Conjugate derived 14-brandenrobinson with a monoclonal antibody or its fragment having antitumor activity and having specificity for the tumor, and a pharmaceutical composition having antitumor activity
(57) Abstract:The invention relates to the field of medicine and relates to conjugates of an antibody derived from a 14-brandenrobinson with antitumor activity. The invention consists in the development of conjugates of monoclonal antetele 14-bromo - 4-demethoxygeldanamycin, while the conjugate contains from 2 to 8 molecules of drugs covalently linked to the antibody or fragment, as well as pharmaceutical compositions containing the given conjugate, as well as acceptable diluent or carrier. The technical result is to increase the specificity of drug delivery to the tumor. 2 C. p. F.-ly, 3 tab., 19 Il. The invention relates to conjugates immunoglobulin, the method of their production and use.The General concept of targeted delivery antineoplastics substances to tumors using monoclonal antibodies (Moab), is known, and at present is determined by its therapeutic value. Usually this approach includes obtaining conjugates of antibodies with a toxic substance capable of selectively localized and kill tumor cells. The focus was on designing immunotoxins of A-C the cells. In fact, many Moab, which was considered to be specific to tumors, were also reactive with respect to the subpopulations of normal cells, and therefore, it may be impossible to use such a strong toxins due to the fact that they have the potential to damage normal tissue. A safe alternative plant toxins is the connection of antibodies with conventional anticancer drugs such as doxorubicin, vindesine, chlorambucil, melphalan and methotrexate. Because of the nonspecific toxicity of commonly used antineoplastic agents attempted to increase their therapeutic index by connecting them with Moab and associated with tumor antigens.Attempts to suppress graft rejection due to produced by the thymus T-cells, often focused on reducing the activity of T-cells by using anticlimatically. Recently with the development of knowledge of monoclonal antibodies has become possible to define subclasses of T-cells in accordance with their functions in vitro and depending on the presence of specific surface antigens defined Moab. This has stimulated the search control mechanism T-CL is toxin/suppressor, the designated L3T4 - and Ly-2 antigens of the mouse. Although ACTS Moab, anti-pan-T-cell reagent, showed potency in vivo, in a recent study we found that 20 Moab were indifferent in experiments in vivo on mice to 20 different antigens of lymphocytes and mouse because they are not useful for in vivo studies. Therefore, you should try out the means by which these highly specific Moab can be made more effective and such that they completely removed the target cells. The use of cytotoxic drugs in connection with Moab is one of such approaches.Clinical trials of the drug conjugates-Moab include immunochemotherapy cancer and facilitating various immunoregulatory diseases and allograft rejections. Many studies showed specific cytotoxicity of toxins and drugs against tumor cells in the case of compounds of these agents with Moab connected with associated with tumor antigens.However, little attention has been placed on in vivo tests the ability of the conjugates of the drug-Moab to destroy T-cells and the study of T-cell immunoregulation during graft rejection, although the conjugates of toxin-antibody widely used in vitro for raznotematicheskih agents, used in cancer chemotherapy with doxorubicin and daunorubicin has demonstrated its effectiveness against solid tumors. However, the connection of daunorubicin and doxorubicin with antibodies resulted in a significant reduction in the effectiveness of drugs, if this connection was made through an amino group. Recently daunorubicin connected with Moab through C-14 using bromoguanosine. These conjugates showed activity in vitro. However, reports of in vivo studies are lacking (Gallego et al., Inl. J. Cancer, 1984, 33, 737 - 744). In addition, it was shown that the conjugates of daunorubicin with Moab have nonspecific toxicity at concentrations of > 10 μg/ml.We found that idarubitsin (4) demethoxygeldanamycin, Ida you can connect with Moab, and these conjugates have a selective and strong antitumor activity in vitro and in vivo. Non-specific toxicity of conjugates of Ida-Moab in vitro was not observed at concentrations of < 8 mg/kgConjugates Ida-Moab have a higher efficiency in vitro and in vivo than the conjugates of daunorubicin with Moab.In addition, we assessed the ability of the conjugates Ida-Moab specific to destroy the population of cells by attaching Ida to Moab, which, as is, is it a surprisingly effective method of destroying cells. For example, anti-Ly-2.1 Moab, which has a strong effect in vivo on Ly-2.1+cells can be turned into an effective cytotoxic agent by connecting to a cytotoxic agent Ida.using Moab to Ly-2 and L3T4-antigens, we showed that the conjugates Ida-Moab able to eliminate target cells in vitro and in vivo. You can get conjugates Ida-Moab, who have a higher ability to destroy specific subclasses of T-cells responsible for graft rejection, compared with antilymphocyte-globulin or anti-pan-T-cell reagents such as ACTS Moab.The invention discloses conjugates of immunoglobulins, including Ida, United with monoclonal antibodies or fragments comprising at least one of the binding sites of the antigen this antibody.Ida described in U.S. patent N 4077988. Preferably 2 to 8 molecules Ida covalently bound to each molecule of the antibody or fragment of antibody, more preferably 2 to 6. Usually, but not necessarily, the molecules Ida attached in position 14 to the monoclonal antibody or antibody fragment. Preferably they are connected directly, the scrap or fragment of the antibody by an amino group. This can be done using decaying catigny connector, dextranase media kislotoustojchivy connector or polyglutamine acid.Usually each antibody or fragment of antibody specific to the antigen on the cell surface, against which we must influence Ida. For example, the antibody or antibody fragment may be specific for the respective target tissue such as neoplasm in humans. Examples of neoplasms the person to which it may be desirable to direct the action of the Ida, are tumors of the breast, colon, lung, prostate, ovary, thymus and other cancers, sarcoma, and leukemia. The antibody and the antibody fragment may be specific to neoplasma animals. You can use the antibody or antibody fragment specific for the transferrin receptor /TFR/ person, which is present in dividing cells, erythroid cells-the precursors and cells of various tumors. Suitable anti-TFR monoclonal antibody can be used against the transferrin receptor on the cell LiCR LON-UHy-2 (HMy-2).If the immunoglobulin conjugates are supposed to be used for the destruction of specific populations of T-lymphocytes, antibody or fragment antitau antibody or antibody fragment may be specific to the population helpers suppressor or cytotoxic T-lymphocytes.Preferably a monoclonal antibody or a fragment of the antibodies of the same class as something against which you want to use conjugate immunoglobulin. Monoclonal antibody or antibody fragment of human or mouse usually therefore used when it is expected to enter a connection to the person. Preferably, the antibody or antibody fragment was class 1C. The antibody fragment may be Fab, Fab' or F(ab)2-fragment.You can also use the monomer IgM, which can be obtained from IgM-antibodies by proteolytic enzyme digestion.Conjugates of an antibody receive according to the present invention the method, which includes the connection Ida with a monoclonal antibody or antibody fragment. Preferably 14-halo-Ida interacts with a monoclonal antibody or antibody fragment. 14-Deputy may be fluorine, chlorine, bromine or iodine, but preferably bromine. 14-bromo-Ida described in U.S. patent N 4125697. Conjugatio, thus, it is possible to carry out the method, which includes
a) a mixture of the monoclonal antibody or antibody fragment with a molar excess of 14-halo-Ida,
b) conductive materials by gelfiltration and
e) removal of the drug (Ide) by absorption chromatography or ion exchange chromatography.Stage (a) is usually carried out in miscible with water, an organic solvent such as N, N-dimethylformamide. Preferably the molar excess of 14-halo-Ida at the stage of (a) from 0 to 50 times. At the stage (b) the reaction is preferably carried out in a period of 1-8 hours Usually the reaction is carried out at room temperature.You can use other ways to obtain a conjugate. If you want conjugate had an inert carrier or a crosslinking agent, located between Ida and monoclonal antibody or the antibody fragment, the media or cross-linking agent is first attached to the carbon atom of C-14 Ida and then attached to an antibody or antibody fragment. As described above, the antibody or antibody fragment may be associated with Ida by an amino group by using a splitting of the peptide compounds of the spacer, dextranomer media, kislotnoschelerngo spacer or polyglutamine acid.Conjugates of an antibody of the present invention can be used for the treatment of cancer in humans and mammals. You can apply a therapeutically effective amount which can be treated with these conjugate, above, you can enter two or more conjugate, in which the monoclonal antibody or the antibody fragment in each conjugate solution has a different specificity.The conjugate can be introduced in the form of injections. It can be used parenterally, for example intravenously. It can be administered locally or directly into the tumor. The amount of conjugate, administered to the patient depends on various factors such as type of treatment to be of the tumor, the patient's condition. The usual dose of 10-200 kg conjugate at 1 m2the patient's body. These conjugates can be used in conjunction with other chemotherapeutic agents or with agents that enhance the activity of these conjugates, such as vasoactive agents or factor protects tumor.Conjugates of an antibody can also be used for specific removal of a subclass of T-lymphocytes from a population of cells. A person or an animal, you can enter a cytotoxic effective amount of the conjugate comprising a monoclonal antibody or antibody fragment directed against a cell surface antigen present on disposal of lymphocytes. In an alternative embodiment, the population of cells can be incubi ernstig antigens. Want to delete lymphocytes can be a helper, suppressor or cytotoxic T cells.This aspect of the present invention can be used to prevent rejection of the transplanted tissue in the recipient. These conjugates can be used as immunosuppressive agents. Recipient enter this amount conjugate, which effectively prevents graft rejection. In this case, it is preferable to remove cytotoxic T-cells.Cancer in animals and humans can be treated by removing T-cells suppressor introduction of the corresponding conjugate. Autoimmune diseases can be treated by introducing conjugate to remove T-cells, helper cells. In each case, apply the above method of administration of the conjugate and dose.Immunoglobulin conjugates prepared as pharmaceutical compositions with a pharmaceutically acceptable carrier or diluent. You can use any suitable carrier or diluent. Suitable carriers or diluents include saline and dextrose ringer.The following examples illustrate the present invention.On the attached ª formula derived anthracycline.In Fig. 2 shows the connection idarubitsina /Ida/ with anti-Ly-2.1 (0.5 mg). The number of moles Ida introduced per mole of anti-Ly-2.1 (1), the left ordinate, the selection of the protein; (2) the right ordinate is shown as a function of the number of nmoles Ida in the reaction mixture (abscissa).In Fig. 3 shows the titer of antibodies as % racecourses cells (ordinate) depending on dilution (10-1) (abscissa) HRP-anti-Ly-2.1 on the target cells ITT (I) 75 NS E3. Serial dilutions of a solution of 0.5 mg/ml anti-Ly-2.1 (3) or anti-Ly-2.1 was performed using solution 2 (4) or 8 (5) mol Ida mol of antibody.In Fig. 4 shows the inhibitory activity of Ida (6) or Ida-anti-Ly-2.1, 5 mol Lda/mol antibody (7), E3-cells during the 24-hour test, which shows the % inhibition of the introduction of [3H]-thymidine (ordinate) depending on the concentration of Ida(M) (abscissa).In Fig. 5 shows the inhibitory activity of Ida (8), Ida-anti-Ly-2.1, 5 mol Ida/mol antibodies (9) or Ida anti-TFR, 5 mol antibodies (10) on /Ly-2+/ E3-cells in a 30-minute test, which shows the % inhibition of the introduction of [3H] -thymidine (ordinate) depending on the concentration of Ida (M) (abscissa).In Fig. 6 shows the inhibitory activity of Ida-anti-Ly-2.1, 5 mol Ida mol antibodies (11) the introduction of [3H] thymidine (ordinate) depending on the concentration of Ida- (N) (abscissa).In Fig. 7 presents the growth thymoma E3 mice CBF1who was subcutaneously injected 2106cells. Groups of 10 mice intravenously introduced drugs, indicated by the arrow; PBS (13), Ida (14), anti-Ly-2.1 (15), Ida-anti-TFR (16) or Ida-anti-Ly-2.1(17), the Average tumor size (cm2) (the ordinate) is shown depending on the number of days after injection of tumor cells (abscissa). Histogram of error indicates the standard error of the mean.In Fig. 8 presents the individual curves of tumor growth in mice CBF1that subcutaneously introduced 2,0106cells E3 tumors and which are not 4 - and 5-day intravenous introduced Ida-anti-Ly-2.1-conjugate. Tumor size (cm2) (the ordinate) is shown depending on the number of days after injection of tumor cells (the abscissa).In Fig. 9 presents the growth of timony E3 in CBF1-mice, which was subcutaneously injected 3,0106cells. Groups of 10 mice intravenously introduced drugs, as shown by the arrow; PBS (18), anti-Ly-2.1 (19), Ida (20) or Ida-anti-Ly-2.1 conjugate (21). Average tumor size (cm2) (the ordinate) is presented depending on the number of days since rst CLASS="ptx2">In Fig. 10 shows the growth of timony E3 mice CBF, which was subcutaneously injected 3,0106cells. Groups of 10 mice were administered intravenous drugs, is shown by an arrow: PBS (22), anti-Ly-2.1 (23), Ida (24) or Ida-anti-Ly-2.1 conjugate (25). The average tumor size (cm2) (the ordinate) is presented depending on the number of day after injection of tumor cells (abscissa). Histogram of error indicates the standard error of the mean.In Fig. 11 shows the growth of xenograft tumors COLO 205 human in hairless mice, which was subcutaneously injected 2106cells. Groups of 10 mice intravenously injected drugs, arrows: PBS (26), free Ida (27), Ida-250-30,6-conjugate (28), a mixture of Ida and 250-30,6 (29) and 250-30,5 (30). The mean tumor size (cm2) (the ordinate) is presented depending on the number of days after injection of tumor cells (abscissa). The histogram represents the standard error of the mean size of the tumor.In Fig. 12 presents the individual growth curves of tumors, xenotransplantion hairless mice, which were treated intravenously (arrow) Ida-250-30,6-conjugate. The dashed line indicates the average tumor size in mice treated with PBS. The size of the closed cells (abscissa).In Fig. 13 shows the connection idarubitsina (Ida) with anti-L3T4 (0.5 mg). The number of moles Ida introduced per mole of anti-L3T4 (31) (left ordinate), and the allocation of the protein (32) (right ordinate) is shown as a function of the number n of moles of the Ida in the reaction mixture (abscissa).In Fig. 14 shows the titer of antibodies as % rosette cells (ordinate) depending on dilution of antibody (10-1) (abscissa) anti-TLy-I-conjugates ITT (1) 75 NS at E3 target cells. A series of dilutions was carried out on a solution of 1 mg/ml anti-TLy-I (33) or conjugate solution 1 (34), 4 (35) or 7 (36) mol Ida/mol anti-TLy-I.In Fig. 15 presents the impact of Ida-Moab and Moab on the number of L3T4+- and Ly-2+-cells in the spleen of mice treated Ida-anti-L3T4 (37), anti-L3T4 (38), Ida-anti-Ly-2.1 (39), anti-Ly-2.1 (40) or untreated (41), where % rosette cells (ordinate) is shown in dependence on time in days (abscissa).In Fig. 16 pre-impact joint processing Ida-Moab-conjugate to the survival of transplant P388D1 tumors (difference H-2 and non-H-2) in CBA mice. Groups of 10-15 mice subcutaneously introduced 8106cells P388D 1 tumors and intravenous (arrows) one of the drugs: (I) PBS (42). (II) anti-L3T4 and anti-Ly-2.1 (43). (III) Ida-anti-L3T4 (44). (IV) Ida-anti-Ly-2.1 (45) is esta days after injection of tumor cells (abscissa). Histogram of error represents the standard error of the mean.In Fig. 17 presents the impact of Ida-anti-i.l.y bit-1 is conjugate to the survival of transplant tumor P388D1 in mice CBA. Groups of 10 mice subcutaneously introduced 1106tumor cells P383D1 and intravenous (arrows) one of the drugs: (I) PBS (47). (II) anti-TLy-1 (48) and (III) Ida-anti-TLy-1- (49). The average size (cm2tumor presents (ordinate) depending on the number of days after injection of tumor cells (abscissa). Histogram of error represents the standard error of the mean.In Fig. 18 presents the growth of COLO205 xenograft (30,6+, 17,1+) bezwolosykh mice, which introduced 8106cells of each mouse. Groups of 10 mice was intraperitoneally injected (arrows) PBS (50), 17,1-Ida (51), 30,6-Ida (52) or a mixture of 30.6-Ida and 17.1-Ida (53). The average tumor size (cm2) (the ordinate) is presented depending on the number of days after injection of tumor cells (abscissa). Histogram of error indicates the standard error of the mean. The total dose Ida amounted to 200 mcg.In Fig. 19 shows the growth of xenograft tumors obolochnoy intestine of man at bezwolosykh mice, transplanted in the form of a fragment Ida (58). The mean tumor size (cm2) (the ordinate) is presented depending on the number of days after transplantation of the tumor (abscissa). Histogram of error represents the standard error of the mean. The total dose Ida amounted to 80 mcg.Example 1. The materials and methods. Tumor cells.The cell line studied in this example included an option [E3] III(1)NSE3(Ly-2+) murine thymoma (Snuth ctal. , S. Natl Cancel 1985, 76, 503-510) ELy(Ly-2-, AR-), INFAMY (norowtz et al., Science 1968, 160, 533-535), CEM-line (TFR+human cells (Foley et al., Cancer 1955, 18, 522-529) and line (-250-30,6+human COLO205 cells. Cells were maintained in vitro in medium Needle, modified, Dulbecco (DME), or in medium RPMI 1640 ( Flow laboratories, Sydney, Avstralia), to which was added 10% heated newborn calf serum (Flaw), 2 mm glutamine (Commoniwealth Screen Laboratories, Sydney), 100 μg/ml of streptomycin (Glaco, Melbauro, Australia) and 100 I. E./ml penicillin (Commoniwealth Screen Laboratoris). Tumor E3 was maintained in vivo by serial passage in (C57BL(6BALB/c/F1mouse/mouse CBF1. Cells from ascitic fluid was washed and centrifuged (400g5 min) twice in phosphate-buffered saline (PBS), pH 7.3, re-suspended in PBS and subcutaneously introduced (S,c) in the abdominal wall of the mouse: these operations is Brabec and daily measured subsequent size of tumors using calibre-square, measured along the perpendicular axes of the tumors. Data were recorded as mean tumor size (the product of the diameters of the root mean square error).Mouse. CBA (C57BL)6BALB/c/CBP1mouse and naked mice (nu/nu) were obtained in the Departament Pothology, University of Melbone. Experimental groups of 8-10 mice of the same sex and age were used in each experiment.Monoclonal antibodies. Used the following monoclonal antibodies: (I) anti-Ly-2.1(units igc1), responsive to mouse Ly-2.1 specificity (Uogarth et al Immunology, 1982, 46, 135-144), (ii)A3C6 (anti-TFR) (units igc1), responsive to tranfering receptor human (TFR) (Panaccio et al., Immunology and Cell Biology) and (iii) the antibody labeled 250-30,6, reacting with the antigen present on cells of carcinoma of the colon of man.Moab was isolated from ascitic fluid by precipitation with 40% ammonium sulfate, dissolved in PBS and dialyzed in the same buffer solution. These crude preparations or absorbed on protein-A-sepharose (Pharmacia Inc Piscataway, New Lersey), intensively washed with PBS (pH 7.3) and were diluted with 0.2 M glycine/HCl (pH 2,8), or passed through the blue column Afligel (Bio-Rad Zaboratories Ptz Ltd Sydney). After neutralizing Moab was subjected to dialysis against PBS, took aliquot samples and keep the weeks cells LiCR-LON-HMy-2(HMy-2) (OCT+Vlin the number 2106by removing the spleen in three days after the last injection and merging cells P3-NS1-AC4-1(NS-1).Obtaining and quantifying conjugates.The intact anti-Ly-2.1, anti-IFR Moab or 250 - 30,6 (1 to 2 mg/ml in Cambridge buffer solution, pH 8) was mixed with a molar excess (1 to 50) 14-bromo-4-demethoxygeldanamycin (Br-Ida), dissolved in N,N-dimethylformamide (DMF) at 10 mg/ml of the Reaction was maintained at room temperature for 4 h before centrifugation (400 g5 min) to remove any precipitate. Free Br-Ida and other source materials were removed by gel-filtration chromatography using a column of Sephadex C-25 (PD-10, Pharmacia), and conjugates then pass through the column Porapax Q (Millipore) to remove any absorbed drug (Miederevieser et al. J Chromatog, 1971, 54, 215 - 223). The number of Ida that are included in the conjugates of the drug - Moab, was determined by absorption spectrometry at 483 mm /E498= 3,4103M-1cm-1and protein estimation (Bredford, Biochem Anol 1976, 72, 248 - 253).The activity of antibodies.Test rosethorne using sheep antimachine immunoglobulin (SANG), used for determining the activity of antibodies Ida-Moab-conjugates compared to free monoclonal ol Method, 1978, 20, 173 - 183).The activity of the drug.(a) 24-hour test suppression: 100 μl of cells (2 - 5106ml) made in microtechnology Cup with a flat bottom and were grown for 1 h at 37oC. Free idarubitsin (Ida), dissolved in PBS, and conjugates Ida-Moab is aseptically filtered in sterile PBS was performed dilute 50 ál of the free Ida or conjugate added to the cells, using dual wells for each sample; control wells received 50 μl of PBC, and cells were grown at 37oC, 7% CO2within 24 hours(b) a 30-minute test for suppression: 200 μl of cells (2 - 5106ml) was collected in sterile Eppendorf tubes, re-suspended in sterile drug or conjugate solution and mixed for 30 min at 37oC. Then the cells were centrifuged (400 g5 min), re-suspended in the medium of cultivation and 100 ál aliquots were inoculated into microtiter Cup, using dual wells for each sample before incubation period of 16 to 24 hours After the incubation period in both of these samples were added to 50 μg of a nutrient medium containing I and Ci[3H] -thymidine (specific activity 5Ci/mmol); Amersham/ and cups were grown 2 - 4 h Cells were then collected, obezbedili, a single sample was divided and considered in beasts the different samples. The standard error for any point was determined by dual definitions and did not exceed 5% for any given experimental point.The toxicity. Groups of 10 - 20 CBA mice were given a single intravenous injection of different doses of Ida or Ida-anti-Ly-2.1 and observed a survival rate of mice in relation to the dose expressed in mg/kg Organs of these mice were removed and weighed before fixation in formalin and staining with hematoxylin and eosin.Results. Obtaining and characterization conjugates.Br-ida (Fig. 1) covalently joined with several Moab against human TER, against antigens present on cancer cells of the colon of a person (antibody 250 - 30,6), and against Ly-2-alloantigen mouse. Reaction conditions established for conjugation by changing the molar excess Br-Ida, added in Moab, and compromise between the higher introduction Ida and lower protein secretions. Ida-anti-Ly-2.1 (Fig. 2), Ida-anti-TER and Ida-250-30,6 (data not shown) included 3 - 5 molecules Ida when protein secretions more than 50%. The reaction of Br-Ida with monoclonal antibodies (Moab) can provide two types of links (Fig. 1C and D). To establish what was presented, the conjugates were subjected to pH 4.5 and the Yali spectrometry. 50% of the bound drug was released when exposed to a base (pH 9), while at pH 4.5 loss was not observed. This suggests that at least 50% of medicines have ester bond (Fig. 1D), since the ester bond is sensitive to basic conditions, while amine the relationship is sustainable.The activity of antibodies.Titers of antibodies before and after conjugation was measured by the method of rosethorne and they were determined as the dilution at which 50% of target cells were Demetrashvili outlet. Ida-anti-Ly-2.1-conjugates containing 2 and 8 molecules Ida, had titers of antibodies against the cells B3 1:56000 1:33000, respectively, while the titer deconjugation antibodies was equal to 1:8000 (Fig. 3). Ida-250-30,6 - conjugates containing 2 and 6 molecules Ida, had antibody titers against COLO 205 cells 1:16,000 and 1:11000, respectively, while the titer deconjugation antibodies was 1:33000. Thus, there is some loss of antibody activity due to the conjugation procedure; compared with less than 6 molecules Ida-molecule Moab was used for in vitro and in vivo. It was noted that the solubility and activity of the antibodies Ida-anti-Ly-2.1-conjugates are significantly reduced below these levels of addition (data not p is evaluating antibodies.In vitro activity and conjugates idarubicin and idarubicin-Moab.In vitro-cytoxicity Ida and two Ida-Moab-conjugates on cell line mouse ITT (1) 75 NSE 3 (Ly2+TFR-) and cell line CEM man (Ly - 2-TFR+) was measured in 24-hour suppression test and determined I. D.50(50% suppression when making standards [3H]-thymidine). The results are shown in Fig. 4 and table. 1. I. D.50for Ida was in the range of 1.0 - 2,510-7M check for both cell lines (Fig. 4, PL. 1). I. D.50for Ida-anti-Ly-2.1 at E3 was 4 times higher (Fig. 4) and Ida-anti-TER on the CEM values I. D.50were 1 to 2 times larger than for the free Ida (PL. 1). Therefore, free Ida was more cytotoxic towards E3 and CEM than Ida-anti-Ly-2.1 and Ida-anti-TFR, respectively. However, these conjugates Ida-Moab are 10-fold lower cytotoxicity to areagirls the cell lines (table. 1), noting in such a way that their cytotoxic effect was specific and was a result of the delay activity of the antibodies (Fig. 3).1 I. D.50- 50% suppression with the introduction of standards [3H]-thymidine;
2, the number of tested drugs.Conjugates Ida-250-30,6 were slightly less active than the free Ida. is OK target COLO 205.For the study, are the conjugates of selectivity in their cytotoxic activity against target cells Ida-anti-LY-2.1 and Ida-anit-TFR grew 30 min with E3 (Ly-2+) cells before pouring the unbound conjugate and measurement of cytotoxicity. Ida-anti-Ly-2.1 conjugate had I. D.506,2 10-7M compared with I. D.505.2 10-7M for free Ida (Fig. 5). In contrast nerealiausi Ida-anti-TFR-conjugate had I. D.505 10-6M, i.e. 10 times more than for the free Ida, showing that the activity of antibody binding Ida-anti-Ly-2.1-conjugate leads to its selective cytotoxicity. Similarly, Ida-250-30,6-conjugate and free drug were grown for 30 min with COLO 205 (250-30,6+Ve) and E3 (250-30,6-Ve) cell lines re-washed and tested for cytotoxicity. Both cell lines showed a similar response to the dose of the free drug, i.e., 9,2 10-7M for COLO 205 and 9.8 10-7M for E3. However, Ida-250-30,6-conjugate was 4 times more toxic compared in COLO 205, than to non-reactive cell line E3 antibody. Similar results were obtained using the cell line CEM and Ida-anti-Ly-2.1 as non-standard (data not shown). For additional conviction that the cytotoxicity of conjugates of Ida-Moa inhibition of cytotoxicity of the conjugate, using free Moab. When the concentration of the Ida 4 10-6M (2 µg anti-Ly-2.1) the cytotoxicity of the conjugate anti-Ly-2.1 E3-cells was reduced by 70% after the addition of 50 mg (250 mcg/ml), anti-Ly-2.1. (Fig. 6), indicating that the cytotoxicity of the conjugate Ida-anti-Ly-2.1 directly related to its characteristic of antibody binding. Similar to the control results obtained with 260-30,6. It should be noted that in all of the tests available anti-Ly-2.1, anti-TFR and 250 - 30,6 were nectocarcinus (data not shown).In vitro treatment of cells murine thymoma ITT(1) 75 NS T3.To assess inhibition of growth of solid tumors in mice groups CBF1(10 animals per group) immunized 2 106R3-cells subcutaneously in the abdominal area, were treated with intravenous injections of one of the following drugs: (I) PBS); (U) anti-Ly-2/1; (iii) (Ida; (iv) Ida-anti-TFR, or (v) Ida-anti-Ly-2/1. Mice received 20 µg Ida and/or 1200 µg anti-Ly-2.1, respectively, for 4 - and 5-th day (tumour size 0.1 cm2) after inoculation.Within 24 h of the first processing processed Ida anti-Ly-2.1 mice had a mean tumor size was equal to 20% of tumors treated with PBS mice (i.e., 80% reduction in tumor mass (Fig. 7)). Obviously, one anti-Ly-2.1 and Ida, covalently associated with non-specific anti-TFR and M is able, while others had a 25% reduction of body weight. Individual growth curves of the tumors of mice receiving Ida-anti-Ly-2.1, showed a decrease of 9 out of 10 tumors during the course of treatment (Fig. 8); 5 of the 10 tumors completely regressed and did not appear again (200 days) and those tumors that continued to grow after treatment (5 of 10), grew more slowly than tumors of mice that were treated with PBS and Ida-anti-TFR. There was conducted an additional experiment to evaluate the intravenous treatment of solid tumors using Ida-anti-Ly-2.1. Groups CBF1mice (10 animals per group) were inoculable 3 106E3-cells, and mouse then received 15 mg and 900 µg Ida and Inti-Ly-2.1, respectively, at 6 (tumor size 0.2 cm2and the 7th day after inoculation of the tumor (Fig. 9). Mice treated Ida-anti-Ly-2, had an average tumor size at 50% less than mice treated with PBS, and 66% of the tumors of mice treated with Ida on the seventh day. This trend continued until the end of the study (18-th day). Individual growth curves of tumors 10 mice CBF1taking Ida-anti-Ly-2.1, show that there were 4 regression and one complete disappearance of the tumor mass ( >200 days. data not presented). Therefore, Ida-anti-Ly-2.1 was effective against large tumors, and in both exp vitro treatment of tumors of the colon human COLO 205.The impact of Ida-anti-250-30,6-conjugate was evaluated on naked mice (PI/PI), generating COLO 205 xenografts.Injections subcutaneously in the abdominal wall 2 of 106cells led to palpable tumors after 4 days (approximately 0.1 cm2). Groups of 10 mice were then treated by intravenous injection of one of the following drugs: (I) PBS; (ii) 250 - 306; (iii) Ida plus 250 - 30,6 (unconjugated); (vI) Ida; (v) conjugate Ida-250-30,6. Just introduced 275 µg Ida series of intravenous injections (5) 4-, 5-, 6-, 10-, and 12-day after inoculation of the tumor.In groups of mice that were treated with PBS or conjugaison 250-30,6, was not observed therapeutic effect. When using Ida 2 out of 10 mice survived, whereas all the mice of the treated group conjugatively Ida plus 250-30,6, was characterized by a sharp decrease in tumor size. These results are shown in Fig. 11. In opposite to this growth curves of tumors for individual mice (Fig. 12) show that 5 out of 10 mice had tumors that regressed on the 7th day, these tumors then continued to grow, although 2 out of 10 mice were left without tumors. These mice showed no symptoms of toxicity.Intratumoral ESA and animals. Therefore, have conducted studies to characterize the antitumor activity of conjugates of Ida-anti-Ly-2.1 when you enter them directly in the hard E3 tumor. Groups of 10 mice CBF1that subcutaneously implanted 3 106E3-cells developed tumors (0.1 to 0.2 cm2on the 5 th day after inoculation of the tumor. Treatment consisted of 2 injections of 5 - and 5-th day after implantation of the tumor, the mice received one of the following treatments: (1) PBS; (2) Ida; (3) anti-Ly-2.1 and (4) Ida-anti-Ly-2.1 (total number of Ida 30 µg). Ida-anti-Ly-2.1 showed the greatest antitumor activity, free Ida and only anti-Ly-2.1 showed no effect on tumor growth when introduced directly into the tumor. Mice that received Ida-anti-Ly-2.1, had an average tumor size equal to 60% of the tumors of mice that received PBS for 8 days, and 30% of the tumors of mice that received PBS on the 13th day (Fig. 10). Individual growth curves of the tumors (data one full regression, while the remaining mice showed a delayed decrease of tumor growth on the 3rd day after treatment.The toxicity.For the experiments on acute toxicity groups of 10 CBA (Ly-2.1+) mice were given a single injection of different doses of Ida, Ida-anti-Ly-2.1 or Ida-anti-TFR. In what by any conjugate Ida-Moab, was not observed weight loss. In table. 2 shows the toxicity of Ida and Ida-Moab-conjugates, expressed in values of L. D.50and L. D.10. As shown, the summer Palace10Ida-anti-Ly-2.1 was 10 mg/kg Ida compared to only 0.75 mg/kg for free Ida. In addition, the conjugate Ida-anti-TFR had a summer Palace108 mg/kg. Conjugate Ida-Moab has not been tested on HP DV50- the dose. These results show the large therapeutic index conjugates Ida-Moab compared with the free Ida.Histopathological results.Acute effect of intravenous administration of free Ida (1 mg/kg) results in atrophy of the white pulp in the spleen on the 15th day after treatment and some hypertrophy of cardiac muscle pulled (data not shown). In contrast, a single dose Ida-anti-Ly-2.1 (2.4 mg/kg) did not cause nonspecific tissue toxicity after 15 and 30 days, although some swelling hepatocytes was observed on the 15th day.Example 2. Substances and methods.Mouse. Department of pathology the University of Melbourne received the type mice DBA/2 x BALB/c/F1and 6BA. In each experiment used a group of 10-20 mice of the same sex and age.Outlive cells.The cell line studied in the 1968, 160, 533-535), COL0 carcinoma of the colon of a person (Semple et al, Cancer EN 1976, 38, 1345-355) and CEM legkaya T-cells (Ly-2-. L3T4-) (Foley et al., Cancer, 1965, 18, 522-529). Cells were maintained in vitro as described in example 1. Line macrophages P338D1 was maintained in vivo by serial passage in mice (DBA/2 BA/B/c/F1. Cells from ascitic fluid was washed and centrifuged (400g, 5 min) twice in phosphatebuffered saline (PBS, pH 7.3), re-suspended in PES and were injected subcutaneously in the abdominal wall of the mouse, where they have evolved in palpable tumor grafts. Mice were then subjected to a series of intravenous treatments, the size of tumors was measured daily using calibre-square, measuring tumor on perpendicular axes. Data were recorded as mean tumor size (product of two diameters standard error.Monoclonal antibodies.Monoclonal antibodies to murine cell surface antigens L3T4, Ly-2 and ThY-1, which characterize individual subpopulations of lymphocytes were used as a model.We used the following Moab: (i) anti-Ly-2.1 (mouse IgC2a), reactive with the specificity of the murine Ly-2.1 (Horgarth et al, Immunology 1982, 46, 135-144), (ii) H129.19 (anti-L3T4) (IgC2 rats), reactive with h et al. , J. Immunology 1979, 122, 2491-2497). Antibodies were isolated, purified, and stored as in example 1. The activity of the antibodies was determined by a test on rosethorne with sheep artemisinin immunoglobulin (CAMC), as described in example 1.Obtaining conjugates idarubitsin-monoclonal antibody.Moa b (1-2 mg/ml) was combined with 5-20 molar excess of 14-bromo-4-demethoxygeldanamycin (Br-Ida), dissolved in N, N-dimethylformamide (10 mg/ml) for 4 h at pH 8 (of 0.05 M borate buffer solution) at room temperature. Spent the reaction and treatment, and the number of Ida in the resulting conjugate was determined as described in example 1.The activity of the drug.Two tests (24 h and 30 min) was conducted to evaluate the activity of drugs similar to example 1, using cells at a concentration of 1-5106mg/mlSerology.To determine the titer of antibodies and the number of L3T4 cells+and Ly-2+in these experiments used rosethorne. This method involves the binding of SAMG and it also defines the immunoglobulin rats (Ig) associated with sheep red blood cells (SRC) to detect antibodies on the surface of lymphoid cells. Ig+splenic cells were surface Ig removed using locks with SAMG (25 the disgust resynthesis of immunoglobulin.Results.The study was conducted in separate phases:
(a) in vitro determination of the characteristics of the three different Ida-Moab-conjugates;
(b) subsequent demonstration of their use in the active destruction of a subclass of T-cells before and during allograft rejection of tumor cells.Preparation and characterization of conjugates.Br-Ida covalently linked with Moab against murine L3T4, Ly-2 and Thy-1 antigens. Reaction conditions were established for conjugation by changing the molar excess Br-Ida added to Moab and keeping a compromise between the higher introduction Ida and lower allocations of the protein. Ida-anti-L3T4 (Fig. 13), Ida-anti-Ly-2.1 and Ida-anti-Thy-1 (data not shown) included 3-6 molecules Ida discharge of the protein more than 50%.The activity of antibodies.Antibody titers before and after conjugation was measured by the method of rosethorne and was defined as the dilution at which 50% of target cells E3 form rosettes. Ida-anti-Thy-1-conjugate containing 1,4 and 7 molecules Ida, had antibody titers of 1:425000, 1:170000 1:130000, respectively, while the titer of unmodified antibody was equal to 1:of 550,000 (Fig. 14). Thus, after conjugation with Ida there is some loss of aldovini. Similarly, the activity of the antibodies of both conjugates Ida-anti-Ly-2.1 and Ida-anti-L3T4 (data not shown) decreases sharply with the introduction of more than 6 molecules Ida-molecules with Moab.The activity of drugs in vitro.Cytotoxicity of conjugates of Ida-Moab tested on various reactive target cells, using the 24-hour test, and compared with the free Ida. The activity of the free Ida 4-10 times higher than Ida-Moab-conjugates with I. D. 50(50% inhibition in the introduction of standards [3H]-thymidine) free Ida, which occur when 6,6-9,010-8N, against the scanned lines of tumor cells. It was clear that Ida-anti-Ly-2.1 conjugate is the most cytotoxic conjugate (I. D.50-4,310-7M) when verifying against the cell line ITT(I) 75NS E3, which is a variant of a native cell line, superior to antigen Ly-2. To study the selectivity of the effects of the conjugate to target cells Ida-Moab-conjugates were grown for 3 min with target cells, then the unbound conjugate is washed and measured the cytotoxicity. Using this 30-minute test revealed that non-reactive Ida-Moab-conjugates had I. D.5010-50 times more than I. D.50free Ida, showed that the binding of antibodies is essential for titwala cytotoxic effects on target cells in vitro in the absence of complement.The results are given in table. 3.In vivo potency of conjugates idarubitsin-monoclonal antibody.In vivo immunosuppressive potency of conjugates of Ida-Moab were compared with this feature one Moab by testing the ability of these conjugates selectively removed from spleen cells L3T4+or Ly-2+. Mouse CBA received 4 intravenous injections of anti-Ly-2.1 or anti-L3T4-conjugate (30 µg Ida/1.5 mg Moab) in the 0-, 2-, 5 - and 10-th day, and the number of Ly-2+or L3T4+- cells in the spleen was controlled using test rosethorne. For each of the treatment cells spleens of two mice who received the drug were studied in each day and the results were averaged (Fig. 15). There was a sharp decline in the number of L3T4+cells from approximately 30% of the total number of splenic cells from normal mice on day 0 to about 4% of the mice that received the Ida-anti-L3T4, on the 20th day. These L3T4+cells remained depleted for more than 60 days before he was to be a gradual increase. Exhaustion after in vivo treatment one anti-L3T4 also led to a sharp decline in the number of L3T4 cells+(from 30 to 5%).Conjugate Ida-anti-Ly-2.1 reduced the number of spleen cells Ly-2+from 25 to day. It was interesting to note that one anti-Ly-2.1 Moab was unable to drain Ly-2+cells significantly. Fluctuations in the number of cells Ly-2+and L374+untreated control mice, as expected, associated with natural variations between individuals. Thus, it became clear that both Ida-anti-L374 and Ida-anti-Ly-2.1 can remove L3T4+- or Ly-2+cells from the spleens of treated mice more effectively than one Moab. The impact of Ida-anti-Ly-2.1-conjugate important as anti-Ly-2.1 Moab has no effect when using only him, but could turn into a potential immunosuppressive agent in connection with the Ida. Depletion supported by the conjugates of medicine - Moab, was, therefore, is suitable for studying the role of cell Ly-2+and L3T4+in vivo rejection of the transplant.The impact of Ida-anti-L374, Ida-anti-Ly-2.1 and Ida-anti-TLy-1 on the survival of tumor transplant.In these experiments, Ida-anti-L374-, Ida-anti-Ly-2.1 - and Ida-anti-TLy-1-conjugates were used to increase the survival time of tumor grafts P388D1 in CBA mice. The allografts were performed on H-2 (class 1 and 11) and non-H-2 districts.(a). Combined treatment and intravenous-1-, 0 (the day of tumor inoculation)-, 3-, 5 - and 10-th day one of the following drugs: (I) PBS, (ii) anti-L3T4 and anti-Ly-2.1, (iii) Ida-anti-L3T4, (iv) Ida-anti-Ly-2.1 and (v) Ida-anti-L3T4 and Ida-anti-Ly-2.1. The total number of entered Ida and Moab was 125 μg and 5.75 MK, respectively.Tumor grafts mice that received PBS and Moab, surviving within 14-17 days with a maximum average size of tumors 0,31 cm2showed that both unconjugated monoclonal antibodies together unable to effectively Deplete L3T4+- and Ly-2+cells to tumor transplant to survive (Fig. 16). In addition Ida-anti-Ly-2.1 or Ida-anti-L3T4 separately were only able to prolong the survival of the graft (20-28 days) with a maximum average size of tumors 0,40-0,50 cm2. Apparently, one Ida-anti-Ly-2.1 or Ida-anti-L3T4 may not remove all T-cells, and a strong antigenic infection (as seen with all the differences MHC) can cause the proliferation of the remaining Ly-2+and L3T4+cells and a rejection reaction. In contrast, the combination of Ida-anti-Ly-2.1 - and Ida-anti-L3T4-conjugates allowed 14/15 tumor transplantation P388D1 to avoid exclusion and to increase in size, while mice do not slew (50-day - 2 cm2). It was noted that while 8-16-s females; however, the constant depletion of L3T4+- and Ly-2+cells (10-day) allowed tumor grafts P388D1 to establish persistent tumor growth.(b) Ida-anti-TLy-1-conjugate.Groups of 10 mice CBA) was subcutaneously injected tumor cells P388D1 in the amount of 107and intravenous-1-, 0-, 5 - and 6-th day one of the following drugs: (I) PBS, (ii) anti-TLy-1 and (iii) Ida-anti-TLy-1. The total number of entered Ida and anti-TLy-1 was 130 μg and 5.4 MK, respectively, and tumor grafts PBS treated mice lived for 15 days. One anti-TLy-1 Moab supported the survival of tumor transplant for 28-32 days at maximum average tumor size of 0.53 cm2(6 days) (Fig. 17). The mice that received the Ida-anti-Thy-1 30% of tumor grafts were completely rejected on the 40th day, while the remaining 70% continued to grow, which ultimately (32 days), allowed to increase the average tumor size of the group. Therefore, Ida-anti-Thy-1 is similar to the combination of Ida-anti-Ly-2.1 and Ida-anti-L3T4 was able to Deplete Ly-2+and L3T4+cells so that the majority of tumor grafts P388D1 survived CBA mice in the absence of normal rejection reactions.Example 3. Preparation of conjugates in Sona antibody 30,6. Cell carcinoma COLO 205 colon man (30,6+, 17,1+) were subcutaneously inoculable hairless mice in the amount of 8106cells/mouse as described in example 2. Then inoculated mice were subjected to a series of intraperitoneal injections. Tumor size was measured with a caliber-square, measuring along the perpendicular axes of the tumors. Data were recorded as mean tumor size (the product of two squares standard error). The results are shown in Fig. 18.Example 4. Prepared conjugates in accordance with the procedure described in example 1, between Ida and monoclonal antibody 17,1, between Ida and monoclonal antibody ICT-13, between Ida and monoclonal antibody 27,1 and between Ida and monoclonal antibody 30,6. Hairless mice implanted xenograft tumor: colon man LIM2210 (1-5). Then implanted mice made a series of intravenous injections. Tumor size was measured with a caliber-square, measuring along the perpendicular axes of the tumors. Data were recorded as mean tumor size (the product of two diameters standard error). The results are shown in Fig. 19. 1. Conjugate derived 14-brandenrobinson with a monoclonal antibody or a fragment of what it contains 14-bromo-4-demethoxygeldanamycin, conjugated with the antibody or its fragment having at least one binding site with the antigen and conjugate contains from 2 to 8 molecules of drugs covalently linked to the antibody or fragment.2. Pharmaceutical composition having antitumor activity, including derivative anthracycline and a physiologically acceptable carrier or diluent, wherein as derived anthracycline it contains conjugate under item 1, taken in an effective amount, and as a diluent saline or dextrose ringer.
< / BR>where in the case of the racemate and the enantiomers R1means cyclohexyl, or in the case of enantiomers R1also means cycloheptyl, as well as their salt
FIELD: organic chemistry, pharmacy.
SUBSTANCE: invention relates to new derivatives of benzimidazole represented by the following formula (I) or its salt:
wherein R1 represents (lower)-alkyl group; R2 represents aromatic (lower)-alkyl group that can be substituted with one or more groups taken among halogen atom, alkyl group, halogen-(lower)-alkyl group, nitro-group, aromatic group, aromatic (lower)-alkoxy-group, (lower)-cycloalkyloxy-(lower)-alkyl group, aromatic (lower)-alkyl group, aromatic (lower)-alkenyl group, aromatic (lower)-alkynyl group, aromatic oxy-(lower)-alkyl group, (lower)-cycloalkyl-(lower)-alkoxy-group, alkenyl group, (lower)-alkoxy-group, (lower)-alkylthio-group and (lower)-alkanesulfonylcarbamoyl group; R3 represents alkyl group, hydroxy-(lower)-alkyl group, alkenyl group, aromatic group, halogenated aromatic group, (lower)-alkyl aromatic group, (lower)-alkenyl aromatic group or aromatic (lower)-alkenyl group; -X- represents cross-linking group represented by one of the following formulas: (II) , (III) , (IV) , (V) . Also, invention relates to pharmaceutical compositions eliciting activity that reduces blood glucose level based on this compound. Invention provides preparing new compounds and pharmaceutical compositions based on thereof used for prophylaxis and treatment of damaged tolerance to glucose, diabetes mellitus, insulin-resistance syndrome, vascular failures syndrome, hyperlipidemia and cardiovascular disorders.
EFFECT: valuable medicinal properties of compounds and compositions.
16 cl, 1 tbl, 86 ex
FIELD: organic chemistry, medicine, pharmacy.
SUBSTANCE: invention describes compound of the formula (I):
as a free form or salt wherein Ar means group of the formula (II):
wherein R1 means hydrogen atom or hydroxy-group; R2 and R3 each means independently of one another hydrogen atom or (C1-C4)-alkyl; R4, R5, R6 and R7 each means independently of one another hydrogen atom, (C1-C4)-alkoxy-group, (C1-C4)-alkyl or (C1-C4)-alkyl substituted with (C1-C4)-alkoxy-group; or R5 and R6 in common with carbon atoms to which they are joined mean 6-membered cycloaliphatic ring or 6-membered heterocyclic ring comprising two oxygen atoms; R8 means -NHR13 wherein R13 means hydrogen atom, (C1-C4)-alkyl or -COR14 wherein R14 means hydrogen atom; or R13 means -SO2R17 wherein R17 means (C1-C4)-alkyl; R9 means hydrogen atom; or R8 means -NHR18 wherein -NHR18 and R9 in common with carbon atoms to which they are joined mean 6-membered heterocycle; R10 means -OH; X means (C1-C4)-alkyl; Y means carbon atom; n = 1 or 2; p = 1; q = 1; r = 0 or 1. Also, invention describes pharmaceutical composition based on compound of the formula (I), a method for preparing compound of the formula (I) and intermediate compound that is used in the method for preparing. Compounds elicit the positive stimulating effect of β2-adrenoceptor.
EFFECT: improved preparing method, valuable medicinal properties of compounds.
13 cl, 3 tbl, 35 ex
FIELD: medicine, oncohematology.
SUBSTANCE: the present innovation deals with treating elderly patients with chronic lympholeukosis accompanied with cardiovascular failure. The method deals with applying chemopreparations and cytoprotector. Moreover, 1 wk before the onset of chemotherapeutic therapy one should prescribe preductal at the dosage of 105 mg daily. At this background one should sample blood out of elbow vein at the volume of 200 ml into a vial with glugicir to centrifuge it, isolate plasma, divide into two portions, add into the 1st vial - cyclophosphan 600-800 mg/sq. m, vincristin 1.4 mg/sq. m, into the 2nd vial - adriamycin 50 mg/sq. m to be incubated for 30 min at 37 C and intravenously injected by drops for patients. Simultaneously, the intake of prednisolone should be prescribed at the dosage of 60 mg/sq. m since the 1st d and during the next 5 d and preductal at the dosage of 105 mg daily during a week, and then 2 wk more at the dosage of 60 mg daily. All the procedures should be repeated in above-mentioned sequence 4-6 times. The method enables to decrease toxic manifestations of chemotherapy while applying adequate dosages of cytostatics, anthracycline antibiotics, among them, at no great manifestations of their toxicity due to preductal's cardioprotective action.
EFFECT: higher efficiency of therapy.
1 ex, 5 tbl
FIELD: pharmaceutical industry.
SUBSTANCE: invention provides antibacterial drug in the form of enveloped tablet with its nucleus containing ofloxacin as active substance and auxiliary ingredients: silica powder, calcium stearate, collidone, sodium carboxymethylcellulose, milk sugar, talc, and microcrystalline cellulose. Envelop of tablet contains collidone, hydroxypropylcellulose, talc, and titanium dioxide. Manufacture of tablet comprises mixing ofloxacin, silica powder, milk sugar, and microcrystalline cellulose; moistening resulting mixture with collidone solution; wet granulation; drying; dry granulation; and powdering of granules with mixture of calcium stearate and sodium carboxymethylcellulose. Afterward, granules are enveloped.
EFFECT: increased storage stability.
FIELD: organic chemistry, antibacterial agents.
SUBSTANCE: invention describes 8-cyano-1-cyclopropyl-7-(1S,6S)-2,8-diazabicyclo-[4.3.0]-nonane-8-yl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid of the formula (I): with the crystalline modification A and a drug eliciting effect against pathogenic microorganisms. The prepared crystalline modification shows stability and doesn't transform to another crystalline modification or amorphous form being even at prolonged storage.
EFFECT: improved and valuable properties of compound.
4 cl, 4 dwg, 6 ex
FIELD: pharmaceutical industry.
SUBSTANCE: invention provides composite therapeutical agent exhibiting antituberculous effect and made in the form of solid dosage form containing as active principle combination of lomefloxacin, isoniazid, pyrazinamide, ethambutol hydrochloride, and pyridoxine hydrochloride plus auxiliaries.
EFFECT: increased assortment of antituberculous drugs.
4 cl, 1 tbl, 4 ex
FIELD: pharmaceutical industry.
SUBSTANCE: invention provides high-activity antituberculous formulation made in the form of solid dosage form containing as active principle combination of lomefloxacin, protionamide, pyrazinamide, ethambutol hydrochloride, and pyridoxine hydrochloride plus pharmaceutically acceptable auxiliaries.
EFFECT: increased assortment of antituberculous drugs.
4 cl, 1 tbl, 4 ex
SUBSTANCE: the present innovation deals with medicinal preparations designed as solution and indicated for therapeutic needs. Eye drops contain ciprofloxacin hydrochloride monohydrate being equivalent to 0.3% free foundation, a buffer system that keeps pH within 3.5-5.5 interval, as a conserving agent - benzalconium chloride and a s a stabilizer - the salt of disodium ethylenediamine tetraacetic acid, moreover, their range of osmolality values correspond to 150-450 mM/kg H2O. Eye drops should be obtained by preparing buffer system in which mannitol should be dissolved followed by the salt of disodium ethylenediamine tetraacetic acid, benzalconium chloride, ciprofloxacin hydrochloride. Then one should perform the control for the quality of obtained solution to be then filtered by applying sterilizing elements and packed. This innovation provides treatment of eyes at creating the pressure in an eye and at certain desired osmolality.
EFFECT: higher efficiency of therapy.
4 cl, 1 ex
FIELD: organic chemistry, medicine.
SUBSTANCE: invention describes 8-cyano-1-cyclopropyl-7-(1S,6S-2,8-diazabicyclo-[4.3.0]-nonane-8-yl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid of the formula (I):
in the crystalline modification B and a medicinal agent based on thereof that elicits effect against pathogenic microorganisms. Indicated modification of compound of the formula (I) shows stability and insignificant absorption of air moisture ant doesn't convert to another crystalline modification or amorphous form being even in the prolonged storage.
EFFECT: valuable properties of agent.
4 cl, 4 dwg, 6 ex