Pharmaceutical composition for prevention or treatment of diseases caused by the formation of il-6

 

(57) Abstract:

The invention can be used for preparation of drugs suitable for the treatment of diseases caused by disorders of the blood, immunological diseases, and antitumor actions, and other diseases caused by the formation of IL-6. The pharmaceutical composition comprises the antibody to the receptor of interleukin-6. In particular, diseases, for the prevention and treatment which is intended composition, are: plasmacytes, hyperimmunoglobulinemia, anemia, nephritis, cachexia, rheumatism, disease of Castellana, mesangial proliferative nephritis. The antibody in the composition may be a monoclonal antibody, antibody RM-1, a chimeric antibody, a human antibody, a modified human antibody RM-1. The composition is effective against many diseases caused by the formation of IL-6. In the case of normal levels of IL-6, it has no effect. 13 C.p. f-crystals, 4 tab., 18 Il.

The invention relates to pharmaceutical compositions suitable for the prevention and treatment of diseases caused by the formation of interleukin-6 (IL-6), including antibody (antibody to IL-6R) to the receptor inter is involved in various immunodeficiency, technological, hematological and acute reactions (Taga, T. et al., Critical Reviews in Immunol., 1992, 11, 265-280), and also acts as a growth factor in the development of multiple myeloma and diseases involving plasmacytosis, such as rheumatism (Harada, T. et al., Eur.J. Immunol., 1988, 18, 1797-1801; Houssiau, F. A. et al. , Arth, Rheum., 1988, 31 784-788), a disease of Castleman [Yoshizaki, K. et al. , Blood, 1989, 74, 1360-1367; Brant, S. J. et al., J. Clin. Invest., 1990, 86 592-599), proliferating mesangial jade (Ohta, K. et al., Clin. Nephrol. (Germany), 1992, 38, 185-189; Fukatsu, A. et al., Lab. Invest., 1991, 65, 61-66; Horii, Y. et al., J. Immunol., 1989, 143, 3949-3955), and the state of cachexia accompanied by the growth of the tumor (Strassmann, G. et al., J. Clin. Jnvest.,1992, 89, 1681-1684), etc.

In transgenic mice (H-2LdhIL-6 (IL-6Tgm), which due to genetic manipulation Express high levels of human IL-6(hIL-6), were awarded the IgG1 plasmacytes, jade due to cell proliferation mesange, anemia, thrombocytopenia, the appearance of autoantibodies and other (Miyai, T. et al., report on the 21st Congress of the Japanese Immunological Society "Hematological changes in N-2LdhIL-6 transgenic mice during aging", 1991), which suggests the involvement of IL-6 in the pathogenesis of many diseases. However, nothing is known about the effectiveness of the antibody to the receptor of interleukin-6 against diseases, vizuelna pharmaceutical composition, suitable for the prevention and treatment of diseases caused by the formation of interleukin-6. To solve the above problems, the present invention offers a pharmaceutical composition suitable for the prevention and treatment of diseases caused by the formation of interleukin-6, which include the antibody to the receptor of interleukin-6.

In Fig. 1 depicts a graph showing the increase in body weight of animals in each group.

In Fig. 2 depicts a graph showing the change in the percentage of protein in the urine. This characteristic is zero in all groups except group 1 and group 3.

In Fig. 3 depicts a graph showing changes in the level of hemoglobin in each group of animals.

In Fig. 4 depicts a graph showing changes in the number of red blood cells in each group of animals.

In Fig. 5 depicts a graph showing the change in the number of platelets in each group of animals.

In Fig. 6 depicts a graph showing the change in the number of cells for each group of animals.

In Fig. 7 depicts a graph showing changes in the concentration of IgG1 in the serum in each group of animals.

On phocasa the result of sorting cells using control IgG antibody and Gr-I antibodies in animals in groups 1 and 2 by the method of fluorescent antibodies.

In Fig. 10 shows the result of sorting cells using control IgG antibody and Gr-I antibodies in animals in groups 6 and 7 by the method of fluorescent antibodies.

In Fig. 11 depicts a graph showing the weight of the spleen of animals in each group at the end of the experiment.

In Fig. 12 depicts a graph showing the change in body weight of animals in each group.

In Fig. 13 depicts a graph showing the concentration of triglyceride in the blood of mice on day 11 of the experiment.

In Fig. 14 depicts a graph showing the concentration of glucose in the blood of mice on day 15 of the experiment.

In Fig. 15 depicts a graph showing the concentration of ionized calcium in the blood of mice on the 11th day of the experiment.

In Fig. 16 depicts a graph showing the level of survival of control mice having a tumor.

In Fig. 17 depicts a graph showing the body weight of mice at 10 and 12 day from the beginning of the experiment.

In Fig. 18 depicts a graph showing the concentration of ionized calcium in the blood of mice in the 10-th and 12-th day from the beginning of the experiment.

Diseases caused by the formation of interleukin-6, include, in particular, preferire mesangial jade, cachexia, etc.

Used according to the method of the present invention the antibody to the receptor of interleukin-6 may be of any origin and any type (monoclonal, polyclonal), important only its ability to block the signal transduction under the action of IL-6 and to inhibit the biological activity of IL-6. Preferably, it was a monoclonal antibody derived from a mammal. The antibody blocks the signal transduction that occur under the influence of IL-6 and inhibits the biological activity of IL-6 by inhibiting the binding of IL-6 to IL-6R.

The cells of animals, suitable for production of monoclonal antibodies, may refer to mammalian cells, these antibody can be a human antibody or an antibody derived from a different animal than human. Preferably, in the case of a monoclonal antibody derived from an animal other than human, so it was a rabbit or rodent due to ease of production in these animals. In a preferred embodiment, the rodents include, but are not limited to, mice, rats, hamsters, etc.

The above-mentioned antibody to the receptor of interleukin includes, in particular, the antibody MR16-1 (Tamura, Tantala can be obtained known in the art method. So, you can get them using IL-6R as the immunizing antigen, which is already used in the traditional technique of immunization, and then merge, thus obtained immune cells with the parent cell using a known method of cell fusion and subsequent screening normal for this case, the method of antibody-producing cells.

More specific, monoclonal antibodies receive the following way. So, for example, the immunizing antigen can be obtained using the gene sequence of the human IL-6R, as described in the application for the European patent EP 325474. Then the genetic sequence of the human IL-6R is inserted into the system of the expression vector in a suitable host cell for transformation, after which the desired protein is IL-6R is recovered from the host cells or the culture supernatant and purified for subsequent use of such purified protein IL-6R as the immunizing antigen.

Furthermore, the said immunizing antigen can be isolated from mice using mouse gene sequence IL-6R, which was described in Japanese laid out the proposal will not pass the examination, 3(1991)-155795, with the antigen, as IL-6R, expressed on the cell membrane, can also be used and the sequence (sIL-6R), which, apparently, are separated from the cell membrane, sIL-6R includes mainly the extracellular domain of IL-6R associated with the cellular membrane, differing from the membrane-bound IL-6R fact that it does not contain a transmembrane domain or contains no transmembrane domain or intracellular domain.

Regarding mammals that undergo immunization with said immunizing antigen, although not necessarily, but it is preferable to consider their compatibility with the parent cell used for the merge, and typically they include mice, rats, hamsters, rabbits, etc.

Immunization of the animal described by immunizing antigen is known in the art method. Thus, the General method involves intraperitoneal or subcutaneous administration of the above-mentioned antigen in the body of a mammal. More specifically, the immunizing antigen, diluted or suspended in the FBI (phosphate buffer solution), saline solution, and so on, up to the appropriate volume, mix, if necessary, with the correct number corresponding to hell is feeding several times, every day 4-21. In addition, the immunization mentioned immunizing antigen may be used in appropriate media.

After immunization of an animal as described above carry out the determination of antibodies in the serum to confirm increase its level to a desired value, extracted from animal immunocytes and carry out cell fusion. As the preferred immunocyte note cell of the spleen.

The myeloma cell used in the present invention as the parent cell to merge with immunocytoma, is preferably one of the well-known cell lines such as P3 (Rad.653) (J. Immunol., 1978, 123, 1548), p3-Ul (Current Topics in Microbiology and Immunology, 1978, 81, 1-7), NS-1 (Eur. J. Immunol., 1976,6, 511-519), MPC-11 (Cell.,1976, 8, 405-415), SP2/0 (Nature, 1978, 276, 269-270), FO [J. Immunol. Meth., 1980, 35, 1-21), S194 (J. Exp. Med. , 1978, 148, 313-323), R210 [Nature, 1979, 277, 131-133), etc.

The cell fusion of the above-mentioned immunocyte with a myeloma cell carried out essentially in accordance with the known technique described by Milshtein et al. (Milstein et al., Methods Enzymol., 1981, 73, 3-46).

More specifically, consider the cell fusion can be carried out in the presence of, for example, agent, accelerating cell fusion, Provost polyethylene glycol (PEG), The Sendai virus (HVJ) and the other, and if desired, to increase the efficiency of cell fusion can be directly on Wednesday introduced adjuvant such as dimethyl sulfoxide, etc.

The ratio used immunocytes and myeloma cells is preferably from 1 to 10 times more immune cells than the myeloma cells. As liquid culture media that can be used to implement the above cell fusion, it is possible to mention such as liquid medium RPMI 1640 liquid medium MEM, which is the most favorable for the growth of myeloma cell lines, and for the growth of cell cultures can be used conventional culture broths, in addition, may be made of serum supplements such as amniotic calf serum (FCS) and other

The desired fused cells (hybridoma) can be obtained by mixing predetermined amounts of the above-mentioned immune cells with myeloma cells in the specified nutrient broth, with concomitant addition of PEG solution, preheated to a temperature of 37oC, in particular of a solution of PEG having an average molecular weight in the range from 1000 to 6000 at a concentration of from 30% to 60% (weight/volume). Next, the value of the supernatant, and agents made earlier for cell fusion and which are undesirable for the growth of hybridoma.

Described hybridoma can be further selected during cultivation on common objectives of the breeding environment, such as liquid culture medium HAT (liquid culture medium containing gipoksantin, aminopterin and thymidine). Cultivation in the environment of the HAT is in a period of time sufficient for the cells other than hybridoma (i.e. not merged cells), were killed, it usually takes from several days to several weeks. Then screening using the traditional method of limiting dilutions to determine hybridoma producing the desired antibody and receive on the basis of monoclonal lines.

Hybridoma producing the desired monoclonal antibodies can be subjected to subculturing in traditional liquid medium, after which they can be stored in liquid nitrogen for a long period of time.

To obtain on the basis of the specified hybridoma monoclonal antibodies using various methods, in particular, one in which hybridoma cultivated according to traditional methods, applied the lowland compatible mammal and grow it to generate antibodies formed from ascitic fluid, etc. The first of these methods is applicable for obtaining highly purified antibodies, whereas the second method is suitable for producing large quantities of antibodies.

Next, the monoclonal antibodies obtained using the above methods can be purified by conventional procedures such as salting out, gel filtration, affinity chromatography, etc.

The ability so prepared antibodies to recognize antigen with high affinity and high accuracy is confirmed by conventional immunological methods such as radioimmunoassay, enzyme immunoassay (EIA, ELISA), a method of measuring fluorescence antibody (immunofluorescence assay), etc.

Monoclonal antibody used according to the present invention is not limited to the antibody produced by hybridomas, and may represent an artificial antibody to reduce its heterogeneity for a person. So, for example, can be used chimeric antibody comprising variable plots mouse monoclonal antibody and the constant parts of the human antibody. Such chimeric antibodies can be obtained using a known method used in the statutory invention can be used in a human antibody with a modified form. It represents such an antibody in which the complementary sections defining regions of a human antibody is replaced with complementary areas of characteristic regions in another antibody of a mammal other than human, such as mouse antibodies, the primary method of genetic engineering, used for such manipulation, known from the prior art. Using this method, you can obtain a human antibody with a modified form suitable for the present invention.

If necessary amino acids in the framework regions (FS) of the variable segment antibodies can be subjected to substitution, so comlementary sites that define the region recovered human antibody may form a binding site for the corresponding antigen (Sato et al., Cancer Res., 1993, 53, 1-6). As a preferred example of such a modified human antibodies can be noted received from the human body antibody PM-1 (hPM-1) (see International patent application 9219759).

Genes encoding fragments of antibodies, such as Fab or Fv in a single chain Fv (scFv), which plots Fv H chain or L chain are connected through going in order to use them for the above purposes, because these fragments bind to the antigen and inhibit the activity of IL-6 (see , for example, Bird et al., TIBTECH, 1991, 9, 132-137); Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883). In addition, the aforementioned modified V region of the antibody can be used genome Fv H chain and L chain to obtain scFv.

The pharmaceutical composition used for the prevention or treatment of diseases caused by the formation of IL-6, which comprise as an active ingredient the antibody to the receptor of IL-6 of the present invention can be used according to the method of the present invention, because they have the ability to block signal transmission of IL-6 and is effective against diseases caused by the formation of IL-6.

The pharmaceutical composition used for the prevention or treatment of diseases caused by the formation of IL-6, preferably injected parenterally, in particular by intravenous, intramuscular, intraperitoneal or subcutaneous injection, and others, both systemically and topically. In addition, they can be a pharmaceutical composition or kit used in combination with at least one carrier or diluent.

Although the dosage of pharmaceuti drug it is necessary however to choose the appropriate number to use. For example, a number in the range from 1 to 1000 mg per patient can be entered in four separate doses. Alternatively, they can be introduced in an amount of from 1 to 10 mg/kg/week. The use of pharmaceutical compositions of the present invention for prevention or treatment is not limited to the above-mentioned doses.

The pharmaceutical compositions of the present invention can be prepared using known conventional methods. So, for example, parenteral preparations can be obtained by dissolving purified antibody to IL-6R in a solvent such as saline solution, buffer solution, etc. to which add antiadherent, i.e., tween 80, gelatin, human serum albumin (HSA) and others, or the composition may be in dried form, which immediately before use dilute. As fillers for lyophilization can be used a sugar alcohol such as mannitol, glucose, etc. or sugars.

Examples

The following are examples, including reference, for a more detailed explanation of the present invention, however their NS="ptx2">

An example of a song

Antibody to the receptor of interleukin-6 - 2.5 mg/ml

Polysorbate 80 - 0,005%

D-beckons - 0,5%

Sodium phosphate - 19 mm (pH 6.5)

Sodium chloride - 120 mm

Reference example 1

Design B6LdIL-6' transgenic mouse

The Sphl fragment-Xhol (Ld-IL-6) size of 3.3 KB, bearing human IL-6 cDNA, which is connected with the H-2Ldpromoter (Suematsu et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 7547), is injected into the pronucleus of a fertilized egg of a mouse C57BL/6J (B6) (Nihon Clea) through microinjection technique according to the methods described (Yamamura et al., J. Biochem., 1984, 96, 357).

A fertilized egg is implanted in the oviduct of the female mouse strain ICR, which before that had been subjected to processing that mimic pregnancy. After that newborn mice conducted a study that integrate hiL-6 cDNA into the genome by southern blotting analysis, using as source EcoRl-split tail DNA as probe Tagl-Banll the cDNA fragment of human IL-6, labeled32R. Animal, which was shown to integration, grew together with B6 mice to establish lines that have the same genotype.

Reference example 2

Getting in rats antibodies to IL-6R

On method is. the entrances incubated at 37oC in MEM medium containing 5% amniotic calf serum (FBS) in a humidified atmosphere containing 5% CO2. Air-conditioned environment regenerate and used as drug murine slL-6. The concentration of murine sIL-6 in the environment determined by the method of enzyme-linked immunosorbent assay (sandwich ELISA) using monoclonal antibodies RS15 to mouse IL-6R (Saito et al., J. Immunol., 1991, 147, 168-173) and polyclonal rabbit antibodies to mouse IL-6R.

Mouse sIL-6R cleaned, using as a source of murine preparation of sIL-6R and applying for this column for affinity chromatography, which absorbed monoclonal antibody to mouse IL-6R (RS12). Fifty micrograms of purified murine sIL-6R in complete Freund's adjuvant is injected subcutaneously rat Wistar, then two weeks later the rat repeatedly subjected to immunization four times subcutaneous injection of 50 μg of mouse sIL-6R in incomplete Freund's adjuvant with a frequency of once a week. A week after the first booster injection, the rats injected with 50 μg of mouse sIL-6R in 100 μl of phosphate buffer solution (FBI).

After three days in rats deprived of the spleen and spend the merger of splenocytes of rats with Milani the e nights at a temperature of 37oC in 100 ál of medium RPM1 1640 containing 10% serum amniotic calf, and then add 100 ál of medium containing gipoksantin/aminopterin/thymidine (HAT). Within four days, daily replacing half of the medium on HAT medium.

After seven days with a test for the binding of murine sIL-6R (ELISA) select hybridoma producing antibody to mouse sIL-6R. In General the procedure is as follows: incubate 100 ál of culture supernatant on a plateau, in a cell which has previously been introduced at a concentration of 1 μg/ml polyclonal antibody to IgG rabbit rats. The plate is washed and then incubated with 100 μg/ml murine sIL-6R. After rinsing, add up to a concentration of 2 μg/ml polyclonal antibody rabbit to mouse IL-6R, the plate is washed, and then for 60 minutes to conduct the incubation with the complex, including alkaline phosphatase, conjugate with a polyclonal goat antibody to rabbit IgG (special prizes were also given).

And finally, after laundering, conduct incubation with the substrate Alp (Sigma 104: p-nitrophenylphosphate) and measured at a wavelength of 405 nm using register Torah plate (Toso). Hybridoma that recognizes murine sIL-6R, double-clone, PRA, three days later, spend intraperitoneal injection of installed cells hybridoma in the amount of 3106. After 10-20 days collecting ascites, which is isolated and then clean monoclonal antibody MR16-1 using a column with protein G (Oncogene Science).

Negate the effects of IL-6 antibody produced by MR16-1, was investigated with the inclusion of3H-thymidine MN. BSF2 cells (Matsuda et al., Eur. J. Immunol. , 1988, 18, 951-956). Cells MN. BSF2 select the sample in the amount of 1104cells/200 μl/cell 96-breeding plateau, then to each cell type mouse IL-6 (10 ng/ml) and antibody MR16-1 or RS12, and then carry out incubation of cells at 37 ° oC for 44 hours in an atmosphere containing 5% CO2. After 4 hours add to each cell3H-thymidine (1 mCi../cell) and investigate the inclusion of3H-thymidine.

Example 1

In accordance with the method of reference example 1 was prepared and further processed 31 transgenic mouse, which contains the cDNA of human IL-6, reproduced on the basis of (B6 IL-6) transgenic mice (B6 IL-6 Tgm), and together with them, use 11 normal animals, not carrying cDNA of human IL-6 (all animals were 4 weeks of age; males). B6 IL-6 Tgm share n the t seven animals. Normal animals of broods also divided into several groups: group 6 from 5 mice and the group 7 consisting of six mice.

Was used following the injection mode:

Group 1 (B6 IL-6 Tgm): at 4 weeks of age, i.e. (on the first day of the experiment) injected rat IgGI antibody (KN) (control antibody) at a dose of 2 mg/0.2 ml, and 5 weeks of age (8th day of the experiment) and later injected subcutaneously twice a week with 100 µg of antibody KN (injection carried out every 3-4 days).

Group 2 (B6 IL-6 Tgm): At 4 weeks of age animals injected intravenously at a dose of 2 mg/0.2 ml MR16-1 antibody, and 5 weeks of age and later, 100 μg MR16-1 injected subcutaneously 2 times a week.

Group 3 (B6 IL-6 Tgm): At 4 weeks of age animals injected intravenously with 0.2 ml of a phosphate buffer solution, and 5 weeks of age and later twice a week injected subcutaneously with 100 µg of R16-1.

Group 4 (B6 IL-6 Tgm): At 4 weeks of age the animals injected intravenously with 2 mg/0.2 ml MR16-1 and 5 weeks of age and later injected subcutaneously once a week for 400 mcg MR16-1.

Group 5 (B6 IL-6 Tgm): At 4 weeks of age the animals injected intravenously with 2 mg/0.2 ml MR16-1 and 5 weeks of age and later injected subcutaneously 1 mciroy intravenous 2 mg/0.2 ml of the control antibody CN, and 5 weeks of age and later twice a week they injected subcutaneously with 100 μg CN.

Group 7 (B6 normal animals): 4 weeks of age the animals injected intravenously with 2 mg/0.2 ml MR16-1, and 5 weeks of age and later injected subcutaneously twice a week for 100 mcg MR16-1.

Testing is carried out as follows.

Determination of body weight and determination of protein in urine: daily weighing and determination of protein in urine using indicator paper on urine protein (Combistics San-kyo). A three-fold excess of protein in the urine (100 to 300 mg/DL) was taken as positive values in this test.

Sampling blood from the back-orbital sinus blood samples were collected regularly through the week, with the beginning of the experiment (4 weeks of age), and at the end of the experiment (18 weeks) was selected from all the blood through the inferior Vena cava.

Blood analysis: using microscience cells (Sysmex F-800) determine the number of leukocytes (L), erythrocytes (e) and platelets (T), and hemoglobin (Hb). By the end of the experiment for a number of groups (groups 1, 2, 6 and 7) prepare blood smears and calculated on the basis of the percentage of leukocytes.

Measuring the Ohm protein of mouse IgG.

The concentration of IL-6 in the blood: the measurement is carried out according to the method of TYPHOID fever with the use of hIL-6.

Determination in blood titer antibodies to mouse IgG (IgG class): because the injected antibody is a mouse heterogeneous, the formation of antibodies to introduce heterogeneous antibody is determined according to the method of TYPHOID using as antigen rat IgG. The result expressed in units using standard IL-6 Tgm serum of an adult animal, which gave a rat antibody.

Determination of chemical properties of blood: at the end of the experiment, mice in groups 1, 2, 3, 6, and 7 using an automated analyzer (COBAS FARA 11, Roche) measure the level of total protein (TP), albumin (Alb), glucose (Glu), triglyceride (TG), creatinine [BAP (Cre)], nitrogen in the blood and urine (BUN), calcium (CA), Alp (ALP), glutamylcyclotransferase [SFT (GOT)] and glutamyltransferase [GCT (GPT)].

Flourescently analysis (FACS analysis) bone marrow and splenocytes: at the end of the experiment, animals in groups 1, 2, 6 and 7 receive the bone marrow and splenocytes that are analyzed for the presence of antigens on the cell surface using fluorescence-activated analyzer FACScan (Beckton Dickensian). On the 220 (splenocytes).

Autopsy: at the end of the experiment, performed the autopsy and measure the weight of the spleen, and other major organs visually examined.

Body weight: Fig. 1 shows changes in body weight of animals in groups 1 and 3. Between groups there was no difference in weight change phone

Protein urine: in group 1 animals that are evaluated positively by the presence of protein in the urine, begin to appear with 13 weeks of age (Fig. 2), while seven were in this group of animals 4 animals (2 to 16 weeks of age and 2 to 17 weeks of age) died during the autopsy. Whereas in the other groups was not observed deaths. In group 3 by the end of the experiment, two of the six animals were positive for the presence of protein in the urine, but in other groups positive on this criterion animals was not found.

Hematological indices: in group 1 decreased levels of hemoglobin (Fig. 3) and the number e (Fig. 4), and the severity of these changes increases with age. The number of platelets (Fig. 5) temporarily increases, but then decreases rapidly. In group 3 was noted a similar trend, albeit with some delay in comparison with group 1. On the other hand, in groups 2, 4 and 5 was not a decrease in their number. The differential counting of blood cells in the blood smears in group 1 was found increased levels of neutrophils and monocytes and a corresponding reduction in the fraction of lymphocytes, but for group 2 were obtained normal values (table 1). Between groups 6 and 7 also did not reveal significant differences.

The concentration of IgGI in the blood: in group 1 for the concentration of IgGI in blood has been shown a significant increase immediately after the start of the experiment, which reached almost 100-fold excess over its concentration in normal mice (Fig. 7). In group 3 increased concentrations of IgGI occurs somewhat later than in group 1. In contrast, in groups 2, 4, and 5 is not marked increase in the concentration IgGI, which remains throughout the experiment almost on the same level. On the other hand, in normal mice is not observed changes associated with the introduction of antibodies.

The concentration of hIL-6: concentration of hIL-6 in the blood (Fig. 8) varies in the same way that the concentration of IgGI, demonstrating greater in groups 1 and 3, but remaining almost at the same level during the whole experiment in the other groups.

The titer of antibody to rat IgG in the blood: antibodies to Krysanova as in group 6 only two of the five animals showed a higher titer. On the other hand, other groups have not observed a significant increase.

Determination of chemical parameters of blood: in groups 1 and 3 there is an increase in the number of platelets and decreased albumin levels. The level of platelets and Alp decreased in groups 1 and 3, while in group 1 also decreases glucose. Whereas in group 2 was not detected such changes.

Fluorescent analysis (FACS analysis): Analysis of bone marrow cells (KM) and splenocytes (SP) in groups 1,2,6 and 7 showed a very strong increase in the share Gr-I positive cells, which are precursors of granulocytes in the bone marrow cells in animals in group 1 (Fig. 9 and Fig. 10), however, values similar characteristics to group 2 close to the results obtained for normal animals. This was not observed significant difference between groups 6 and 7. On the proportion of CD4-, CD8 - and 220V-positive cells in splenocytes was shown between groups no differences, except for group 1, for which we found a decrease in the proportion of CD8 - and 220V-positive cells due to increased numbers of plasma cells (table 4).

The autopsy results: in groups 1 and 3 is, rather, the sun is. what was also noted a slight increase in the liver. In other groups such changes have been identified, the only significant change was a slight increase in splenic size in comparison with normal animals, and it was noted in groups 2, 4, and 5.

Below is an explanation of the experimental results. In the group of IL-6 Tgm (group 1), which was used during the introduction of the control antibodies, there are a lot of symptoms, such as IgGI plasmacytes, anemia, thrombocytosis, thrombocytopenia, renal failure, abnormal chemical characteristics of blood, etc. However, it became obvious that all of these symptoms completely suppresses MR16-1.

It is known that IL-6 leads to the fact that cells differentiate at the final stage in plasma cells (Muraguchi A. et al., J. Exp. Med. 1988, 167, 332-344), in the case of IL-6 Tgm education IL-6 causes an increase in the concentration of IgGI in the blood, as well as an increase in the concentration of platelets and a decrease in the concentration of albumin in serum. These data indicate the beginning of IgGI plasmacytosis.

Strong growth system lymphatic tissues, such as lymph nodes and spleen, marked in this case, leads to an increase in body weight, whereas in groups 1 and 3 hudsen disease, but also suppresses the increase in blood concentrations of IL-6. Thus, it is confirmed that age-related, as in the case of IL-b Tgm, increasing the concentration of IL-6 is directly related to the development of plasmacytosis. Therefore proliferating plasma cells, most likely, actively secrete IL-6, which then, in turn, enhances the growth of plasma cells, which results in the fact that IL-6 is produced in large quantities.

As well as the effects of IL-6 on the formed elements of blood, a well-known influence on the increase in the number of platelets (Ishibashi T. et al., Proc. Natl. Acad. Sci. , USA, 1989, 86, 5953-5957; T. Ishibashi et al., Blood, 1989, 74, 1241-1244) and its inducing effect on the development of macrocytic anemia (Hawley, R. G. et al., J. Exp.Med., 1992, 176, 1149-1163). In addition, the group IL-6 Tgm observed thrombocytopenia associated with aging, which, apparently, is related to autoimmunity, defined activation In cells (Miyai, Tatsuya et al., ibid).

MR16-1 completely inhibited the direct and indirect effects of IL-6 on the formed elements of the blood, but does not affect the number of blood cells in normal animals. Thus, it is confirmed that the antibody to the receptor of IL-6 are not affected is to precursors of granulocytes, as well as the proportion of peripheral blood neutrophils, the mechanism of which is still not fully understood. It was also discovered in this study that this effect takes place in the bone marrow at the level of cells predecessors. In addition, the same study it was found that MR16-1 completely inhibited the effect of IL-6, but does not affect the level of neutrophils in the bone marrow and in peripheral blood.

MR16-6 suppresses the development of nephritis seen in the IL-6 Tqm. In the literature there is a message indicating that IL-6 is closely associated with the launch of proliferation mesange when jade as autocrine growth factor cells mesange. Despite the fact that jade is in the group of IL-6 Tgm is a nephritis associated with proliferation mesange, we cannot exclude the participation in this process, the immune system, strengthen under the action of IL-6 (Katsumo, Asao et al. paper presented at the 21st Congress of the Japanese society of immunologists "Characterization of SCID x (SCID x H-2LdhIL-6 transgenic mice"), 1991]. In any case, as is the suppression of the appearance of protein in the urine and no deaths, it is clear that the antibody against the receptor of IL-6 now, in terms of suppressing the onset of nephritis caused by the formation of IL-6.

In the group of IL-6 Tgm observed mean is xperimenta demonstrated the efficiency MR16-6 to reduce cachexia, because group 1 is the decrease in the values of the concentrations of glucose and platelets, whereas in group 2 these parameters are changed, taking values are practically on the same level as in normal animals.

Because MR16-6 is a rat IgGI, which is for mice foreign protein, one should expect the formation of antibodies against the entered antibodies, which can make the introduced antibody ineffective.

With the intention of inducing in the experiment, the immunological tolerance through the introduction when the first sensitization large number of antigen groups organized so that when it is first administered intravenously to give 2 mg/mouse. Among the groups that received MR16-6 (groups 1, 4 and 5), not detected by the production of significant quantities of antibodies against rat IgG, regardless of input dose and the interval between injections, which leads to a complete suppression of disease development in the beginning. However, group 3, which is a group introducing the control antibody, demonstrates the gradual emergence of the same symptoms as in group 1, although it shows an increased level of antibodies against rat IgG, and the development of price this processing is effective from the point of view of inducing immunological tolerance, however, the antibody against rat IgG was also found in all animals in group 1 and in 2/5 animals in group 6, which was obtained in the same mode control antibody. Since the development of plasmacytosis in IL-6 Tgm induces polyclonal activation In cells, it is impossible to conclude that found in groups 1 and 3 antibodies against rat IgG specific to that introduced antibodies. However, it was concluded that immunological tolerance generated by exposure to large amounts of antigen in the first sensitization groups 2, 4 and 5, has an inducing effect, which, combined with the inhibiting effect of education in response to the introduction of a large number MR16-6-specific antibodies, leads to induction of complete tolerance.

This experiment showed that the antibody against the receptor of IL-6 is highly effective against many diseases caused by the formation of IL-6, in the case of a normal level it has no effect.

Example 2

Examine the impact of antibodies to murine receptor of IL-6 on the model induced cachexia in colon 26. The experiment used a 6-week male BALB/C mice, in which bnta immediately before implantation of colon 26 animals were injected with 2 mg/mouse antibody MR16-6 by murine receptor IL-6 (see reference example 2), and then on day 7, 11, 14 and 18 (n = 7) as given in the dose of 0.5 mg/mouse. In the previous experiment has already been confirmed that neutralizing antibodies against the foreign protein is not easily formed in the conditions of this method. In the same mode (n = 7) control group bearing a tumor injected with the control antibody to rat IgGI (CN). Control group not containing the tumor (n = 7), was administered by the FBI. Since the start of the experiment, every day weigh animals and 11 and on the 15th day after the beginning of the experiment determine the chemical characteristics of the blood and the amount of ionized calcium.

On the 10th day and later in the group bearing a tumor, there is a marked reduction in body weight compared to the group without tumors, whereas in the treated group, MR16-6, marked partially vast reduction in body weight effects (Fig. 12). In Fig. 13 and Fig. 14 shows respectively the concentration of triglycerides in the blood on day 11 and glucose concentrations on day 15. These values show a marked reduction in the control group, not bearing tumors, whereas in the treated group, MR16-6, observed suppressor trend in glucose and a significant suppressor effect in relation to TRIG the Noah group, bearing a tumor, in comparison with a control group that does not contain the tumor, whereas in the treated group, MR16-6, there is a significant suppressor effect (Fig. 15).

In the same mode (n = 10) was carried out the experiment, confirming the effect on survival time. It was shown that in the group receiving MR16-6, survival time is affected by the input antibodies (Fig. 16).

Example 3

Examine the impact of antibodies to receptor of IL-6 on the model OSS-1-induced cachexia accompanied by hypercalcemia. In the experiment using 6-week male Nude mice. On the first day of the experiment in the side to mice implanted line squamous cancer cells. Antibody MR16-6 receptor murine IL-6 (see reference example 2) was injected intravenously at a dose of 2 mg/mouse immediately before implantation OSS-1 on the first day of the experiment, and then on day 7 and 10 (n = 6) mice injected his subcutaneously at a dose of 100 μg/mouse. In previous experiments it has been shown that neutralizing antibodies against the foreign protein-rat antibodies, is not easily formed in the conditions of this method. To the control group bearing a tumor, in the same mode (n = 6) injected with control antibody to krysi the live animals and determine the blood levels of ionized calcium.

In a group bearing a tumor, there is a decrease in body weight, while in the treated group, MR16 6 demonstrated changes similar to those observed in the control group not containing the tumor, indicating suppression of the reduction of body weight (Fig. 17).

The concentration of calcium ions in the blood significantly increased in the control group bearing a tumor, in comparison with a control group that does not contain the tumor, whereas in the treated group, MR16-6, this increase markedly inhibited (Fig. 18).

1. Pharmaceutical composition for prevention or treatment of diseases caused by the formation of interleukin-6, which includes the antibody to the receptor of interleukin-6.

2. Pharmaceutical composition for prevention or treatment under item 1, wherein the disease caused by the formation of the specified interleukin-6, is plasmacytes.

3. Pharmaceutical composition for prevention or treatment under item 1, wherein the disease caused by the formation of the specified interleukin-6, is hyperimmunoglobulinemia.

4. Pharmaceutical composition for prevention or treatment under item 1, characterized in that zabolevaniya composition for prevention or treatment under item 1, characterized in that the disease is caused by the formation of the specified interleukin-6, is a jade.

6. Pharmaceutical composition for prevention or treatment under item 1, wherein the disease caused by the formation of the specified interleukin-6, is a cachexia.

7. Pharmaceutical composition for prevention or treatment under item 2, characterized in that the specified plasmacytes induced by rheumatism.

8 Pharmaceutical composition for prevention or treatment. under item 1, wherein the disease caused by the formation of interleukin-6, is a disease of Castellana.

9. Pharmaceutical composition for prevention or treatment under item 5, characterized in that the jade is mesangial proliferative nephritis.

10. Pharmaceutical composition for prevention or treatment according to any one of paragraphs. 1 to 9, characterized in that said antibody is a monoclonal antibody.

11. Pharmaceutical composition for prevention or treatment under item 10, characterized in that said antibody is an antibody RM-1.

12. Pharmaceutical company is Noah antibody.

13. Pharmaceutical composition for prevention or treatment under item 10, characterized in that said antibody is a modified human antibody.

14. Pharmaceutical composition for prevention or treatment on p. 13, characterized in that said antibody is a modified human antibody RM-1.

 

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