitable tool for HPLC equipped with UV detector (wavelength detection 280 nm) and column Bond GF-250 (a 9.4×250 mm, Agilent) or TSKgel G3000 SWXL (of 7.8×300 mm); mobile phase used a 200 mm solution of sodium phosphate with a pH of 7.0 or 250 mm solution of potassium chloride in 200 mm solution of potassium phosphate pH 7.0.

Ion exchange chromatography (IEC) was carried out to identify the products of chemical degradation, changing the total charge HuMab < CD20> in the compositions. This method was applied with a suitable tool for HPLC equipped with UV detector (wavelength detection at 220 and 280 nm) and column Dionex Propac WCX-10 (4×250 mm). As mobile phase a and b are used respectively 10 mm sodium phosphate buffer in water with a pH of 6.0 and 10 mm sodium phosphate buffer pH 6.0+0,75 NaCl with a flow rate of 1.0 ml/min

To determine the concentration of protein was performed by UV-spectroscopy at spectropho is metre Varian Cary Bio or Perkin Elmer at a wavelength of 280 nm.

To determine the turbidity was measured opalescence in FTU (units turbidity) using turbidimeter HACH 2100AN at room temperature.

The samples were analyzed for the presence of invisible particles using a HIAC Royco PharmaSpec (HRLD-150), as well as visible particles with a tool for visual inspection Seidenader V90-T.

1. The composition in lyophilized form containing:
10 mg/ml HuMab < CD20>
of 0.02% (wt./about.) Polysorbate 20,
20 mm L-histidine, and
240 mm trehalose
at pH 6.0.

2. The composition containing:
25 mg/ml HuMab < CD20>
of 0.02% (wt./about.) poloxamer 188™,
20 mm L-histidine, and
240 mm trehalose
at pH 6.0;
or
25 mg/ml HuMab < CD20>
0,01% (wt./about.) poloxamer 188™,
20 mm L-histidine, and
240 mm trehalose
at pH 6.0;
or
25 mg/ml HuMab < CD20>
0,1% (wt./about.) poloxamer 188™,
20 mm L-histidine, and
240 mm trehalose
at pH 6.0;
or
25 mg/ml HuMab < CD20>
of 0.02% (wt./about.) Polysorbate 80,
20 mm L-histidine, and
240 mm trehalose at pH 6.0;
or
30 mg/ml HuMab < CD20>
0,01% (wt./about.) poloxamer 188™,
20 mm L-histidine, and
200 mm trehalose at pH 6.5.

3. The composition according to claim 2, where it is in liquid form and contains:
25 mg/ml HuMab< CD20>
of 0.02% (wt./about.) poloxamer 188™,
20 mm L-histidine, and
240 mm trehalose
at pH 6.0.

4. Applying a composition according to any one of claims 1 to 3 for the manufacture of medicaments used to treat D20-related diseases.

5. The use according to claim 4, where the disease is selected from the group consisting of b-cell non-Hodgkin lymphoma (NHL), lymphoma mantle cell (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), b-cell diffuse both lymphoma (DLCL), Burkitt lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, lymphoma marginal zone, post-transplantation lymphoproliferative disorder (PTLD), HIV-associated lymphoma, macroglobulinemia waldenstrom or primary CNS lymphoma.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to chemical-pharmaceutical industry and represents the use of lanost-8,24-dien-3-ols for preparing pharmaceutical compositions inhibiting higher activation of serine/threonine protein kinases, or modulated transcription factors.

EFFECT: invention provides the use of lanost-8,24-dien-3-ols as anti-inflammatory, anticancer, analgesic agents acting by inhibition of enzymes activity of which is related with proliferation of specific types of cancer.

15 cl, 5 ex, 10 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quaternary ammonium salts of meso-tetra[1-(4'-bromobutyl)-3-pyridyl]bacteriocholine of general formula where

EFFECT: compounds possess high photoinduced activity and may be used for the photodynamic therapy of malignant growths as a photosensitiser.

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SUBSTANCE: group of inventions refers to pharmacology and medicine. The group of inventions involves pharmaceutical compositions containing 5-azacytidine for the oral introduction wherein the compositions release a cytidine analogue substantially in stomach, a method of treating an individual suffering a disease associated with abnormal cell proliferation which involves the oral introduction of the pharmaceutical composition into the individual, using 5-azacytidine for preparing the pharmaceutical composition for treating the disease associated with abnormal cell proliferation.

EFFECT: invention provides higher clinical effectiveness.

9 tbl, 9 ex, 23 dwg

FIELD: medicine.

SUBSTANCE: method includes application of homeopathic preparation, obtained by double successive dilution with physiological solution to 1: 104 of heterologic liquor of patients with brain tumours after radical operation without development of recurrences at the moment of liquor obtaining. Said preparation is introduced to sexually mature non-pedigree rats with inoculated sarcoma 45, when tumours reach the dimensions 0.7-1.3 cm3, into subclavian vein in volume 0.3-0.4 ml depending on animal's weight. Introduction is performed 2 times within 2 weeks.

EFFECT: method ensures anti-tumour effect, being experimental basis for studying application of liquor of a different species in oncology.

5 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula I:

,

where X1 denotes a bond, NR8 or S; Y1 denotes O or NR ; R1 denotes C1-10alkyl, C6-10aryl or a 5-10-member heteroaryl containing 1-3 heteroatoms which are independently selected from N or S; where said R1 is optionally substituted with 0-2 J1; R2 denotes H or C1-10alkyl; each of R3, R4, R5 and R6 independently denotes H or C1-10alkyl; and R7 denotes C1-10alkyl, C3-10cycloalkyl, phenyl, 5-6-member heterocyclyl containing 1-3 heteroatoms independently selected from O and N, - (C1-6alkyl) -(C3-10cycloalkyl), - (C1-6alkyl) - (phenyl) or -(C1-6alkyl)-(6-member heterocyclyl containing 2 heteroatoms selected from O and N); where said R7 is optionally substituted with 0-5 J7; or R3 and R4, together with a carbon atom with which they are bonded, optionally form a 3-4-member saturated or partially unsaturated monocyclic fragment; R3 and R5, together with carbon atoms with which they are bonded, optionally form a 5-member monocyclic fragment; R8 denotes H; R9 denotes H or unsubstituted C1-6alkyl; or R2 and R9, together with atoms with which they are bonded, optionally form a 5-member aromatic monocyclic fragment containing 3 nitrogen atoms; each J1 independently denotes C1-6halogenalkyl, halogen, NO2, CN, Q or -Z-Q; or two J1 together can optionally form =O; Z denotes C1-6alkyl, wherein 0-3 carbon atoms are optionally substituted with -NR-, -O-, -C(O)- or -SO2-; wherein each Z is optionally substituted with 0-2 J2; Q denotes H; C1-6alkyl; 3-8-member aromatic or non-aromatic monocyclic fragment containing 0-3 heteroatoms independently selected from O, N and S; or an 8-10-member aromatic bicyclic system; each Q is optionally substituted with 0-2 JQ; each J7 independently denotes C1-6alkyl or halogen(C1-4alkyl); each of JQ and J7 independently denotes M or -Y-M; each Y independently denotes an unsubstituted C1-6alkyl, wherein 0-3 carbon atoms are optionally substituted with -O-, -C(O)- or -SO2-; each M independently denotes H, C1-6alkyl, C3-6cycloalkyl; halogen (C1-6alkyl), phenyl, halogen, CN, OH, OR1; or two M together can optionally form =O; R denotes H or an unsubstituted C1-6alkyl; R' denotes an unsubstituted C1-6alkyl. The invention also relates to methods of producing said compounds and a pharmaceutical composition for inhibiting PLK based on said compounds.

EFFECT: novel compounds which can be used in medicine as inhibitors of protein kinase are obtained.

34 cl, 1 tbl, 279 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to particular compounds, which demonstrate inhibiting activity with respect to ERK, whose structure formula is given in description, to their pharmaceutically acceptable salts, based on them pharmaceutical composition and their application for treatment of cancer, mediated by ERK activity.

EFFECT: obtaining compounds, which demonstrate inhibiting activity with respect to ERK.

5 cl

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SUBSTANCE: claimed invention relates to novel compounds of formula (V), compounds of formula (IX), compounds of formula (XIII) or their tautomers or pharmaceutically acceptable salts, which are capable of inducing Hsp70, as well as to pharmaceutical composition, which contains claimed compounds.

Value of substituents in formulas (V), (IX) and (XIII) are such, as claimed in invention formula.

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12 cl, 12 ex, 36 dwg, 2 tbl

FIELD: medicine.

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1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to new heteroaryl compounds of general formula (I) and their pharmaceutically acceptable salts possessing the properties of protein kinase inhibitor, such as mTOR, IKK-2, Tyk2, Syk-kinase. In formula (I) R1 represents substituted or unsubstituted C1-8alkyl, substituted or unsubstituted aryl, specified in phenyl, substituted or unsubstituted 5-6-member heteroaryl with 1-3 nitrogen atoms in a cycle specified in pyridine, pyrazole, indole, indazole, triazole, benzimidazole, 2-(1H-imidazo-[4,5-b]pyridine, substituted or unsubstituted 5-7- member cycloalkyl or substituted or unsubstituted heterocycloalkyl specified in pyrrolidinyl; -X-A-B-Y- taken together form -N(R2)CH2C(O)NH-, -N(R2)C(O)CH2NH-, -N(R2)C(O)NH-, -N(R2)C=N- or -C(R2)=CHNH-; L represents a direct bond, NH or O; R2 represents substituted or unsubstituted C1-8alkyl, substituted or unsubstituted aryl specified in phenyl, tetrahydronaphthalene, unsubstituted 5-7- member mono- or 8- member bicycloalkyl; and R3 and R4 independently represent H or C1-8alkyl. The substitutes in the substituted groups are specified in one or more halogen, C1-8alkyl, hydroxyl, amino, nitro, thiol, C1-4alkyl thioether, cyano, carboxyl, C1-4alkyl ester, halogen alkyl, C6cycloalkyl or heteroaryl specified in pyridyl, triazole, O-lower alkyl, aryl specified in phenyl, phenyl-lower alkyl, CO2CH3, CONH2, OCHF2, CF3 or OCF3 groups wherein CONH2 group may be substituted by cyclohexyl.

EFFECT: compounds can find application for treating or preventing cancer, inflammatory pathological conditions, metabolic pathological conditions.

24 cl, 8 dwg, 2 tbl, 169 ex

FIELD: medicine.

SUBSTANCE: invention refers to crystalline forms of 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid (formula I), pharmaceutical compositions and dosage forms containing these crystalline forms, as well as to methods for preparing such crystalline forms and methods for using them for treating, preventing a disease or a disorder associated with premature terminating codon. There are prepared new crystalline forms of 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid which are non-absorbent and which can find application in medicine for treating or preventing such diseases or disorders as type III mucopolysaccharidosis, hemophilia A, hemophilia B, neurofibromatosis 1, neurofibromatosis 2, Parkinson's disease, cystic fibrosis, macular degeneration, cephalooculocutaneous telangiectasis, retinitis pigmentosa, tuberous sclerosis, Duchenne muscular dystrophy and Marfan's syndrome, cancer.

EFFECT: higher effectiveness of using the compounds and a method of treating.

46 cl, 11 dwg, 9 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine and described a water-based liquid composition for activation of intraocular penetration of a compound of formula (I) containing an amide compound, and an agent forming an ionic pair. What is also described is a method for activation of intraocular penetration of the compound presented by formula (I), and using the compound prepared by formula (I).

EFFECT: invention provides activation of penetration of the compound of formula (I), as well as maintaining the pre-set concentration of the drug preparation, even if a periodicity of the composition administration is reduced.

14 cl, 7 tbl, 2 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to pharmaceutical compositions for preparing infusion solutions of antibacterial and antimycotic preparations, and to methods for preparing them. The declared pharmaceutical compositions are presented in the form of powder, contain sodium chloride and dextrose and particles of colloidal silicone dioxide among which a portion of particles of 5 mcm and less makes less than 35%, in certain weight proportions. A method for preparing said pharmaceutical compositions consists in the fact that sodium chloride in the form of powder and dextrose in the form of powder are mixed with powdered colloidal silicone dioxide in certain proportions; the prepared mixture is mechanically treated by impact abrasion to increase a weight portion of fine particles of silicone dioxide of 5 mcm or less to min. 35%.

EFFECT: group of inventions provides the intensified therapeutic effectiveness of parenteral forms of the antibacterial and antimycotic preparations.

4 cl, 4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to a device for assisting substance oral administration comprising an internal tube (110) encasing a first cavity (120), an external tube (170) encasing a second cavity (180) with the second cavity accommodating a space section (182) adjoining an inlet of the external tube and containing a substance; a separation space (130) is presented in the second cavity with the separation space adjoining the space section and the first cavity; there is also a connective passage (160) of selective action between the space section and a pressure compensation space for selective supply of a substance flow from the space section through the first cavity. Besides, the invention refers to a kit of parts comprising the device according to the invention, a pre-set amount of the substance in a cavity with the cavity being a part of the kit, or a pre-set amount of the substance in the space section, as well as a package leaflet.

EFFECT: invention concerns a method for filling the device according to the invention, as well as a method for oral administration with the use of the device according to the invention.

15 cl, 10 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, particularly, to an oral liquid pharmaceutical composition containing desmopressin, and to its use in treating central diabetes insipidus, primary nocturnal enuresis, bleedings in the patients suffering hemophilia A and von Willebrand's disease, and postoperative complications.

EFFECT: invention provides stability of the pharmaceutical composition containing desmopressin, enables flexible dosage, causes no mucosal irritation.

14 cl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to pharmaceutics, particularly an aqueous composition containing water and protein of molecular weight more than approximately 47 kDa, and to methods for preparing said composition. The aqueous composition under the present invention is rich in proteins and has the low conduction levels ensured by the low content of ionic excipients. The amount of the invention also involves such compositions containing water and proteins which have a low osmolarity or hydrodynamic diameter which is less approximately by 50% thereof in a buffer solution in the given concentration with protein in the composition having a molecular weight more than approximately 47 kDa.

EFFECT: invention provides protein stability in the solution.

42 cl, 31 ex, 77 tbl

FIELD: medicine.

SUBSTANCE: invention relates to pharmacology and medicine, in particular, to gastroenterology, and represents medication for intestinal lavage, which includes sodium chloride, potassium chloride, sodium phosphate disubstituted, citric acid, sodium acetate, magnesium sulphate and boiled water, characterised by the fact that it additionally contains sorbite, with medication components being in specified ratio.

EFFECT: invention ensures achievement of comfortable, predicted complete emptying of intestine, which does not cause unpleasant sensations and impairment of electrolytic composition of blood and tissues.

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are offered: light-stable pharmaceutical composition containing vitamin B12, butanol and butaphosphane, its use for preparing a drug; use of butanol and butaphosphane for preparing the drug; use of butanol and butaphosphane for light-stabilising vitamin B12 in the pharmaceutical composition and use of butanol and butaphosphane for preparing the pharmaceutical composition of vitamin B12 with improved light-stability of vitamin B12.

EFFECT: what is shown is improved light-stability of the composition as related to the n-butanol free composition after 3-hour light exposure.

7 cl, 1 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to chemical-pharmaceutical industry and presents a moxifloxacin aqueous formulation for parenteral introduction containing an active substance: moxifloxacin hydrochloride, water for injections and other ingredients used for parenteral introduction differing by the fact that said ingredients are presented by sodium ascorbate and sodium metabisulphite in the following proportions, g/l: moxifloxacin hydrochloride 15-25; sodium ascorbate 0.05-1; sodium metabisulphite 0.1-2; water for injections up to 1 l.

EFFECT: invention provides creating a therapeutic agent providing a high therapeutic effect in prevention and treatment of bacterial infections by reduction of infectious and inflammatory processes in tissues, and reduced length of recovery.

2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: method for preparing a composition for injections containing sodium cevtriaxone and sodium tazobactam involves the following stages: (a) suspension of raw materials, i.e. sodium cevtriaxone, sodium tazobactam, sterilised water for injections, mixed solution of ethyl acetate and isopropyl alcohol, and anhydrous ethanol in mass relation making 3-5:1:2:5:9, with volume relation of ethyl acetate to isopropyl alcohol making 1:2-4; (b) dissolution of sodium cevtriaxone and sodium tazobactam in sterilised water for injections with added activated hydrocarbon and filtration; (c) addition of the mixed solution of ethyl acetate and isopropyl alcohol to the filtrate and agitation of the mixture; addition of a seed crystal of sodium cevtriaxone to the solution for crystallisation initiation; and finally washing of the crystals in anhydrous ethanol and crystal drying; and (d) lyophilisation to form the composition for injections containing sodium cevtriaxone and sodium tazobactam.

EFFECT: composition is characterised by high uniformity, high degree of purity and safety of use.

6 cl, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmacy, namely addictology and may be used for treating human addiction to alcohol, drugs and toxic substances. An intramuscularly injected prolonged-release pharmaceutical composition contains naltrexone as an active substance. Additionally, the composition contains a pharmaceutically acceptable solvent and excipients differing by the fact that the active substance is included in first and second microsphere fractions of polylactide coglycolide. The first microsphere fraction is characterized by the relation of lactide and glycolide monomer links 50 mole %:50 mole % and microsphere size 0.4 to 7 mcm. The second microsphere fraction is characterized by the relation of lactide and glycolide monomer links 75 mole %:25 mole % and microsphere size 20 to 90 mcm. A weight ratio of the first fraction of 0.01 to 0.15, while a weight ratio of the second fraction is 0.99 to 0.85.

EFFECT: preparing the composition for treating human addition to alcohol, drugs and toxic substances.

3 cl, 1 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared group of inventions refers to a pharmacological composition for intranasal introduction for cerebral delivery, and a method for preparing said composition. The declared composition comprises a container base formed by porous particles of calcium carbonate and titanium dioxide of particle size 100-5000 nm and a pharmacologically active component - loperamide. The container surface is modified by surfactants specified in polysorbates, or by polymers specified in a group containing glycosaminoglycanes and polypeptides, or their combination. A method for preparing the pharmacological composition consists in preparing the container base by porous particle synthesis, sorption of loperamide in its pore spaces and modification of the container surface by polymers and surfactants by container incubation in their solutions.

EFFECT: invention provides preparing the pharmacological composition which is applicable for cerebral loperamide delivery after the intranasal introduction.

5 cl, 5 dwg, 1 ex

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