Anticancer pharmaceutical composition (versions) and application thereof

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, specifically to compositions able to inhibit the growth of tumour cells of various histological origins and activated endothelial cells, and can be used in medicine. Components of the specified compositions are non-proteolytic polypeptide fragments of serralysines chosen from ARA1, ARA2, ARA3 and ARA4. Specified fragments of serralysines are also combined with prodigiosins and with antibodies or their antigen-binding fragments directed on antigens of tumour cells that intensify the anticancer effect of the composition.

EFFECT: invention allows preparing the composition with higher antiproliferative activity, in comparison with a composition containing the whole molecules of serralysines.

8 cl, 16 dwg, 7 tbl, 21 ex

 

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates to the field of biotechnology, the pharmaceutical industry and, in particular, to obtain a composition capable of inhibiting growth of tumor cells. This composition comprises polypeptide fragments of serializing obtained by degradation of the intact protein, which have a higher antiproliferative activity than the whole molecule serraline. These fragments belong to the C-end of serializing, from an internal methionine sequence until the end of the molecule, and their combination with prodigiosin enhances the antitumor activity of this composition.

The LEVEL of TECHNOLOGY

Cancer chemotherapy has traditionally focused on the inhibition of proliferation of cancer cells. However, in recent years interest has grown in anticancer products, which induce apoptosis, because cancer, as installed, is a pathology associated with a relative defect in apoptosis, but not excessive proliferation.

Bacteria or their extracts used for the treatment of cancer is almost 100 years old. Most cited message of a physician and surgeon William B. Coley from the Memorial Hospital city of New Cork, called Sloan-Kettering Memorial Hospital. He noticed that many of his patients with several types of cancer felt regression of tumors after infection the Oia pathogenic bacteria (Coley, W.B. 1991 - reprinted from 1893-. Clin. Orthop. 262:3).

Resistance to tumors infected patients or animals attributed antitumor immunity mediated companion cells (Paglia, P. and Guzman, S.A. 1998. Cancer immunol. Immunother. 46:88). The idea that infection by pathogenic bacteria or protozoa can activate the regression of cancer through activation of innate or adaptive immunity, has been recently discussed by Hunter et al. (Hunter, C.A., Yu, D., Gee, M., Ngo, .V., Sevignani, C., Goldschmidt, M., Golovkina, T.V., Evans, S., Lee, W.F. and Thomas-Tekhonenko, A. 2001. J. Immunol. 166:5878). They demonstrated that tissue infected withT. Gondiiproduce some of soluble angiogenic factors, preventing the formation of blood vessels in tumors, which may represent potential therapeutic interest. This process of formation of new capillaries, known as angiogenesis, has become an important focus for the introduction of new methods of treatment of cancer and its metastases. Search antiangiogenic factors is the basis for new anti-cancer therapeutic strategies (Folkman, J. 2003. Seminars in Cancer Biology. 13:159). Recently, the use of anaerobic bacteria as chemotherapy and selective protifasistickych funds has led to a significant regression of subcutaneous (sc) tumors in mice. This treatment is called combined bacteriolytic terap is her (COBALTO) (Dang, L.H., Bettegowda, C., Huso, D.L., Klnzler, K.W. and Vogelstein, B. 2001. Proc Natl Arad Sci USA. 98: 15155). However, live bacteria cause significant toxicity and side effects that limit their use against cancer in humans.

In recent years there have been several reports indicating that anaerobic bacteria produce redox proteins that induce apoptosis of tumor cells (Yamada, T., Goto, M., Punj, V., Zaborina, O. and Chen, M.L. 2002. Proc. Natl. Acad. Sci. USA. 99: 14088; Goto, M., Yamada, T., Kimbara, K., Homer, J., Newcomb, M., Gupta, AS and Chakrabarty, A.M. 2003. Environ Mol. 47:549). Suggested that redox proteins can be included in the group of soluble proteins that secretively ancestors prokaryotic cells, and their functions could be the elimination of the ancestors of eukaryotic cells (Punj, V., and Chakrabarty, A.M. 2003. Cellular Microbiology. 5:225). In General, little is known regarding the establishment of soluble secreted factors data prokaryotic organisms that can specific way to act on cancer cells, causing their death and, in parallel, regression of tumors.

Serratia marcescensis a facultative anaerobic bacterium. From several strains received several drugs with anti-tumor properties; the most studied are: [a] ImuVert®(Budagov, R.S. and Ulianova, L.P. 2001. Radiats Biol Radioecol Russian. 41:38), the drug ribosomal membranes, is the quiet activate the immune system of the patient; [b] protease fromSerratiaMr 56000 (Wu, J., Akaike, T., Hayashida, K., Okamoto, T., Okuyama, A. and Maeda, H. 2001. Jpn. J. Cancer Res. 92:439), which induces cell death through necrosis, depending on the expression of α-2-macroglobulin; and [with] prodigiosin, a family of pigments that act as immunosuppressants and anticancerogenic through induction of apoptosis (Montaner, B. and Tomas Perez, R. 2003. Curr Cancer Drug Targets. 3:57; Tomas Perez, R. y Montaner, B. 2003. Histol Histopathol. 18:379). The authors of the present invention has been nefrotoksicheskoe fragments of serializing with higher cytotoxic activity than the whole molecule. This allows you to combine these fragments with low doses of prodigiosin, reducing the toxicity reported for pigment, and increasing antiproliferative effect on tumor cells.

DETAILED description of the INVENTION

The compositions of this invention are able to inhibit the growth of tumor cells and are composed of polypeptide fragments of serializing with a higher antiproliferative activity than the whole molecule serializing, which can be combined with prodigiosin that enhances their antitumor activity.

The present invention describes a drug MG2327, which together are present as polypeptides and prodigiosin, which has a broad spectrum of cytotoxic activity in lines of malignant cells, and the election effect on the transformed tumor cells and, in particular, cells that have been activated for growth. The sensitivity study on different cell lines, tumor or not, demonstrates that the line of normal cells are only slabocuvstivenami to the drug MG2327, while cells derived from melanoma, carcinoma of the larynx, fibrosarcoma, carcinomas and liver carcinomas of the cervix (positive or not for human papilloma virus) is very sensitive. Carcinoma hematopoietic origin are less sensitive. Cells HUVEC activated for growth are more sensitive to MG2327 than nonactivated. The drug MG2327 able to act on specific factor, expressed or sverkhekspressiya in the process of cell division. These factors form a therapeutic target against cancer and other proliferative diseases of origin. Moreover, these factors also constitute targets for early diagnosis of diseases caused by excess proliferation, or uncontrolled proliferation of differentiated or undifferentiated cells. Formulations with controlled release containing these molecules, can be used for data proliferating target, acting a specific way for them because normal cells are more resistant to their action.

To show p is tibooburra drug activity MG2327, mice BALB/c mice were injected intraperitoneal inoculation of tumor cells CB Hep.1 myeloid origin that can cause ascitic tumors in mice (Fontirrochi, G., Duenas, M., Fernandez de Cossio, M.E., Fuentes, P., Perez, M., Mainet, D., Ayala, M., Gavilondo, J.V. and Duarte, C. 1993. Biotecnol Aplic. 10: 24-30). After 10 days the mice were intraperitoneally injected with the drug MG2327 or PBS. 60% of the animals treated at the rate of 1 mg/kg survived, while only 25% of controls remained alive after 45 days after the start of treatment (Fig). Complete regression of tumors was observed in treated surviving animals demonstrated a healthy condition, while at the controls of the tumor progressed, forming large dense formations and mice showed a deterioration of the General condition.

Mouse BALB/c mice, with tumors of myeloid origin, treated with a single dose of the drug MG2327 rate of 1 mg/kg, survived with complete regression. The same dose increases the survival rate, with a significant decrease in tumor volume, BALB/c mice with tumors derived from transformed fibroblast E6/E7. MG2327 protects BALB/c mice from engraftment of myeloid tumors.

The drug MG2327 received as a result of optimization of cultivation conditions for the production antiproliferative molecules, which form a protective substance from engraftment and development of malignant tumors, thus, ka is este inductor develop antiproliferative, apoptotic and antiangiogenic molecules in normal and tumor cells that can be successfully used in the prevention and treatment of cancer, moreover, other diseases associated with these phenomena. Strain CMIB 4202 sverkhekspressiya soluble proteins in the range of 45-50 20-30 kDa (50 and 25 kDa by SDS-PAGE, with a coefficient of determination 0,984). For fractions of 25 kDa, identified as p25, showed dose-dependent strong antiproliferative activity in the experiment, performed on the cell line HEp-2, incubated with EDTA, while the fraction of 50 kDa, identified as p50, did not inhibit growth. However, incubation with 5 μm Zn2SO4for faction p50 showed antiproliferative activity, but less strong than for the faction p25. IC50fractions p25 and p50 consisted of 0.48 nm and 16 nm, respectively.

Selected protein biomolecules with antiproliferative action (polypeptides and prodigiosin) was obtained according to the present invention by means of only one phase chromatography: ion exchange using step gradient of NaCl. Applied matrix of sepharose Fast Flow DEAE or QAE, equilibrated with 50 mm phosphate buffer, pH 8,00. The elution was carried out stepwise gradient of NaCl: 50 mm phosphate buffer-0.1 M NaCl, pH 8,00; 50 mm phosphate buffer and 0.2 M NaCl, pH 8,00; 50 mm-2 M NaCl, pH 8,00, and in the end was suirable fraction of the pigment absorbed the and matrix, ethanol from absolute to 70%. The results confirm that the protein component of the drug 25 kDa has a greater capacity than the protein component of 50 kDa of the same drug to inhibit growth of the cell tumors, and both havein vitrobiological activity in individual form.

Degradation p50 led to the emergence of parts of two or more molecular masses, which included fragments of 25 kDa. Increased degradation of p50 were obtained by high temperature, and the formation of p25 was in proportion to this increase of temperature, while the temperature rise was reduced p50. Polyclonal antibodies to p50, obtained in sheep, learned p25 in the analysis of Western blot testing; thus, p25 occurs as a product of degradation of protein p50. The amount of degradation products were increased proportionally antiproliferative activity of degradation products. These results confirm that the autolysis p50 able to give fragments of degradation with antiproliferative activity stronger than the original molecules p50. In addition, p25 protein also induces complete regression of malignant tumors of myeloid origin. Fragments p50 can genetically konjugierte by means of some already known methodology fragments of antibodies and create immunotoxins applicable for the treatment of ill is of proliferative etiology. Also this fragment by itself or in combination with other protein molecules can be used as a carrier from the inside of cells or specific receptors. Fragment of p50 may also be exposed to the external environment regulated system release to maintain directional control on specific targets.

Proteolytic activity inhibited p50 7 mm EDTA and demonstrated that p50 is metalloproteases belonging to the family of serializing, as identified by mass spectrometry.

The main similarity was found to have form ID PRZN_SERSP and PRZN_SERMA in the database Swissprot proteins. Protein p25, purified by chromatography, did not possess enzymatic activity, and it corresponds to the non-catalytic carboxykinase area serializing.

Protein components and prodigiosin was in the same composition, which was significantly increased (p<0,005), inhibitory effect compared with the individual components of this composition. The song has received, maintaining the same ratio of protein and prodigiosin, which is used to evaluate the individual components. In such compositions prodigiosin can be detected in a concentration of 0.1-100 nm and fragments serraline between 0.1-150 mg/ml

A composition containing the polypeptide fragments derived from from the rulesyou, with an increased antiproliferative effect compared to intact molecules serializing, and the combination of fragments of serializing and prodigiosin, which selectively enhances the biological activity of the composition.

In addition, these polypeptide fragments have apoptotic effects on cancer cells. This apoptotic action includes fragmentation of mitochondria, microtubules and DNA, amplifying the signal of programmed cell death, with the use of reduced doses of this composition. These events were also observed with combined composition of the polypeptide and prodigiosin.

Antiangiogenic action MG2327 and antiproliferative fragments of the polypeptides was estimated by the method of forming tubular structures in Matrigel. Microvascular endothelial cells (HMEC) were incubated with narutoxsasuke concentrations MG2327 and its fractions p25 and P50. The final result was evaluated, taking into account the length of the formed tubular structures and the number of connections between them. Here we used the image processing program Pro Express 4.5. The result confirms that the processing composition MG2327 allows to explain that the processing of the protein and its antiproliferative polypeptides inhibited significantly (p<0,05, ANOVA) differentiation or maturation of endothelial what's cells, demonstrating that MG2327, and separated from it polypeptides possess antiangiogenic activity.

Apoptotic, angiogenic activity and selectivity are some of the characteristics that are most important to consider the composition of this invention, as a potential medicinal and protective agent against cancer.

Demonstrated that the combination of fragments of serializing and prodigiosin is stronger and more selective than the individual components, as anti-cancer remedies. These polypeptides can be used to obtain recombinant toxins and immunotoxins for the prevention and treatment of cancer or other diseases associated with proliferation of endothelial and transformed cells.

These polypeptides and their possible combination with prodigiosin applicable in the pharmaceutical industry to obtain vaccines, medicines and diagnostic tools for use in humans or animals against cancer and other pathologies of the proliferative type, which are highly selective and have a broad spectrum of action.

Description of the DRAWINGS

Figure 1. InteractionS. marcescenswith tumor cells CB Hep.1 get bacterial strains with high proliferative ability and modifica vannoy expression of the protein. A - cell Survival. After 72 hours, cell survival was estimated by the MTT method. For strain CMIB 4202 showed a strong antiproliferative effect on tumor cells HEp-2, while the effect of strain SM1995 was very weak. These results were averaged for three independent experiments using four replicates per sample. B - Electrophoresis SDS-PAGE (silver staining). CMIB 4202 was sverkhekspressiya soluble proteins that migrated at approximately 25 and 50 kDa.

Figure 2. Antiproliferative activity of fractions 25 and 50 kDa in the cell line HEp-2. Cell viability was determined by MTT method and expressed relative to control cells. Fractions were isolated from gels containing SDS (painted imidazole-zinc) and senatoriable. For fractions of approximately 25 kDa showed strong antiproliferative activity (IC500.35 nm/ml), whereas the p50 was unable to inhibit growth. Adding 5 μm of Zn4SO2to p50 observed increase in antiproliferative activity (IC5045 nm/ml), although lower than that of p25. Curves constructed on the basis of average values of five independent experiments and drew with the corresponding standard deviation (SD).

Figure 3. The kinetics of expression of protein and prodigiosin strain CBMI 4202. Expression of prodigiosin in the cultural environment of origin Taiwan is the CIO during the transition from growth phase to stationary phase, where CBMI 4202 reaches a higher doubling time. A - Kinetics doubling time of the cells. B - the Efficiency of expression of prodigiosin (product/biomass). C - Kinetics antiproliferative actions on cells Hep-2, determined by the MTT method. D - the growth kinetics of the cells, determined by optical density and protein biosynthesis.

Figure 4. The sensitivity of normal and tumor human cells to treatment MG2327. Normal cells have low sensitivity, and cells of hematopoietic origin are less sensitive than the rest of the analyzed cell lines. At the same time, cells that have been activated for growth (HUVEC bFGF), and cells derived from tumor malignant lesions are more sensitive.

Figure 5. Analyses of survival in BALB/c mice after treatment or without treatment drug MG2327 and the introduction of tumor CBHep1. A Dose of 1 mg/kg induced regression of tumors in 60% of treated animals, while 100% of the untreated animals died within 60 days. B - forty-five days after inoculation of tumor cells, untreated animals showed an extremely weakened state, with the presence of solid tumors and ascites, whereas animals treated at the rate of 1 mg/kg showed no signs of tumors and lived longer than 300 days.

6. Antitumor activity MG2327 on wholeway model 3T316. A - graph of daily average values for each group revealed statistically significant differences between the volumes of the tumors of the treated and untreated animals (p<0,003). Analysis of tumor growth over time showed no significant variations (p=0,109) for the group treated MG2327, while the negative control group showed a significant increase in this parameter (p=0.04). B - Survival of treated and untreated animals.

7. The selection of protein active fractions from MG2327. (A) Electrophoresis SDS-PAGE (12.5 percent). The results of staining on Kumasi. On track 1 shows the sample corresponding to the elution of 0.2 M NaCl, pH 8,00, where the protein band corresponding to 50 kDa, may be worthwhile. On track 2 shows a protein band of 25 kDa. Staining was carried out according to the method of Kumasi. (B) an Antiproliferative effect on HEp-2 proteins p50 and p25 on tumor human cells. The ratio of the dose-effect for p25, p50 and MG2327 were expressed as the concentration of total protein.

Fig. Antiproliferative effects of active fractions isolated from MG2327, cells HEp-2. For proteins of 50 and 25 kDa, contained in MG2327, shows the activity of inhibiting growth. The combination of these proteins with prodigiosin reduced dose, is able to inhibit the growth of 50% of tumor cells when compared with control (IC50).

Fig.9. SDS-PAGE and Western-blotty is, (A) a protein Sample obtained from MG2327. The sample was analyzed by gel SDS-PAGE (12%) and stained with silver. (B) Protein band of approximately 50 and 25 kDa (arrows) were cut from a similar gel, stained imidazole-zinc, senatoriable and again shared in the new SDS-PAGE. Proteins were transferred to nitrocellulose membrane and Western blotting demonstrated using antibodies to p50. Polyclonal antibody produced in goat, was able to learn p25 and degradation products p50, and learn stripes unrelated proteins (Neg C).

Figure 10. The autolysis p50. A - Electrophoresis SDS-PAGE: 1-24°C, 2-37°C, 3-45°C, 4-60°C, 5-4°C. B - Densitometric analysis of p50 and p25 showed that with increasing the incubation temperature decreases the intensity of the band p50, while increasing the intensity of the band p25.

11. p50, purified by chromatography on sepharose Fast Flow DEAE. p50 (suirvey of 0.2 M NaCl) gives the degradation products with a higher antiproliferative activity than the parent molecule.

Fig. The enzymatic activity of p25 and p50, obtained by different chromatography. A - For p25 not demonstrated activity, while for p50 shows enzymatic activity. B - 7 mm EDTA completely inhibited the enzymatic activity of p50.

Fig. MG2327 induced time-dependent fragmentation of the DNA of tumor cells P3X63Ag8. After 6 hours of incubation on lugati oligonucleosome fragments, and their number increases with time, reaching normal apoptotic profile with oligonucleosome fragments of 180-200 base pairs to 24 hours.

Fig. The ultrastructure of cells of the mouse myeloma P3X63AG8, treated and untreated (control cells) drug MG based on 22 µg/ml (A) electron micrographs of control cells murine myeloma P3X63Ag8 shows the cytoplasm and nucleus of these cells, shown by ultrastructure, normal for normal cells, and ultrastructure of mitochondria, normal for normal cells: mitochondrial matrix had a higher density than the surrounding cytoplasm(B) To 2 hours after treatment by MG in the cytoplasm were present vacuolation Taurus (which partly originate from modified mitochondriaswelling of mitochondria and destruction Cristnormal kernel(C, D) Aggregation of mitochondria and individual Christa, which become fusedalso observed after two hours of treatment in (). (E) Electron micrograph with apoptotic morphology: condensation(marginalia and fragmentation of chromatinand apoptotic cells (Δ) for 6 hours after treatment through the om MG2327. (F) Apoptosis were identified by compaction of chromatin in the nucleus was observed peripheral patches of condensed chromatin. {Please note that the swelling of mitochondria and the destruction of Christ, all of the cytoplasmic membrane was present at all different points in time}. Magnification: ×6000 (E), ×10000 (A, B, D) ×15000 (C) and ×40000 (F).

Fig. Influence MG2327, p25 and p50 (purified by chromatography) and their fractions on the differentiation of endothelial cells in Matrigel. The HMEC cells were cultured in activation (10 ng/ml EGF, 1 μg/ml hydrocortisone) in the presence of similar concentrations MG2327 (A), p50 (B) and p25 (C), without processing (E) and without activation (D). In the diagram (F) grouped the results of 3 independent experiments, demonstrating inhibitory activity MG2327 and its components on the formation of tubular networks in Matrigel. As MG2327 and p50 fully paid-induced activation to the level of unactivated cells (ANOVA MG2327, p50 and CN p>0,05. Cells treated p25, showed a lower rate of formation of the network than observed for MG2327 and p50 (unpaired t p=0,0107 and p=0,0498 respectively).

Fig. Survival of BALB/c mice, immunized or not MG2327, and with the entered cell myeloma X63. When using 3 and 6 dose rate of 1 mg/kg MG2327, at 100% of the immunized animals occurred rejection myeloid tumors.

EXAMPLES

Example 1. Obtaining strain CMB 4202

To obtain strains of bacteria that produce anticancer molecules, wild typeSerratia MarcescensSM1995 isolated from the ventral surface of BALB/c mice, mixed with tumor cells CBHEp.1 (Aleman, M.R., Valdes, R., Perez, M., Ibarra, N., Reyes, C., Gonzalez, M., Mendoza, A., Padilla, S., Agraz, A. and Rodriguez, M.P. 2000. Biopharm 13:48-52) and inoculable intraperitoneally to BALB/c mice previously inoculated with 10 days earlier heavy liquid petrolatum. Eight days after inoculation with mixtures of cells were made every 2 days extract from ascites. Analyzed the kinetics of tumor growth, and carried out the microbiological control of ascites each animal, giving an overview of regression of the tumor.

The selected bacteria were grown in different media and cultivation conditions. Supernatant culture were sterilized by filtration using membrane filters of 0.22 μm, and their toxicity was evaluated on cells CBHEp.1. The strain with higher cytotoxicity was stored under inventory number CMIB4202 in the Collection of Microorganism with Biotechnological Importance of the Center of Genetic Engineering and Biotechnology, Habana city, Cuba. CMIB4202 and its parent strain SM1995 were cultured in parallel in the fermenters 5 l in media with peptone-glycerol at 28°C. For sterile filtrate CMIB 4202 shown activity, dose-dependent, while the filtrate SM1995 has only marginal activity on the line Raco what s in human cells HEp-2 (figa) in the analysis of antiproliferative using MTT method (Skehan, P., Storeng, R., Scudiero, D., Monks, A., Mcmahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S. and Boyd, M.R. 1990. J. Natl. Cancer. Inst. 82:1107).

Strain CMIB 4202 was sverkhekspressiya soluble proteins in the range of 45-50 20-30 kDa (50 and 25 kDa as determined by SDS-PAGE, with a coefficient of determination 0,984), figv.

For faction p25 isolated from bands present in the gels with SDS, painted imidazole-zinc (Hardy, E., Santana, H., Sosa, A., Hernandez, L., Fernandez-Padron, C. and Castellanos-Serra, L. 1996. Analytical Biochemistry. 240:150), showed strong antiproliferative activity on HEp-2, dose-dependent, while the fraction p50 did not inhibit the growth of cells.

However, for p50 during incubation with 5 μm Zn2SO4demonstrated antiproliferative activity, but lower than observed for p25 (figure 2). IC50fractions p25 and p50 were of 0.48 nm and 16 nm, respectively.

In order to compare the ability of both strains to Express the proteins used factorial ANOVA. The probability value of the interaction was not significant (p=0,93). On the other hand, the probability of both main effects showed that both protein expressed at significantly different levels (p=0.01) and that this number is strongly dependent on strain (p=0,0004). Moreover, there were significant differences in the expression of these proteins between the two strains (p<0,001).

Example 2. Getting antiproliferative drug MG2327

For p the receipt of antiproliferative drug side strain S. marcenscesCMIB 4202 received 1 l of the culture of the microorganism and culture medium optimized to receive the necessary molecules (figure 3). Culture CMIB 4202 was centrifuged at 12000 g and 4°C for 30 minutes. The supernatant was collected and then filtered through a molecular filter of 0.2 μm in sterile conditions. The volume of the supernatant was reduced in 10 times in the same sterile conditions. The volume of the supernatant was reduced in 10 times, using a membrane with a limit of 10 kDa exclusion and dialigue against PBS at 4°C for 24 hours under physiological conditions. Diarizonae substance was filtered under sterile conditions and distributed in vials of 5 ml of the Preparation was stored at 4°C and called MG2327.

The increase in the loading of the fermenter to 5 l was carried out under the conditions already defined in the screening: medium peptone-glycerol at 28°C for 14 hours, aeration 1 vvm, 250 rpm and an initial optical density of 0.1. the pH of the culture media was brought to physiological and fermentation was carried out at free pH. The rest were carried out in the same form as in the screening.

Example 3. Characterization of the antiproliferative activity of the drug MG2327in vitro

To characterize the antiproliferative activity MG2327in vitroevaluated a panel of human cell lines (table 1). Only 2000 of cells, with the exception of PBMC (20000), were sown in 96-well culture plates and added a different end of the ation MG2327. After 72 hours the number of surviving cells was measured by adding MTT (Skehan, P., Storeng, R., Scudiero, D., Monks, A., Mcmahon, J., Vistica, D., Warren, J.., Bokesch, H., Kenney, S. and Boyd, M.R. 1990. J Natl Cancer Inst. 82:1107). Soluble products formazan were detected at 540 nm in a multi-channel spectrophotometer to read the tablets. For MG2327 showed a wide range of cytotoxic activity against the analyzed human cell lines. IC50was in the range of µg/ml.

Table 1
Cells and culture media used in the study ofin vitro
FIBHUMFIBROBLASTS derived FROM foreskin MAN, P 3-6DMEM, 15% FBS*,INSULIN 30 mcg/ml
H82Carcinoma of the lung manRPMI 1640, 10% FBS
MDA-MB145SAdenocarcinoma of human breastDMEM, 10% FBS
Colo-205Carcinoma human colonRPMI 1640, 10% FBS
HT1080The human fibrosarcomaDMEM, 10% FB
HelaCarcinoma of the cervix manDMEM, 10% FBS
HEp-2Carcinoma of the larynx manMEM, 10% FBS
HepG2The human hepatocarcinomaDMEM, 10% FBS
A375The human melanomaDMEM, 10% FBS
PBMCPeripheral mononuclear cellsRPMI 1640, 10% FBS, IL-2
HuT78T-cell lymphoma of the skin of a personRPMI 1640, 10% FBS
HL60Promyelocytic leukemia personRPMI 1640, 10% FBS
K562Erythroleucus manRPMI 1640, 10% FBS
CRL 1682Adenocarcinoma of the pancreas of a personRPMI 1640, 10% FBS
A 431Adenocarcinoma of the external female genital organs RPMI 1640, 10% FBS
SiHaCarcinoma of the cervix manRPMI 1640, 10% FBS
CaSkiCarcinoma of the cervix manRPMI 1640, 10% FBS
HUVECVascular endothelial cellsM199 30% FBS and 10 ng/ml bFGF
*FBS: Fetal bovine serum

Selectivity MG2327 was compared with the commercial drug doxorubicin (DXR), using the cell line HT1080 (from fibrosarcoma) and primary fibroblasts. Antiproliferative assay was used as described above. Serial cultivation DXR and drug MG2327 used from 10 μg/ml and built curve at five points. The mortality rate was calculated for each point as the ratio between the percentages of mortality for HT1080 and the percentage mortality for primary fibroblasts. Stronger differences were detected at lower test concentrations (MG2327 9:1, DXR is 1.7:1). MG2327 was very selective at concentrations below 2 μg/ml.

Cells HEp-2 originating from carcinoma of the larynx are very resistant to anticancer tools used in the clinic, compared to others who analyzed the cell lines. For this reason, the authors of the present invention was used as a model to studyin vitroeffects of the drug MG2327. Comparison of the cytotoxicity curves constructed for cells HEp-2, treated with known anticancer remedies, have shown similar results (40%) proliferation) at 3 µg/ml for drug MG2327, cisplatin (CDDP), doxorubicin (DXR), vincristine (VC), vinblastine (VB) and Taxol (TX) (p>0.05, the ANOVA criterion). In the same conditions for other anticancer agents, such Ara-C, methotrexate (MTC), bleomycin (Bleo) and cyclophosphamide (CPA), showed no effect. CDDP is one of antineoplastic agents approved by the FDA for the treatment of cancer of the larynx, and the analysis of the survival curves for drug MG2327 and CDDP showed similar values for IC10IC50and IC90.

The sensitivity study of different carcinogenic cell lines and non-cancer (figure 4) showed that the line of normal cells are less sensitive, while melanoma, carcinoma of the larynx, fibrosarcoma, hepatocarcinoma and carcinoma of the cervix (the carrier of human papilloma virus) is very sensitive. Carcinoma hematopoietic origin are less sensitive. Activated for the growth of HUVEC cells are more sensitive to the drug MG2327 than not activated.

Before the previous result showed what drug MG2327 has a broad spectrum of cytotoxic activity in lines of tumor cells with a selective effect on tumor/transformed cells, and cells that have been activated for growth.

Example 4. Antitumor activity of the drug MG2327

To demonstrate the antitumor activity of the drug MG2327 the authors of the present invention was used BALB/c mice, which were inoculated intraperitoneally (I.P. Pavlova.) tumor cells CB Hep.1 myeloid origin that can give rise to murine ascitic tumors (Fontirrochi, G., Duenas, M., Fernandez de Cossio, M.E., Fuentes, P., Perez, M., Mainet, D., Ayala, M., Gavilondo, J.V. and Duarte, C. 1993. Biotecnol Aplic. 10:24-30). After 10 days, mice were injected with I.P. Pavlova. MG2327 or PBS. Sixty percent of the animals treated at the rate of 1 mg/kg survived, while only 25 percent of the controls lived up to 45 days after the initial treatment (figure 5). Complete regression of tumors was observed in all treated the surviving animals, which showed a healthy condition, while at the controls of the tumor progressed, forming large dense formations and animals showed unhealthy overall condition.

In BALB/c mice bearing a tumor of fibroblasts transformed by E6/E7, increased survival after treatment with the drug MG2327, demonstrating a significant reduction in tumor volume.

Also to assess the antitumour activity MG2327 used model of cancer, associated with human papilloma virus (HPV16), developed by Hernandez et al. (Hernandez, P., Merina, N., Lopez-Ocejo, O. and Arana, M.J. 2000. Biochem Biophys Res Commun. 270:119-124). Two groups of BALB/c mice were inoculable subcutaneously (s.c.) 2·106cells T in the left abdominal region. After 48 hours, the dose calculation of 0.75 mg/kg MG2327 or PBS was injected s.c, close to the initial inoculation of the cells in the control group; daily measurements were made of the compass. Tumor volume was calculated using the conventional formula: V=0,52·a2·b, wherearepresents the width, andbrepresents the length of the horizontal perimeter of the tumor (Hernandez, P., Merina, N., Lopez-Ocejo, O. and Arana, M.J. 2000. Biochem Biophys Res Commun. 270:119-124). The nature of the change is shown in Fig.6.

Differences in time of tumor development was statistically significant (p=0,0054) between treated and untreated animals. The attitude of the time:the processing by criterion ANOVA showed that the same value of the difference is not saved between treated and untreated groups; this indicates the existence of differences associated with applied to animal handling.

When applying the criterion of Wilcoxon signed for paired measurements between 21 and 45 days for each group, the authors of the present invention has detected a significant increase in tumor volume for the control group (p=0,043), which has no month is and if treated group (figa). To analyze the existence of significant differences between groups at each point of assessment, the authors present invention was used U-test Mann-Whitney, which were detected significant differences, except for the first point (p<0,01). Also, the authors of the present invention has detected significant difference in the rate of tumor growth. Growth curves resulted in one line, and the slopes were calculated from the equation, the best approximation. Comparison of the slopes shows that the tumor in the control group grew at a rate significantly higher than the observed rate for the treated group (p=0,0088).

Mice of the control group died between 45 and 64 days due to the inoculation of the tumor, while the animals in the treated group began to die at 52 days, with 20% of survivors are left with time (170 days), figv.

When casting data on survival to the Bayesian hierarchical model (the Weibull regression with 500 iterations) the authors of the present invention has been statistically significant difference (p=0,02447), which is confirmed when showed that the confidence intervals of the average time of survival is not fully crossed.

Example 5. Fractionation of the drug MG2327. The allocation of non-protein biomolecules

To determine the composition of the drug MG2327 exercised its molecular fractionation and evaluated is its ability to inhibit in vitrothe growth of the cell line human tumor cells, Hep-2.

Polysaccharide fraction (tr=6.85 minutes) was separated by chromatography in gel Aminex HPX 87-N (size: 300×7.8 mm, elution: 0.5 ml/min). Used samples fructose tr=13,15 minutes, glucose tr=12,12 minutes, disaccharide tr=9,40 minutes, trisaccharide tr=8,24 minutes, polysaccharide tr=7,01 minutes. The fraction of the pigment was separated on a column of butyl-TSK MERCK, balanced 20 mm phosphate, pH=7, in which the balance was retained on the matrix, which was then suirable using absolute ethanol. Absorption spectrum (100% ethanol, pH 5,00) of the obtained product showed a band with a maximum at 470 and 490 nm, and the maximum peak at 537 nm, which correspond to the typical description for monomer and dimer, respectively, with published antiproliferative activity (Perez-Tomas, R. and Montaner, B. 2003 Histol. Histopathol. 18:379-385; Montaner, B., and Perez-Thomas, R. 2003. Curr Cancer Drug Targets. 3:57-65). For the selected polysaccharide showed no inhibitory effect, while the fraction corresponding to prodigiosin showed antiproliferative activity, dose-dependent.

Example 6. Fractionation of the drug MG2327. The selection of protein biomolecules with antiproliferative effect through only one phase chromatography: ion exchange in a stepwise NaCl gradient. Composition

The drug MG2327 were applied to the matrix of sepharose Fast Flow DEAE, uravnoveshennoy mm phosphate buffer, the pH of 8.00. The elution was carried out stepwise gradient of NaCl: 50 mm phosphate buffer-0.1 M NaCl, pH 8,00; 50 mm phosphate buffer and 0.2 M NaCl, pH 8,00; 50 mm phosphate buffer-2 M NaCl, pH 8,00, and in the end was suirable fraction of the pigment absorbed on the matrix, 70%absolute ethanol.

For fractions, corresponding to 0.2 M NaCl, pH 8,00, and the first eluate collected from the faction that did not sarbievius (went), showed antiproliferative activity, dose-dependent, in the already described test. By electrophoresis SDS-PAGE was observed protein band at 50 kDa and a maximum bandwidth (purity >90%) at 25 kDa, respectively (figa). Molecular mass was calculated with the function that links the molecular weight of the commercial sample with the distance of migration of the bands; r2=0,984.

On FIGU shown antiproliferative effect of p50 and P25 in comparison with drug MG2327. For p50 and p25 showed antiproliferative effect on HEp.2.

Table 2 shows the comparative results between p50, p25 and drug MG2327, using a statistical analysis of variants (ANOVA). With his help made the comparison of the reactions at each of the applied dose. It can be observed that there are significant differences between the activities of the three samples. These differences depend on the applied doses. At high concentration of the components of the drug (9 and 18 mg/ml) things which exist significant differences between fractions 25 and 50 kDa, where the fraction of 25 kDa was the most active, being more active at lower concentrations (of 2.25 and 4.5 μg/ml).

Table 2
Comparative results between the protein components and drug MG2327,
using a statistical analysis of variants (ANOVA)
Concentration (µg/ml)ANOVA
Between samples25-5025-MG232750-MG2327
2,250,01100,06490,00890,0958
4,50,03160,06850,00810,3728
90,00400,03180,01510,4271
180,00150,02430,32060,0243

Between the protein component 50 cdai drug MG2327 there was a significant difference for a dose of 18 μg/ml, where the drug was most active, giving a growth inhibition 100% of tumor cells, whereas this protein component 50 kDa gave approximately 80% inhibition of growth. For doses of 2.25, 4.5 and 9 μg/ml did not exist significant differences between the reaction caused by the fraction of 50 kDa and drug MG2327.

However, the activity of the protein component 25 kDa was significantly different from the drug MG2327 for doses of 2.5, 4.5 and 9 μg/ml, where the protein component 25 kDa showed the highest biological activity; and for a dose of 18 μg/ml did not exist significant differences, since both samples gave similar inhibition of 100% of tumor cells.

These results indicate that the protein component of 25 kDa has the basic ability to inhibit the growth of tumor cells than the protein component of 50 kDa, and that both components had biological activityin vitroindependent manner.

Data cleanup procedure was repeated three more times and got consistent results.

Example 7. The composition of the polypeptide serraline with prodigiosin

Protein components and prodigiosin prepared in the same composition, which is significantly (p<0.005 percent) increases the inhibitory effect compared with the effect of an individual component. On Fig shows the IC50antiproliferative biomolecules isolated from drug M2327 and their combinations. The drug MG2327 indicates total protein; the song gained, keeping fixed the same ratio of protein and prodigiosin, which was used to evaluate the individual components. In such compositions prodigiosin can be detected in a concentration of 0.1-100 nm, and fragments serraline in a concentration of 0.1-150 mg/ml

Example 8. p25 is formed from p50

Anti-p50 obtained in sheep, was used to learn about the relationship between p25 and p50. MG2327 inflicted on 12% gel SDS-PAGE and stained the imidazole-zinc (Hardy, E., Santana, H., Sosa, A., Hernandez, L, Fernandez-Padron, and Castellanos-Serra, L. 1996. Analytical biochemistry. 240:150-152). Protein bands of approximately 50 and 25 kDa (figure 9) cut, senatoriable in the gel and were applied to SDS-PAGE. They were transferred to nitrocellulose membrane and carried out Western blotting. Polyclonal antibodies to p50 learned p25; and molecular weight degradation products of p50 was evaluated by means of colored molecular weight markers (Bio-Rad). The Western blot shown is representative of three identical experiments.

Example 9. The products of thermal degradation p50 more active than the p50

p50, obtained in example 6, were incubated at different temperatures and tested its antiproliferative activity on HEp.2 using the MTT method, previously described. Profile of degradation obtained in each condition (4, 37, 45 and 60°C), the amount the state was determined by densitometry.

Education fragment obtained by degradation was directly proportional to the temperature increase, thus decreasing the amount p50 increased the amount of p25, a product of degradation of p50 (figure 10). For degradation products of p50 showed stronger antiproliferative activity than the original p50 (11).

Example 10. p25 causes regression of malignant tumors

Protein p25 obtained by the chromatography described in example 6, was used in reversed-phase chromatography (RP-HPLC)to confirm the homogeneity and purity. It was applied a gradient of acetonitrile 0-100 within 100 minutes. Observed peak protein with a purity of greater than 90%, demonstrating the homogeneity and purity of the eluate.

p25 then were injected with I.P. Pavlova. mice BALB/c mice after 8 days after inoculation and the full development of myeloid tumors P3X63Ag8. Dose p25 based 22 µg/kg of body weight caused complete regression in 80% of treated animals. Negative controls were dead at the end of 30 days, where have already developed solid tumors.

Example 11. p50 is metalloproteases, while the p25 has no proteolytic activity

A modified method of Anson and Mirsky (Anson, M.L., Mirsky, A.E. 1932. J. Gen. Physiol. 16:59) using casein as substrate, in the laboratory of the authors of the present invention adapted for trypsin (y=1,9314x-0,682; R2=0,999). Is white the new faction, obtained by the chromatography described in example 6, were analyzed in this way. For p50 showed proteolytic activity, which is inhibited 7 mm EDTA; thus, p50 is metalloproteases. For p25 showed no enzymatic activity, Fig.

How Imogene using gelatin as substrate (Vacca, A., Iurlaro, M., Ribatti, D., Minischetti, M., Nico, B., Ria R., Pellegrino, A. and Dammacco, F. 1999. Blood. 94:4143-4155) was used to confirm the proteolytic activity of p50 and p25. Furthermore, we analyzed the enzymatic degradation ability p50. In this analysis p50 protein obtained by the chromatography described in example 6 showed enzymatic activity. For the fraction of protein bands in the gel at 25 kDa (from MG2327) showed proteolytic activity, while for p25 obtained by chromatography, did not show this activity.

Example 12. Identification of antiproliferative proteins of 25 kDa from MG2327

To identify proteins with antiproliferative activity present in the band of 25 kDa, this band was cut out of the gel SDS-PAGE (as described in example 8) with printed MG2327. The strip was incubated for 5 minutes in 1 ml of buffer Tris/HCl (100 mm, pH 8.5) to full transparency. The strip was cut into small cubes of approximately 1 mm3produced absorption acetonitrile, rehydratable in the least amount of bicarbonate is ammonium (25 mm), containing trypsin or LEP at a concentration of 12.5 ng/μl. To break down the gel was incubated at 37°C for 18 hours in a thermostatted the stirrer.

The resulting peptides from cleavage by LEP analyzed by MALDI-MS. The signal intensity from the monoisotopic ions were introduced into the program ProFound to identify the protein of interest in a database of sequences. Although the authors of the present invention do not impose any taxonomic constraints on the database search, the 50 kDa protease fromSerratia marcescensEC 3.4.24.40 chose for the greatest similarity. Four peptide (51-57, 58-66, 67-80 and 81-90) belong to the N-terminal region, and one (402-409) belongs to the C-terminal region of the protein. Molecular weight EC 3.4.24.40 exceed the mass present in the analyzed band (approximately 25 kDa as assessed by SDS-PAGE. This discovery suggests that the band of 25 kDa contains two fragments, together migrating in the region of 25 kDa, with similarity to a protein of 50 kDa EC 3.4.24.40 belonging to the family of serializing.

To confirm this hypothesis band of 25 kDa protein was subjected to cleavage by trypsin. The spectrum of the ESI-MS of peptides were subjected to deconvolution, and the signal intensity introduced into the program Profound. In this way discovered a protein that is the same as identified RA is its (EC 3.4.24.40). The sequence covered by cleavage with trypsin (21%)was greater compared to the previous splitting (10%). Coverage map sequence based on seven peptides, which are very well coincided with some fragments of trypsin cleavage of proteins PRZN_SERMA/PRZN_SERSP. Five of them (28-41, 58-66, 67-80, 81-90 and 163-171) corresponded to the N-terminal region, while the remaining two peptide (351-373 and 374-393) correspond to the C-terminal region. These results not only confirm the previous identification of the protein, but that the coverage map suggests the presence of two migratory together fragments of 25 kDa identified the protein as PRZN_SERMA/PRZN_SERSP, in the analyzed band.

Range of ESI-MS/MS, peptides corresponding to the N - and C-terminal regions of the previously mentioned proteins, described manually, and partial sequence allocated to identify, table 3.

On the basis of the applied methods and the obtained results the authors present invention concluded that the analyzed band of 25 kDa is a mixture of proteins, which have fragments with similarity to the N - and C-ends proteins PRZN_SERMA/PRZN_SERSP.

Example 13. Identification p50

To identify p50 protein with antiproliferative activity, protein fraction, corresponding to a protein of 50 kDa, obtained ABM is about cleaning, described in example 6, was treated with endoproteinase Lys-C. identification of the peptides was carried out by sequencing by automated degradation by Admino and two-sector mass spectrometer JMS HX-110, with a gun FAB. These results and alignment are carried out by software Swissprot and PIR, allow us to conclude that this protein belongs to the family of serializing with a molecular mass of 50 kDa. The main similarity was found with form IDs PRZN_SERSP and PRZN_SERMA in the Bank Swissprot. The molecular mass of all peptides analyzed by a mass spectrometer, coincided with the expected theoretical number of peptides of this splitting of proteins by endoproteinase Lys-C.

Example 14. Identification p25, purified by chromatography

To identify the protein of 25 kDa with antiproliferative, apoptotic and antiangiogenic activity, p25, purified DEAE-chromatography described in example 6, were applied to SDS-PAGE. The protein band was washed for five minutes with 500 µl of water after bleaching were washed in 100 mm citric acid solution, then washed with MilliQ water, cut into small cubes of approximately 1 mm3. Next was added acetonitrile to their dehydration and excess removed. Cubes gel completely dehydrational in evaporative centrifuge followed by rehydration solution Bica is Bonita ammonium (50 mm), containing trypsin at a concentration of 12.5 ng/μl. Next incubated in the stirrer, cooled for 30 minutes at 37°C during the night.

Peptides passively separated by adding 20 μl of a solution of ammonium bicarbonate and an additional incubation at 37°C for 45 minutes. Peptides were identified using ZipTipC18™ and then acidified reaction mixture by adding 5 ál of free formic acid, and the newly identified peptides using ZipTipC18™. Peptides associated with ZipTipC18™continuously washed with 5% formic acid, and then separated in the amount of 2-3 µl of 60% solution of acetonitrile containing 1% formic acid.

Peptides obtained by cleavage, were loaded on the borosilicate capillary needle, covered with gold, was introduced in the ion source orthogonal geometric hybrid mass spectrometer equipped with a source of nanospray (QTOF-2™).

Mass spectrum ESI-MS were obtained in the range 350-2000 Yes for 1 second. The signal intensity was chosen from the subsequent sequence ESI-MSMS. As gas collisions used argon and used the energy of the collision, the right to cause extensive fragmentation of the selected peptides, which will allow them to be uniquely identified in the database.

Range of ESI-MS were subjected to deconvolution and eksporterov and in DTA format, and imported into the program MASCOT for the identification of protein databases SWISSPROT and NCBInr through a strategy of mass spectrometric peptide maps (PMF). For exact identication of protein was applied internal calibration to use autoproteolysis trypsin to peptides, and fixed an error in the 0.05 Yes, to search for peptides observed in the spectrum, and chose only the signals that had an intensity exceeding 10% of the intensity of the base peak.

Four peptide present in the analyzed band, sequenced by ESI-MSMS (table 4). These peptides proteins PRZN_SERMA/PRZN_SERSP related to the C-terminal region (marked in red in the sequence table 6), previously identified in the previous examples.

Table 4
Peptides related to p25S. marcescensthat sequenced by ESI-MSMS
No.Amino acid sequencem/z theoretical.m/z exp.Error
1DFLSTTSNSQK1226,511226,580,07
2SAASDSAPGASDWIR1489,751489,680,07
3GGAGNDVLFGGGGADELWGGAGK2060,962060,870,11
4TGDTVYGFNSNTGR1488,651488,600,05

Similarly discovered other signals, which although not sequenced, the values of their masses is in very good agreement with the expected values of the masses of the peptides after cleavage by trypsin, identified as C-terminal region of proteins PRZN_SERMA/PRZN_SERSP, table 5 (marked in blue in table 6). Between these peptides appears intense double the signal, which could correspond to the C-terminal peptide of protein PRZN_SERMA/PRZN_SERSP. Not detected peptides, which could correspond to the typical products of the cleavage of the N-terminal region of proteins PRZN_SERMA/PRZN_SERSP.

Table 5
Peptides after cleavage by trypsin, protein belonging PRZN_SERMA, detected in the spectrum of ESI-MS
No.Amino acid sequencem/z exp.m/z calc.zError
1325SFSDVGGLK333455,20455,2420,04
2417IDLSFFNK424492,24492,2620,02
3475IVGQVDVATDFIV487688,35688,3820,03

Through the methods and the results shown in this example, the authors of the present invention is able to ensure that with a strong antiproliferative effect band p25, purified DEAE-chromatography, there is a C-terminal fragment of the 25 kDa protein PRZN_SERMA/PRZN_SERSP.

Table 6. Identification of p50 and p25, obtained by DEAE-chromatography. In the sequence of detected erroneous amino acids that are not present in the Mature protein; without these amino acids molecular mass proteins PRZN_SERSP and PRZN_SERMA is, accordingly, 50595,4 and 50293,4 Yes. Identification of peptides after cleavage by trypsin, which shifted between molecules suggests that both species may be present and include coexisting (green (italics): identified by mass spectrometry, and maroon (underlined): identified degradation in Admane, inside a continuous rectangle). Peptides marked (and) identified in p25 obtained by the chromatography described in example 6. Peptides marked

(and the underlined identified in p50 obtained by the chromatography described in example 6, and the peptide (italics) identified from a slice of gel.

Table 6

Example 15. Proteins of 25 kDa, corresponding to the N - and C-end

To determine the molecular weight of a protein-degradation products PRZN_SERMA/PRZN_SER that can be in the region of 25 kDa when carrying out SDS-PAGE, the order received and entered into the program GenRun. Fragments of 25 kDa (±2 kDa)according to the corresponding N - and C-Termini, shown in table 7.

Table 7
Protein weight 25±2 kDa,
which degradation products PRZN_SERMA/PRZN_SERSP. The molecular weight was determined by means of the program GenRun, separately for the N - and C-Terminus
ProteinSequence
ARA1
ARA2
ARA3
ARA4

Example 16. Apoptosis induced MG2327

MG2327 induces apoptosis of myeloma cells X63, resulting in fragmentation of the DNA, mitochondria and microtubules

To determine the type of death of tumor cells cells are murine myeloma P3X63Ag8 (2·107handledin vitrothrough MG2327 based 22 µg/ml Treated and untreated cells were prepared for research through ultrastructural transmission electron microscopy at different time points, and genomic DNA was extracted for analysis stepwise degradation of DNA.

Fragmentation On What It was evaluated by electrophoresis in 2% agarose gel. Apoptosis often causes gradual degradation of cellular DNA to 180-200 BP, which corresponds to minucieusemnt distances (Soldatenkov, V.A., Prasad, S., Voloshin, Y., and Dritschilo, A. 1998. Cell Death Differ. 5:307-12). As shown in Fig, observed a significant increase in the number of oligonucleosome corresponding morphological changes in culture and confirmed by electron microscopy.

Mineclearance fragmentation preceded morphological signs of apoptosis, detected by optical microscopy. Moreover, on the micrographs shows the modified cytoplasmic organelles (mitochondria, 2 hours), prior to the condensation of chromatin (4 hours) and mineclearance fragmentation (6 hours).

MG2327 damage microtubules and ultrastructural organization of mitochondria, increasing apoptosis of cells P3X63Ag8

Untreated cells had normal ultrastructure of mitochondria: distinct mitochondrial Christa and mitochondrial matrix high density; mitochondria evenly distributed throughout the cytoplasm (figa).

Cells treated MG2327 have increased the size of mitochondria, reduced density matrix and badly damaged morphology Crist (figw-E). Data ultrastructural changes mainly indicate organelles are degraded.

Moreover, is the second of the present invention observed large cytoplasmic vacuoles, also considering the endoplasmic reticulum as mitochondria and nuclear structure, 2 hours after treatment (pigv).

Different morphological changes observed in the cores to 6 hours after treatment (condensation, mergers and stepwise fragmentation of chromatin). On the micrograph late stage of apoptosis (8 hours) P3X63Ag8 cells treated MG2327, chromatin looks compact (fig.14F). The split was not detected at all time points.

Interestingly, mitochondria P3X63Ag8 cells treated for 2 hours, forming clusters on the periphery of the cytoplasmic membrane (figw-E), resulting in the destruction of microtubules and disruption of transport of mitochondria along the organelles (Schatten, H., and Lewis, M.L. 2001 Acta Astronaut. 49:399-418).

On figs and D shows a larger mitochondria, also with condensed structures attached to the inner mitochondrial membranes. These patterns could be caused by sequential merge Krist. MG2327 could cause apoptosis through the release into the cytosol cytochrome c, after swelling of the outer membrane of mitochondria along with the increase in size, with the subsequent activation of caspases and apoptosis (Green, D.R. and Reed, J.C. 1998. Science. 28:1309-1312. An overview).

No increase in the amount of cytochrome could also completely and quickly released from the damaged Krist mitochondria into the cytosol through the con which acts between the intermembrane space and Christ after the merger (Scorrano, L., Ashiya, M., Buttle, K., Weiler, S., Oakes, S.A., Mannella, C.A. and Korsmeyer, S.J. 2002. Dev Cell. 2:55-67). The described process greatly enhances the signals apoptosis caused MG2327 in the cells.

Example 17. p25 and p50 induce apoptosis

To distinguish the role of p25 and p50 in the event of apoptosis caused MG2327, they were individually introduced P3X63Ag8 cells in different concentrations and analyzed by electron microscopy. All cases were detected condensation of chromatin, mitochondrial Krist and aggregation of mitochondria. Indeed, the induction of apoptosis by MG2327 associated with the action of these two proteins.

Example 18. Antiangiogenic action MG2327 and antiproliferative polypeptides

Development of tubular structures in Matrigel

Endothelial cells derived from the microvasculature person (HMEC) was assessed by the formation of endothelial heavy in Matrigel (Crum R., Szabo, S., Folkman J. 1985. Science. 230:1375-8; Vacca, A., Ribatti, D., Presta, M., Minischettti, M., Iurlaro, M., Ria, R., Albini, A., Bussolino, F. and Dammaacco, F. 1999. Blood 93:3064) after cultivation with nectocarcinus concentrations MG2327, p25 and p50 (Sanz, L., Pascual, M., Munoz, A., Gonzalez, M.A., Salvador C.H., Alvarez-Vallina L. 2002. Microvascular Research 63:335-339). The results considered the length of the tubular structures and the number of connections between them, by calculation using the software package Image-Pro Express 4.5. Showed a significant inhibition (p<0,05, ANOVA) differential and or maturation of endothelial cells (Fig) after spraying MG2327 and its fractions p25 and p50.

Example 19. Indirect effect MG2327 on proliferating cells

MG2327 protects Balb/c mice from engraftment of myeloid tumors

For the analysis of the protective activity MG2327 vaccinated against tumors in mice Balb/c were inoculable (I.P. Pavlova.) at the rate of 1 mg/ml of this anticancer drug by different immunization schemes. Was administered three doses, one dose every week and two doses each week for three weeks, at least three days between doses. The negative control group was inoculable 1× PBS. Two million cells myeloma P3X63Ag8 was inoculable I.P. Pavlova. experimental groups (treated and control) 150 days after the first dose (after 5 months). All mice from the negative control group died within the first 25 days, while 100% of the treated animals survived without tumor (Fig).

Example 20. C-terminal domain of other serializing also has a cytotoxic effect without proteolytic activity

Strain ATCC14756 cultured in similar conditions with strain CMIB4202 according to example 2, and supernatant its culture was processed as described in example 6. In both preparations, the authors present invention watched the squirrels in the area of 50 kDa, which was suirable 50 mm phosphate and 0.2 M NaCl, pH 8,00. Data proteins possessed enzymatic activity, which is inhibited 10 mm EDTA and restoring the Ali 5 μm Zn 2SO4. Both proteins were chemically digested by CNBr, and the nature of the breakdown was the same, giving similar fragments of approximately 25 kDa, corresponding to the C-end of p50, from an internal methionine in the sequence until the end of the molecule.

Fragments derived from cleavage was senatoriable change buffer by dialysis for 48 hours. The biological activity of these samples was tested in the analysis of cytotoxicity, as described here, using cells HEp.2, which were incubated for 72 hours in their presence. The fragments obtained by cleavage and devoid of proteolytic activity in the presence of 5 mm EDTA, had higher cytotoxic activity, which was dose-dependent, approximately 2.5 times more compared to the full molecule p50. These fragments, if their sequences are known, can also be obtained by chemical synthesis or recombinant methods.

Example 21. The combination of fragments of serializing with antibodies or fragments of antibodies

Polypeptide fragments obtained in examples 6 and 20, chemically conjugatively with a monoclonal antibody CB/ior-CEA.1 (Tormo Century. et al. APMIS 97:1073-1080, 1989), with its variable regions, and with a fragment of the antibody obtained through recombinant DNA technology on the basis of its sequence (dimeric antibody) (PA is UNT WO 03/093315). Conjugated biomolecules analyzed for tumor human cell lines LoVo (ATCC CCL-229), AsPC-1 (ATCC CRL-1682) and LS 174T (ATCC CL-188), all of which Express CEA in culture, through antiproliferative assays similar to those described in example 3. Conjugated fragments used in cytotoxic concentrations equivalent to the concentrations of unconjugated fragments, causing the reaction, dose-dependent, while for unconjugated molecules were not observed antiproliferative response. Shown with the use of methods, such as cell ELISA and indirect immunofluorescence (patent WO 03/093315)that conjugated fragments bind CEA on the cell. These results show that the conjugates described herein can be used for therapy and diagnosis of cancer.

1. Antitumor prophylactic or therapeutic pharmaceutical composition comprising one or more nefrotoksicskih polypeptide fragments serraline selected from ARA1, ARA2, ARA3 and ARA4, characterized by the amino acid sequence of what elnett, presented in SEQ ID NO: 1, 2, 3, or 4, respectively, and optionally containing prodigiosin, while the composition has a higher antiproliferative activity compared with the composition containing the whole molecule serializing.

2. Antitumor prophylactic or therapeutic pharmaceutical composition according to claim 1, where these fragments serraline obtained from the supernatant of cell culture recombinant means or chemical synthesis.

3. Antitumor prophylactic or therapeutic pharmaceutical composition according to claim 1, where these fragments are present in the composition separately, conjugated or in a mixture.

4. Antitumor prophylactic or therapeutic pharmaceutical composition according to claim 3, where these fragments serraline obtained from the supernatant of cell culture recombinant means or chemical synthesis.

5. Antitumor prophylactic or therapeutic pharmaceutical composition according to claim 1, where the composition comprises prodigiosin, enhances its antitumor activity.

6. Antitumor prophylactic or therapeutic pharmaceutical composition comprising one or more nefrotoksicskih polypeptide fragments serraline selected from ARA1, ARA2, ARA3 and ARA4, characterized by AMI is kislotno sequence, presented in SEQ ID NO: 1, 2, 3, or 4, respectively, in combination with antibodies or their antihistamine fragments directed to antigens of tumor cells, the composition has a higher antiproliferative activity compared with the composition containing the whole molecule serializing.

7. Antitumor prophylactic or therapeutic pharmaceutical composition according to claim 6, where said components are present in the composition conjugated or in a mixture.

8. The use of a composition according to claim 1 as angiogenic, apoptotic, antiproliferative and as inductor antiproliferative, apoptotic, angiogenic or regulating differentiation factors.



 

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FIELD: chemistry.

SUBSTANCE: in the compound of general formula R1 represents halogen, C1-4alkyl, or C1-4alkoxy; R2 represents hydrogen, halogen; R3 represents hydrogen, or one or several halogens; R4 represents hydrogen, halogen, R8 or Y1R8; Y1 represents -NRa-; Ra and Rb are similar or different, and each represents hydrogen or C1-4alkyl; R8 represents hydrogen, C1-10alkyl; R7 represents hydrogen, hydroxy, trifluoromethyl, amino, C1-6hydroxyalkyl, C1-4alkoxy, C1-4alkylthio, C1-6alkylamino, C1-4alkoxycarbonyl, -S(O)2R, -COOH, -CONH2, or -NRaC(O)R', where R and R' are similar or different, and each represents hydrogen or C1-3alkyl; R5 represents -COOH, Y2R9, Y2R9Y3R10, C1-6alkyl-Y2R9, C1-10alkyl, or unsaturated C3-8carbocycle, said R5 is substituted with one or several, similar or different substituents, represented by R7; R6 represents hydrogen; Y2 represents -O-, -NRa-, -NRaC(O)NRb-, -NRaC(O)-, -C(O)NRa-, -C(O)-, -NRaC(O)O-, or -OC(O)-; R9 represents C1-10alkyldioxolanyl, C1-10alkylthiazolyl, C1-10alkylmorpholinyl, etc.

EFFECT: obtaining novel compounds, represented by formula I, which possess properties of TNF-α or MAP-kinase p38a inhibitors for obtaining medication to be applied as anti-inflammatory or anti-cancer agent.

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FIELD: chemistry.

SUBSTANCE: invention relates to novel anti-tumour compounds which are kahalalide F derivatives, pharmaceutical compositions containing said derivatives and their use in preparing an anti-tumour medicinal agent.

EFFECT: obtaining novel anti-tumour compounds.

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SUBSTANCE: invention relates to biotechnology and is a peptide which induces killer T cells ex vivo and which has an amino acid sequence as shown in one SEQ ID NOS: from 1 to 3. The disclosed peptide is used in an ex vivo agent for inducing anti-tumour immunity, in an ex vivo agent for inducing antigen-presenting cells, in an ex vivo agent which induces tumour-reactive T cells, as well as in an ex vivo pharmaceutical agent when treating or preventing tumours. The invention also relates to an antibody against the said peptide.

EFFECT: disclosed agents enable identification of glypican-3-derivative peptide, which can bond with HLA-A2, and activation of human killer T cells in order to provide an immunotherapy agent which may be effective in approximately 40% Japanese patients suffering from certain types of malignant tumours, accompanied by high level of GPC3 expression.

7 cl, 4 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a pyrimidine nucleoside compound of general formula (1) , in which one of X and Y is a cyano group and the other is hydrogen; R1 is hydrogen, (R3)(R4)(R5)Si- or a carbonyl group which includes an alkyl monosubstituted with an amino group; R2 is hydrogen or (R6)(R7)(R8)Si-, provided that at least one of R1 and R2 is not hydrogen; or R1 and R2 together form a 6-member cyclic group -Si(R9)(R10)-, where each of R9 and R10 is a straight or branched alkyl; R3, R4 and R5 denote a straight or branched alkyl optionally substituted alkoxy, or cycloalkyl; R6, R7 and R8 denote a straight or branched alkyl optionally substituted alkoxy, cycloalkyl or phenyl, or to pharmacologically acceptable salts thereof. The invention also relates to a range of specific compounds of formula (1) or to their pharmacologically acceptable salts: 5'-O-triisopropylsilyl-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 5'-O-diethylisopropylsilyl-2'-cyano-2,-desoxy-1-β-D-arabinofuranosylcytosine; 5'-O-dimethylthexylsilyl-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 5'-O-(dimethyl-n-octylsilyl)-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 3'-O-dimethylthexylsilyl-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 3'-O-diethylisopropylsilyl -2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 3'-O-(tert-butyldimethylsily)-2'-cyano-2'-desoxy-1-β-O-arabinofuranosylcytosine; 3'-O-triisopropylsilyl-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 3'-O-dimethylthexylsilyl-5'-O-(L-valyl)-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; 5'-O-(L-valyl)-3'-O-(tert-butyldimethylsilyl)-2'-cyano-2'-desoxy-1-β-D-arabinofuranosylcytosine; and 3'-O-cyclopropyl-diisopropylsilyl-2'-cyano-2'-desoxy-1-β-D- arabinofuranosylcytosine.

EFFECT: obtaining formula (1) compounds or their pharmacologically acceptable salts for preparing a medicinal agent for treating tumours.

9 cl, 20 tbl, 1 dwg, 73 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I)

, pharmaceutical compositions based on the said compounds, as well as methods of using said compounds in preparing medicinal agents.

EFFECT: obtaining compounds and a composition which can inhibit phosphatase cdc25, particularly phosphatase cdc25-C and can be particularly used for treating cancer.

12 cl, 56 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I and their pharmaceutically acceptable salts. The disclosed compounds have inhibitory effect on CDK1 kinase. In formula I , R1 is hydrogen or R2-(X)n-; X is a lower alkylene or cyclic lower alkylene; R2 denotes ; where denotes phenyl; cycloalkyl containing 3-6 carbon atoms; 4-6-member heterocycloalkyl ring having 3-5 carbon atoms and 1-2 oxygen atoms; R5, R6 and R7 are independently selected from a group containing hydrogen or halide; R4 is hydrogen or -(O)k(CH2CH2O)y-R10; R19 is hydrogen; R20 is hydrogen or -C(O)-R11; R10 and R11 is a lower alkyl; n and k are equal to 0 or 1; y is an integer from 0 to 3.

EFFECT: obtaining a pharmaceutical composition with inhibitory effect on CDK1 kinase, containing one or more of the disclosed compounds.

15 cl, 10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to compounds of formula I and their pharmaceutically acceptable salts and esters. The disclosed compounds have inhibitory effect on cyclin-dependant kinase. In formula I R1 denotes , R3 is selected from a group consisting of H, CO2R6, C(O)R6, SO2R6 and SO2NR5R6, R5 and R6 are each independently selected from a group which includes H and (lower)alkyl, R2 is phenyl which contains one, two or three substitutes independently selected from a group which includes halogen or -O-(lower)alkyl.

EFFECT: preparation of a pharmaceutical composition which contains an effective amount of a formula I compound as an active ingredient.

6 cl, 1 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to experimental medicine and can be applied for amplification of apoptosis or cytolytic activity in cells of mammals. Method and application by invention include bringing mammalian cells in contact with effective quantity of Apo- 2-ligand receptors agonist and resting or unprocessed NK-cells, said agonist is selected from Apo-2-ligand polypeptide, agonistic DR5 antibody and agonistic DR4 antibody.

EFFECT: application of inventions allows amplification of apoptosis and cytolysis induction in tumour cells due to activation by Apo-2-ligand receptors agonist of resting NK-cells.

26 cl, 1 tbl, 18 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, pharmaceutical industry and deals with novel medications used for treatment of dysplastic processes of the cervix and rectum mucosa. Medication for treatment of dysplastic processes of the cervix and rectum mucosa, in form of suppository is characterised by the following: it contains 3,3'-diindolylmethane, epigallocatechin-3-gallate, as well as catalyst of epigallocatechin-3-gallate inhibiting activity with respect to DNA-methyltransferases, representing cations Mg2+ in form of pharmaceutically acceptable magnesium salt, lipophilic base, which contains hard fat, polyvinylpyrrolidone and butylhydroxyanisol and/or butylhydroxytoluol.

EFFECT: medication has efficient impact in case of severe forms of dysplastic injuries.

4 cl, 3 dwg, 5 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and immunology. An antibody against angiopoietin-2 is proposed. Versions of the antibody are disclosed, which are produced by hybridome ATCC PTA-7258, ATCC PTA-7259, ATCC PTA-7260. The corresponding coding nucleic acid and expression vector are disclosed. A host cell which produces the antibody based on the said vector is described. The disclosed antibodies have Kd of the order of 10-10-10-12 M, for the antibody 3.19.3 (from ATCC PTA-7260) IC50=99 nM. The said antibody properties can be used in treating human tumours.

EFFECT: design of a method of treating pathological angiogenesis based on an antibody and use of the antibody to prepare a medicinal agent for treating pathological angiogenesis.

33 cl, 18 dwg, 18 tbl, 24 ex

FIELD: chemistry.

SUBSTANCE: invention relates to N-(2-benzothiazolyl)amide 2-hydroxy-4-oxo-4-(4-chlorophenyl)-2-butenoic acid of formula: , which has antimicrobial and anti-inflammatory activity along with low toxicity.

EFFECT: obtaining a compound which can be used to treat diseases associated with pathogenic microorganisms and inflammation.

1 cl, 2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of organic chemistry, to derivatives of 1,4-diketones, namely to novel biologically active substance -2-(2,4-dichloranilino)-1,4-bis(4-methylphenyl)-2-buten-1,4-dione of formula 1 , which possesses antimicrobial activity and can be applied in medicine.

EFFECT: obtaining novel biologically active substance which possesses antimicrobial activity.

1 cl, 1 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention describes novel benzothiazinone derivatives of formula (I) and their use as antibacterial agents in infectious diseases caused by bacteria, especially mycobacterium tuberculosis (TB) and leprosy, in which R1 and R2 independently denote NO2, CN, CONR7R8, COOR9 CHO, halogen, SO2NR7R8, OCF3, trifluromethyl; R3 and R4 independently denote H or methyl; R5 and R6 independently denote a straight or branched aliphatic radical having 1-8 members in the chain, or R5 and R6 together denote a divalent radical -(CR92)m- or R5 and R6 together denote a divalent radical: R7, R8 and R9 independently denote H or a straight or branched aliphatic radical having 1-7 members in the chain, or phenyl.

EFFECT: design of an efficient method of obtaining benzothiazinone derivatives, a pharmaceutical composition having anti-mycobacterial activity.

12 cl, 6 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: method of producing diphtheria toxin or its mutant or fragment involves a fermentation step where the Corynebacterium diphtheriae strain is grown in a fermenter while stirring in order to maintain a homogenous culture and with limited aeration such that partial pressure of oxygen (pO2) in the culture falls to a level which is 4% lower on the bigger part of the fermentation step. Diphtheria toxin or its mutant or fragment is isolated from the culture. The invention also relates to a method of preparing a pharmaceutical composition for treating or preventing diphtheria, which includes a step for fermentative production of the toxin and mixing it with a pharmaceutically acceptable carrier after isolation.

EFFECT: use of the invention leads to high output of diphtheria toxin or mutant (for example, CRM 197), ensures high output of diphtheria toxin when the culture medium additionally contains iron or complex initial substances of varying quality.

20 cl, 6 dwg, 8 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to pyrido[1,2-a]benzimidazole derivatives of general formula I, where R=alkyl; R1=alkyl, aryl; R2=alkyl. The invention also relates to a method of producing formula I compounds.

EFFECT: novel to pyrido[1,2-a]benzimidazole derivatives with antibacterial activity are obtained.

2 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to treatment of infectious diseases, and can be used in determination of non-specific anti-infectious action of immunomodelling immunobiological preparation (IIP). Essence of method consists in the following: IIP in therapeutically efficient for immunocorrection day dose is perorally introduced to a group of 5-7 monkeys with diarrhea syndrome, in combination with efficient in case of diarrhea syndrome antibiotic, in day dose, which constitutes 1/2 of the dose efficient in case of diarrhea syndrome. After that in case if carried out treatment is inefficient during 2 days, day dose of antibiotic is increased to 3/4 of the dose efficient in case of diarrhea syndrome. Determined is either absence of non-specific anti-infectious IIP action in case when combined application of IIP and antibiotic proves to be inefficient during 7 days, or presence of non-specific anti-infectious IIP action in case when combined application of IIP and antibiotic proves to be efficient.

EFFECT: method allows to determine non-specific anti-infectious IIP action due to determination of minimal efficient in case of diarrhea syndrome day dose of antibiotic in its combination with IIP

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, particularly to pharmaceuticals applied for skin protection against chemical and biological damage factors, bacterial or fungal infections, for prevention of occupational skin diseases, for prevention of contact dermatitis, including allergic dermatitis, and also for first aid and following treatment of wound defects of skin, soft tissues or mucous membranes and can be used in the industry, building, agriculture, medicine and everyday life. Substance of the invention is a therapeutic dermatological composition for local administration which contains an active principle in the form of complex rare-earth salt compounds and a pharmaceutically acceptable carrier.

EFFECT: when applying the pharmaceutical composition based on complex rare-earth salt compounds with polyoxy-compounds in the form of a liquid or soft dosage form, an evident protective and preventive, dermatoprotective and medical effect, no adverse reactions, good tolerance and fast skin repair are observed.

6 cl, 10 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and can be used for integrated treatment of purulent cholangitis by external percutaneous transhepatic drainage and laser and antibiotic therapy. That is ensured by ultrasound-aided percutaneous transhepatic drainage. Then Cefoperazone 2 g in 100 ml of 0.9% saline solution in a plastic package is exposed to low-intensity infra-red pulse laser light at wave length 0.89 mcm, power 200 mWt and frequency 1500 Hz for 6 minutes. Thereafter, the exposed antibiotic substance is introduced intravenously drop-by-drop for 30 minutes. Simultaneously, blood is exposed intravenously to continuous laser light at wave length 0.63 mcm, power 2 mWt for 30 minutes. The therapeutic course of laser and antibiotic therapy is 5-10 days.

EFFECT: method allows preventing purulent-septic complications represented by hepatic abscesses and hepatic failure.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention belongs to vaccine manufacture for infectious diseases prophylaxis. Salmonella vaccine manufacture includes detoxication of exo- and endotoxins in suspention of autoclaved salmonells consecuently with two detoxicants - 0.2% formalin solution at 40±1 C° during 6-7 days and 0.5±0.1% aethonium solution at 41±1 C° during 7-8 days, followed by sterilisation at 1.0 atm during 20 minutes. Derived anatoxins are sorbed on aluminium hydroxide and sterilised at 1 atm during 20 minutes.

EFFECT: anatoxin-vaccine with enhanced effectivity and safety is used for specific prevention of salmonellosis in piglets, calves, carnivorous and poultry.

3 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably to pharmaceutical composition for treatment and prevention of bacterial infections induced by gram-positive bacteria. Composition includes effective doses of cholanic acid or its salt, phosphatidylcholine and neutral lipids. Neutral lipids and phospholipids are associated in lipoprotein-like particles, which does not include proteins or peptides.

EFFECT: composition effectivity is caused by its ability to bind lipoteichoic acid of gram-positive bacteria, neutralising or preventing their pathogenic action.

7 cl, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to application of peptide of formula (I): ((X)1(Y)m)n where peptide contains 3 to 200 amino acids and where 1, m and n represent integers within 0 to 10; X and Y which can be identical or different, represent cationic amino acids chosen from arginine and lysine in preparing drugs for treating fungal infection.

EFFECT: ensured applicability of peptide for preparing a drug for treating fungal infection.

26 cl, 53 dwg, 2 tbl, 19 ex

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