Antitumor peptide preparation based on alpha-fetoprotein fragment, its conjugate, pharmaceutical composition and method for treatment of hormone-dependent tumors

FIELD: medicine, biotechnology, pharmacy.

SUBSTANCE: invention proposes an antitumor peptide preparation representing human alpha-fetoprotein (AFP) fragment with amino acid sequence given in the description (fig. 1) and comprising 243 amino acid residues in primary structure and molecular mass 27 kDa. This AFP fragment is able for binding with AFP receptors, to inhibit estradiol-induced growth of hormone-dependent tumors, and to perform vector function of molecule in delivery of cytotoxic preparation into tumor cells. The preparation is obtained by culturing cells E. coli transformed with plasmid vector comprising cDNA gene of the third domain of human AFP. The peptide product is modified by additional methionine residue (Met) at N-end and by two amino acid residues (Leu-Glu) at C-end. Also, invention relates to the peptide preparation conjugate and cytotoxic agent, pharmaceutical composition and a method of treatment. As a cytotoxic agent the conjugate comprises a substance chosen from the following group: paclitaxel, docetaxel, doxorubicin, vinblastin, diphtherin, ricin and others. Method of treatment involves administration in the patient the conjugate or composition in the effective amount. Invention provides enhancing effectiveness and selectivity in treatment of hormone-dependent tumors.

EFFECT: enhanced effectiveness of treatment, valuable medicinal properties of preparation and pharmaceutical composition.

7 cl, 7 dwg, 7 ex

 

The present invention relates to the field of biopharmacology and medicine and can be used for the preparation of anticancer drugs and therapy of malignant tumors.

The study of the structure, functions and ways of using alpha-fetoprotein (AFP) has its origins and is described in detail in Gearlive ("25 years of study α-fetoprotein", Ontogeny, 1989, t.20, No. 6, str-615). In recent years received a lot of new evidence about the prospects of further studies of this protein.

Alpha-fetoprotein is a large glycoprotein (70 kDa), consisting of a single polypeptide chain, and is one of the main circulating proteins found in mammals and other vertebrates in the process of development of embryo and fetus. Gene AFP together with other genes of the family of albumin is located in the region 4q11-q22 4th chromosome [1]. He is oncofetal marker, because secreted by various tumors, including hepatocellular carcinoma and teratoma. It was also established that changes in the level of AFP during pregnancy can cause fetal development, including down syndrome and spina bifida [2]. Found that the AFP performs many important biological functions, the main of which is transport. It is known that the AFP has a high crods the PTO to fatty acids, the site contains high-affinity binding of bilirubin and effectively binds cations of copper and Nickel [3]. It was also established that this protein participates in the regulation of metabolism and cell division, as well as the interaction of macrophages with T-lymphocytes. In favor of the latter statement data speak about the role of the AFP in suppressing the immune response of the mother to the developing embryo [4].

It is believed that the AFP consists of one polypeptide chain of size 590 amino acid residues, divided into three domains, referred to as domain 1, domain 2 and domain 3. Each domain consists of approximately 195 amino acids, and they are 4, 5 and 6 disulfide bonds, respectively [5].

Patent research has shown that on the basis of AFP developed preparations and methods for their production from natural sources by extraction and purification (RF patent 2121350, 1998, the patent of the Russian Federation 2123009, 1998, the patent of the Russian Federation 2154468, 2000), methods of treatment using such drugs (RF patent 2105567,1998 year).

For therapeutic purposes, required large quantities of protein, which is very difficult to ensure its isolation from natural sources. An urgent task to provide the necessary quantities of AFP is the involvement of genetic engineering and biotechnology. However, the preparation of recombinant AFP is complicated by a number of its features. First, e is the large glycosylated proteins, providing native conformation in which the cells of strains-producers is sometimes an impossible task. Furthermore, formed during the broadcast in the cells of the producer strain denatured AFP contains a large number of free tylnej groups. Therefore, in the process of renaturation of a protein, the formation of both intermolecular and intramolecular disulfide bonds. Education mezhmolekulyarnykh relationships leads to large losses of protein in the education of its large conglomerates and precipitation. The position of the intramolecular bonds formed during renaturation of recombinant protein, often differs from those in the natural AFP. Because the correct formation of disulfide bonds is essential for the formation of native tertiary structure of the protein, violation of the procedure of formation of these linkages in recombinant protein determine the conformational heterogeneity of the drug and lead to the loss of its activity.

Largely to avoid these problems in obtaining recombinant AFP allows the use of genetically engineered structures, ensuring the products are not whole protein, and its biologically active peptide fragments. This can significantly reduce the cost, simplify the procedure for obtaining the preparation is the same and increase the yield of biologically active protein. Modern developments associated with obtaining recombinant molecules AFP and its fragments, described in the publications U.S. application 2002031520, 2002, and the patent 6627440, 2003 are the closest analogues of our invention. Obtained through these works of genetically engineered method fragments AFP designed to enhance cellular immunity in some oncological diseases.

However, the known short-chain fragments AFP is not always sufficiently effective and have not been applied in gormonzavisimykh tumors.

The technical result of the present invention is to remedy these disadvantages, as well as expanding Arsenal of anticancer agents and methods of treatment of malignant diseases.

The technical result is achieved by the fact that the developed anticancer peptide drug on the basis of a fragment of alpha-fetoprotein person representing a peptide with the amino acid sequence represented in figure 1, having the primary structure of 237 amino acid residue and a molecular weight of about 27 kDa, are able to bind to the receptor of alpha-fetoprotein and to inhibit estradiol-induced cell growth gormonzavisimykh tumors, as well as to perform the function of vector molecules for targeted delivery of a cytotoxic drug in the tumor cell is. The drug can be obtained by recombinant method for cultivating cells of E. coli, transformed with plasmid vector containing the cDNA of the gene of the third domain of alpha-fetoprotein person; the resulting peptide product modified incremental methioninol residue (Met) at the N-end and two amino acid residues (Leu-Glu) on the C-end.

Another object of the invention is a conjugate of the peptide of the drug and a cytotoxic agent capable of penetrating into the tumor cell, which represents the product described above, covalently linked to a cytotoxic agent in a molar ratio of from 1:1 to 1:10. Conjugate as a cytotoxic agent may contain a substance selected from the group of: paclitaxel, docetaxel, doxorubicin, daunomycin, karminomitsin, vincristine, vinblastine, melphalan, methotrexate, cytarabine. All these drugs are anticancer or cytotoxic substances that do not have sufficient selective action against tumor cells. In the form of a conjugate with our peptide preparation these substances acquire exceptional selectivity against malignant cells.

On the basis of the peptide drug and conjugate developed pharmaceutical composition having antitumor activity, containing PR is teleophobia component and suitable for intravenous pharmaceutical carrier, at the same time as antitumor component composition contains developed peptide drug or conjugate in an amount sufficient to obtain a therapeutic effect. As a carrier for intravenous administration can be used saline or phosphate-saline, pH 7.4, prepared with water for injection.

The basis of the invention, there are two new substances are developed and obtained peptide-based drug fragment AFP and its conjugate with a cytotoxic agent, which are characterized by high selectivity and efficiency of antitumor activity and can be obtained by using technologically advanced genetic engineering techniques or chemical synthesis.

The invention is illustrated by the following examples:

Example 1. Construction of plasmid vector and expression of the recombinant peptide of the drug (fragment AFP) in the cells of E. coli.

1) Obtaining cDNA gene AFP person

Total RNA was isolated from cells of hepatocellular carcinoma human line HepG2, producing AFP. cDNA gene AFP person synthesized on the mRNA using reverse transcriptase virus myeloblastosis birds (RTAMV). As the seed used primer on the 3'- noncoding region mPHK: 5'-CTTCCCCTGAAGTAAT-3'. The reaction was carried out in 2 stages: 1) annealing of primer, 2) synthesis CDNCA first phase of the mixture, composed of 18 μlH2O, 10 μl 5-fold buffer (50 mM Tris-HCl, pH 8.3, 50 mM MgCl2, 700 mM KCl, 8 μl mixture of four deoxynucleotides dNTP (1.25 mM each dNTP), 10 μl solution mRNA (2 μg/ml) and 25 μM primer, heated to 70°C for 10 min, then slowly cooled to room temperature. To the reaction mixture were added 1 μl of RNase inhibitor RN (40 u/μl) and 2.5 μl RTAMV (12.5 u/μd). The final volume of the mixture was 50 μl. The reaction was conducted at 40°C for 2 hours, and then iactiveaware the reaction mixture keeping at 95°within 5 minutes the Quality of cDNA was verified by PCR using the primers 5'-and 3'-terminal region of the DNA molecule AFP. Band amplification corresponded to the expected size 1773 base pairs (bp).

2) Construction of expression vector.

Previously based on a sequence of AFP mRNA person using a computer program DNA-SUN was obtained restriction map of a DNA sequence AFP to identify missing and unique restriction sites. According to the results of the analysis of these data have been collected and synthesized the primers for amplification of a DNA fragment AFP, corresponding to the third domain of a protein molecule (357 at 590 amino acid). Direct primer 5'ttttCCATGGGATACCAGGAGTTATTG 3' contains the site for the Ncol restrictase. Reverse primer 5 ttttCTCGAGAACTCCCAAAGCAGCAC 3' contains the Xhol site of restrictase. Purified by electrophoresis in 1.2% agarose gel followed by extraction of the DNA fragment embedded in the vector rate(+) (Novagene) restriction sites Ncol and Xhol. This gene construct allows for broadcast to obtain the peptide AFP28D3, which amino acid sequence corresponds to the third domain of the molecule AFP added methionine (Met) at the N-end and several amino acids at the C-end (Leu-Glu-His-His-His-His-His-His). Six end histidinol necessary for the evolution of protein by the method of chelate chromatography. The size of the new peptide is 237 amino acid residues, or about 27 kDa. Cloning was performed by the method of calcium transform DH5a competent cells of E.coli strain with selection on Petri dishes with nutrient agar LB medium containing kanamycin. Selection of colonies with gene construction was performed by polymerase chain reaction using standard vector primers T7prom and T7ter along the length of amplification products. Selected colonies were increased in LB with kanamycin, and from the cell masses were isolated expression plasmids for sequencing. According to the sequencing results (no error amplification) for further work was selected plasmid pAFP28D3.

3) Expression of recombinant peptide fragment AFP in E.coli.

Expressing strain of E.coli BL21(DE3) was transferrable the plasmid pAFP28D. Overnight culture of bacterial cells were diluted 50-fold in LB medium containing kanamycin (30 μg/ml), and increased the cells at 37°to the level of turbidity of culture medium 0.6-0.8. Then IPTG was added to final concentration of 0.4 mm, and incubated in cell suspension for 3 hours under vigorous stirring. The cell precipitates were collected by centrifugation and stored at -30°C.

Example 2. The selection of the final product (peptide drug) of the producer strain E. coli.

1) Preparation of buffer solutions

Prepared solutions A, B, C, D, E. F:

A: 0.1 M Tris-HCl buffer, 6 M guanidinium, pH 8.0.

In: 0.02 M Tris-HCl. 0.3 M NaCl, pH 8.0.

From: 0.05 M Tris-HCl buffer, 6 M urea, 0.4 M NaCl and 0.02 M imidazole, pH 8.0.

D: 0.05 M Tris-HCl buffer, 6 M urea, 0.4 M NaCl and 0.3 M imidazole, pH 8.0.

E: 0.1 M Tris-HCl buffer, 0.5 M NaCl and 2 mm EDTA, pH 8.0.

F: 0.01 M Tris-HCl buffer, 0.15 M NaCl, pH 8.0.

2) Isolation of peptide drug AFP (PEP-AFP) from biomass.

The biomass of 600 ml of culture medium was added 3 ml of buffer with 2 mm PMSF. The suspension was treated with ultrasound on the cage High Intensity Ultrasonic Processor 750 Watt Series at 0°C. the Suspension was centrifuged 10 min (12000 g, 4°C). The supernatant was discarded, sediments suspended in 5 ml of buffer and centrifuged under the above conditions. The sediment washing was repeated 5 times. To the washed precipitate Taurus inclusion was added 4 ml of buffer A, on relatively ultrasound to dissolve the protein and centrifuged 30 min (12000 g, 4°). The supernatant was applied to a column (diameter 0.5 cm) with 1 ml iminodiacetate-sepharose Chelating Sepharose CL-6B, rich Nickel ions (2+) and washed with 2 ml buffer A. the Sorbent is then washed with 10 ml buffer a and 10 ml of buffer C. Protein was suirable 6 ml of buffer D. the eluate was added 42 ál β-mercaptoethanol (to a final concentration of 0.1 M) and the solution was stirred 1 h at room temperature. The solution recovered protein was poured with stirring in 60 ml of cold (4° (C) buffer E. After 48 h protein deliberately first against 3 l of buffer E (48 h, 4°C), then against 3 l of buffer F (24 h, 4°). Dialysate was concentrated on Amicon cell (membrane PM-10), 400 μl of the concentrate was fractionally on a column of Superdex™ 200 10/30, elwira buffer F with a flow rate of 0.5 ml/min and detection at 280 nm. There are two factions - dimer (fraction I) and the monomer (fraction II). The elution profile of total protein is shown in figure 2 (I - dimer, II - monomer). As can be seen in the figure, the efficiency of obtaining monomer II reaches 40% of the total protein. The concentration of protein was determined by the method of ICA (ICA-set Sigma), exit - 3 mg. Conducted electrophoresis selected protein fractions in regenerating and non conditions in a 13%polyacrylamide gel according to laemmli's method. The target fraction II was concentrated on Amicon cell (membrane PM-10) up to 1.5 ml.

Figure 3 presents electro is ores 13%SDS page of fractions, corresponding to the dimer and monomer PEP-AFP, reducing and non conditions (lanes 1,2 - without recovery; lanes 3-5 with recovery β-mercaptoethanol; 1,4 - fraction I (dimer); 2,5 - fraction II (monomer); 3 - molecular weight markers).

Example 3. The analysis of binding and capture of FITC-labeled peptide preparation (PEP-AFP) in tumor cells and lymphocytes using flow cytofluorimetry.

1) preparation of FITC-labeled derived PEP-AFP.

To a solution of 1 mg PEP-AFP in 1 ml of 0.1 M sodium bicarbonate, pH 8.5, was added to 6.5 μl (4-fold molar excess) of a solution of FITC (fluorescein-isothiocyanate) in DMSO (10 mg/ml). The reaction was carried out in the dark for 1 h at room temperature, then 18 h at +4°after that he applied on the column (h cm) with Sephadex G-25, equilibrated PBS, pH 7.4, and was suirable the specified buffer when the velocity of the flow 1 ml/min and detection at 280 nm. Collected protein fraction in the free volume, concentrated to 1 ml protein Concentration was determined using bicinchoninic acid, the concentration of FITC - by absorption in the area of 498 nm. The molar ratio of FITC - peptide was 1.4:1.

2) analysis of the binding and capture of FITC-labeled PEP-AFP in tumor cells and lymphocytes using flow cytofluorimetry.

Cell adenocarcinoma breast cancer line MCF-7 and ovarian adenocarcinoma man is the AC line SKOV3 were cultured in plastic flasks (Coming Costar) in DMEM (ICN), containing 10% FBS, Gibco) and 50 μg/ml gentamicin (ICN) in CO2-incubator at 37°C in humidified atmosphere containing 5% CO2. The peripheral blood lymphocytes of healthy volunteers were isolated by centrifugation of blood through the gradient of the solution ficoll-Pak according to the method of Boyum [(Boyum, A., llin. Invest. 21: 9-18, 1968]. Cells subcultured in 6-hole tablets a day before the experiment. Before the experiment the cells were incubated for 2 hours in DMEM without FBS. To analyze the binding of PEP-AFP-FITC in the concentration range 30-4000 nm) was added to cell suspension and incubated at 4°C for 1 hour. In the study of endocytosis incubation was carried out at 37°C. After incubation, cells are washed three times with cold PBS and fixed with 2% paraformaldehyde. The fluorescence intensity was measured on a flow cytometer EPICS XL. For fluorescence excitation used argon laser (λ488 nm, bandwidth of 520 nm). Each sample (105cells) was determined by the average value of the fluorescence intensity and percentage of cells, the fluorescence of which exceeded the autofluorescence. The specificity of binding of PEP-AFP-FITC with AFP receptor was determined by incubation of cells with PEP-AFP-FITC in the presence of 30-fold excess AFP.

It was shown that PEP-AFP-FITC binds to tumor cells (MCF-7 and SKOV3) in the profile is from peripheral blood lymphocytes (figa). However, the intensity of fluorescence of cells treated with PEP-AFP-FITC concentrations up to 1 μm, was close to the fluorescence of cells treated with AFP-FITC. These studies show the selectivity of our preparation against gormonzavisimykh tumors.

The study capture PEP-AFP-FITC tumor cells and lymphocytes of peripheral blood showed that tumor lines MCF-7 and SKOV3 actively endocytic PEP-AFP-FITC (figb), in contrast to lymphocytes. In lymphocytes endocytosis PEP-AFP-FITC was practically absent. To study the binding specificity, SKOV3 cells were incubated with PEP-AFP-FITC with the addition of 30-fold excess unlabeled AFP in the same conditions. AFP significantly inhibited the binding of PEP-AFP-FITC cells (figure 5). These data confirm that our product (fragment PEP-AFP) is indeed associated with the AFP receptor on tumor cells and competes with the intact protein AFP for the binding site.

Example 4. The study gormonzawisimah activity of the new peptide.

Cells MCF-7 were sown in 96-well tablets with a density of 4×105cells/well. The next day, change the medium to DMEM without phenol red with 5% calf serum, and incubated the cells under standard conditions to achieve a dense monolayer (4 days). Next to the cells was added hormone 17 β-variety of the diol (E 2) at a concentration of 10-9M, PEP-AFP and AFP (both at concentrations of 10-11-10-6M). After 3 days in the hole made [3H]-thymidine (1 MKS/ml, specific activity 50 Ci/mmol) for 2 hours, after which cells were collected on filters and measured the radioactivity in a liquid scintillation counter RackBeta. After 72 hours incubation to stimulate cell proliferation was assessed by incorporation of [3H]-thymidine into DNA. Proliferation averaged 165% of control - 100%). Simultaneous with E2adding to the cells AFP or PEP-AFP in the concentration range 10-11-10-6M the inclusion of [3H]-thymidine was significantly decreased (117±5.85% for AFP and 120±4.8% for PEP-AFP), as shown in Fig.6.

Thus, PEP-AFP inhibits 17 β-estradiol-induced growth gormonzawisimah cell line MCF-7.

Example 5. Getting conjugate PEP-AFP with paclitaxel.

0.63 mg of paclitaxel and 1.2 mg of N,N-carbonyldiimidazole were mixed in 20 ml of dimethylformamide. The solution was kept for 20 min at 55°With, then added with stirring to 1 ml PEP-AFP (2 mg) in phosphate-buffered saline (PBS), pH 7.4. The reaction mixture was stirred over night at room temperature, then centrifuged for 5 min at 13 400 rpm, the Supernatant was separated and was applied to a chromatographic column with medium Sephadex G-25, balanced PBS. The fractions containing the conjugate were collected and concentrated to a final volume of 1 ml To determine the molar ratio of PEP-AFP/paclitaxel separately determined the concentration of each component of the conjugate. The concentration of PEP-AFP in the conjugate was determined by the method of Lowry. To determine the content of paclitaxel in the conjugate aliquot of the concentrate was transferred into a 0.2 M solution of sodium acetate, pH 4.0, and was kept for 48 h at room temperature. In these conditions, paclitaxel was completely separated from the protein and precipitated. Fallen paclitaxel was extracted with chloroform and dried in air. Dry matter was dissolved in acetonitrile and conducted quantitative determination of paclitaxel using high-performance liquid chromatography. The molar ratio of PEP-AFP/paclitaxel in the resulting conjugate was 1:1.

Testing of antitumor activity were conducted on models and confirmed by the following examples.

Example 6. The effectiveness of the conjugate PEP-AFP with paclitaxel in the cells of breast carcinoma line MCF-7.

Cells were cultured in plastic bottles ("Costar") in RPMI medium (Sigma)containing 10% fetal bovine serum (Sigma), 100 u/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO2at 37°C. the Cells subcultured in 96-LUN is cnie plates (Costar) at 10 thousand cells/well, adding investigational drugs in different concentrations and incubated For 72 h 2-4 h before the end of the incubation, each well was added 50 μl of a solution (1 mg/ml) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) in the medium for cell culture. After color development, the medium was removed, dissolved crystals formazan in 150 μl of dimethyl sulfoxide and spectrophotometrically measured the colour intensity of the absorption at 540 nm. The survival of cells exposed to paclitaxel and the conjugate was evaluated in percent, assuming a 100% survival rate of the control culture.

The values of the IC20was 21 nm for the free paclitaxel and 13 nm for the conjugate. It should be noted that the proposed conjugate PEP-AFP with paclitaxel has a much higher cytotoxic activity against tumor cells of the wild type than the free drug paclitaxel.

Similar studies were performed with each substance from the group of: docetaxel, doxorubicin, daunomycin, karminomitsin, vincristine, vinblastine, melphalan, methotrexate, cytarabine.

When using a new peptide drug and its conjugate was developed a method of treatment of malignant tumors, while experimental animals with gormonzavisimym tumors have introduced our anticancer p is ptiny the drug or its conjugate with a cytotoxic agent. For treatment was also used in the pharmaceutical composition described above effective therapeutic result number. In therapy you can optionally enter adjuvants, other anticancer drugs, various immunomodulators or antiangiogenic funds. As immunomodulators can be applied interferon-αinterferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12, polyoxidonium, as antiangiogenic funds - angiostatin, endostatin, thalidomide, vitaxin, pentosan, suramin, fumagillin, squalamine, combretastatin, prinomastat, marimastat, neovastat.

Example 7. The antitumor activity of conjugate PEP-AFP-paclitaxel on the model of transplantable tumors in mice.

For comparative evaluation of therapeutic activity of the conjugate PEP-AFP-paclitaxel against solid tumors received subcutaneous injection of mice tumor cell line In 16 used intravenous our conjugate. The drugs were injected at doses of 0.2 mg/kg) paclitaxel according to the scheme once in 2 days, a total of five injections, starting from the 3rd day after inoculation of the tumor. Data on the inhibition of tumor growth in mice with administration of drugs presented in Fig.7. As can be seen from the drawing, the conjugate, in contrast to free paclitaxel significantly slowed BP is me the appearance of tumors and their growth rate. In addition, the introduction of conjugate increased the average life span of animals by 37%.

Thus developed a complete system designed to ensure gormonzavisimykh tumors, based on the peptide fragment AFP that contains the new peptide drug, its conjugate with a cytotoxic agent, a pharmaceutical composition and method of treatment. The developed system against gormonzavisimykh tumors highly selective and effective against malignant cells.

LITERATURE

1. Yang, F. et al., Nucleic Acids Res. 13: 8007-8017, 1985.

2. Deutsch H.F. et al., Adv. Cancer Res. 56: 253-312, 1991.

3. Hisa J. et al., J. Biol. Chem. 255: 4224-4227, 1980.

4. E. Dudich et al., Eur. J. Biochem. 266: 750-761, 1999.

5. Mizejewski, G., Proc. Soc. Exp.Biol. Med. 215: 333-362, 1997.

1. Antitumor peptide drug on the basis of a fragment of alpha-fetoprotein (AFP) person representing the peptide with the amino acid sequence represented in figure 1, having the primary structure of 237 amino acid residue and a molecular weight of about 27 kDa, are able to bind to the receptor of alpha-fetoprotein and to inhibit estradiol-induced cell growth gormonzavisimykh tumors, as well as to perform the function of vector molecules for targeted delivery of a cytotoxic drug in the tumor cells.

2. The preparation according to claim 1, characterized in that can be obtained by recombinant method is ri cultivation of cells of E. coli, transformed with plasmid vector containing the cDNA of the gene of the third domain of alpha-fetoprotein person; the resulting peptide product modified incremental methioninol residue (Met) at the N-end and two amino acid residues (Leu-Glu) on the C-end.

3. The conjugate of the peptide of the drug and a cytotoxic agent capable of penetrating into the tumor cell, which represents the product described in claim 1 or 2, covalently linked to a cytotoxic agent in a molar ratio of from 1:1 to 1:10.

4. The conjugate according to claim 3, which as a cytotoxic agent contains a substance selected from the group of paclitaxel, docetaxel, doxorubicin, daunomycin, karminomitsin, vincristine, vinblastine, melphalan, methotrexate, cytarabine.

5. Pharmaceutical composition having antitumor activity, containing antitumor component and suitable for intravenous pharmaceutical carrier, characterized in that as an antitumor component contains a drug, characterized in claims 1 and 2 or conjugate, characterized in PP and 4, in an effective amount.

6. The method of treatment of malignant tumors, including the introduction of an antitumor agent, wherein the patient with gormonzawisimah tumor is administered pharmaceutical composition is, characterized in claim 5, in effective amounts.

7. The method of treatment according to claim 6, wherein the patient is additionally administered adjuvants, other anticancer drugs, immunomodulators, or antiangiogenic tools.



 

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27 cl, 13 dwg, 5 ex

FIELD: organic chemistry of natural compounds, medicine, oncology.

SUBSTANCE: invention relates to a novel crystalline form of (1S,2S,3R,4S,5R,8R,9S,10R,13S)-4-acetoxy-2-benzoyloxy-9,10-[(1S)-2-(dimethylamino)ethylideneoxy]-5,20-epoxy-1-hydroxytax-11-ene-13-yl-(2R,3S)-3-(tert.-butoxycarbonylamino)-3-(3-fluoro-2-pyridyl)-2-hydroxypropionate that shows the diffraction picture of roentgen rays in powder with characteristic peaks at diffraction angles (θ)= 6.2o, 10.3o, 10.7, 11.4o and 12.0, and a method for its preparing. Method involves carrying out the crystallization step by using organic solvent chosen from group consisting of ketone type solvent, nitrile solvent type and their mixture, or mixture of said solvent and water. Also, invention relates to an antitumor agent based on the prepared crystalline form. Invention provides the stable quality of a medicinal agent based on its lower hygroscopicity.

EFFECT: improved and valuable properties of compounds.

8 cl, 5 ex

FIELD: organic chemistry, chemical technology, medicine, pharmacy.

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

or their pharmaceutically acceptable salts or esters hydrolyzing in vivo and possessing activity inhibiting the cellular cycle and selective with respect to CDK-2, CDK-4 and CDK-6. Compounds can be used in cancer treatment. In the formula (I) R1 represents halogen atom, amino-group, (C1-C)-alkyl, (C1-C6)-alkoxy-group; p = 0-4 wherein values R1 can be similar or different; R2 represents sulfamoyl or group Ra-Rb-; q = 0-2 wherein values R2 can be similar or different and wherein p + q = 0-5; R3 represents halogen atom or cyano-group; n = 0-2 wherein values R3 can be similar or different; R4 represents hydrogen atom, (C1-C6)-alkyl, (C2-C6)-alkenyl, (C2-C6)-alkynyl, (C3-C8)-cycloalkyl, phenyl or heterocyclic group bound with carbon atom wherein R4 can be optionally substituted at carbon atom with one or some groups Rd; R5 and R6 are chosen independently from hydrogen, halogen atom, (C1-C)-alkyl, (C2-C6)-alkenyl or (C3-C8)-cycloalkyl wherein R5 and R6 can be substituted at carbon atom independently of one another with one or some groups Re; Ra is chosen from (C1-C6)-alkyl, (C2-C6)-alkenyl, (C2-C6)-alkynyl, (C3-C8)-cycloalkyl, (C3-C8)-cycloalkyl-(C1-C6)-alkyl, phenyl, heterocyclic group, phenyl-(C1-C)-alkyl or (heterocyclic group)-(C1-C6)-alkyl wherein Ra can be substituted optionally at carbon atom with one or some groups Rg and wherein if indicated heterocyclic group comprises residue -NH- then its nitrogen atom can be optionally substituted with group chosen from the group Rh; Rb represents -N(Rm)C(O)-, -C(O)N(Rm)-, -S(O)r-, -OC(O)N(Rm)SO2-, -SO2N(Rm)- or -N(Rm)SO2- wherein Rm represents hydrogen atom or (C1-C6)-alkyl, and r = 1-2. Also, invention relates to methods for synthesis of these compounds, a pharmaceutical composition, method for inhibition and using these compounds.

EFFECT: improved preparing method, valuable medicinal properties of compounds and pharmaceutical compositions.

24 cl, 3 sch, 166 ex

FIELD: immunology, cell therapy.

SUBSTANCE: claimed composition contains heat shock protein of HSP family or fragment thereof and tumor-specific peptide selected from group containing MAGE-A1, -A2, -A3, gp100, HER2/NEU, EGFRvIII or fragment thereof non-covalently bound to said protein, wherein non-covalent bond is provided by preliminary incubation of components under physiological conditions with adenosine diphosphate. As heat shock protein composition contains protein selected from group containing DnaK from M. tuberculosis, M. bovis or E. coli or human HSP 70 protein. Tumor-specific peptide fragments are described in table represented in description. Additionally composition contains immunomodulator selected from group containing INFα, INFγ, TNFα, IL-2, -4, -6 or combination thereof and pharmaceutical carrier. Also disclosed are cell preparation which represents isolated and washed dendrite leucocytes activated by incubation with new composition as well as method for tumor immunotherapy or immunoprophylaxis by parenteral administration of composition or cell preparation in effective dose.

EFFECT: new antitumor preparation of improved effectiveness.

8 cl, 1 tbl, 8 ex, 3 dwg

FIELD: molecular biopharmacology.

SUBSTANCE: method for production of stress protein (heat shock protein) preparations includes such protein gene expression in E.coli cells followed by isolation, refolding and purification. Gene expression is carried out in structure of vector cDNA containing T7 promoter, kanamycin resistance gene, replicative pUC ori origin, and gene encoding lac-inductor. Vector DNA is cultured in structure of E.coli cells BL21 in nutrient medium with addition of kanamycin (100 mug/ml) and glucose (0.2 %) up to turbidity level of 0.5-0.6 OD600. Then 0.02mM isopropyl beta-D-thiogalactoside is added into medium and culturing is carried out for 1-3 h to accumulate finish product. Target product is purified by anion exchange chromatography. In said method vector DNA encoding proteins: HSP 100-200; HSP 100; HSP 90; Lon; HSP 70; HSP 60; TF 55; HSP 40; FKBPl cyclophylin; HSP 2-30; ClpP; GrpE; HSP 10, etc. of any gene are used. Invention also related to stress protein (heat shock protein) preparation obtained by abovementioned method. Protein represents recombinant HSP protein having molecular weight of about 70 kDa, which is isolated after incubation of E.coli cells in growth medium LB for 12 h at 37°C with addition of kanamycin (100 mug/ml) and glucose (0.2 %) up to turbidity level of 0.5-0.6 OD600, wherein transformed cell contains pBSH2-HSP70 plasmide DNA, carrying cDNA of heat shock protein gene, whish has size of 5118 n.p. and represents pBSH2 promoter containing T7 promoter, kanamycin resistance gene, replicative pUC ori origin, and gene encoding lac-inductor. Finished protein product has purity of at least 98 %.

EFFECT: stress proteins of high activity and high purity.

8 cl, 5 ex, 5 dwg

FIELD: molecular pharmacology, in particular human endostatin preparation and method for production thereof.

SUBSTANCE: claimed preparation is isolated after cell incubation E.coli BL21 (DE3) in cultural medium up to dense level of 0.4-0.6 OD6-00. Cell contains plasmide vector pBSH-ED15 or pBSH-ED16 having size of 6675 n.p. and gene of canamicine resistance gene; vector contains replicative sites pUS ori and M13 and gene encoding lac-repressor controlled by T7-promoter, and finished product has purity of 99 % and apyretic properties. Claimed preparation is obtained by expression of endostatin gene using plasmide DNA containing in E.coli cells; product refolding and purification, wherein expression is carried out in E.coli BL21 (DE3) cells, transformed by recombinant plasmide DNA pBSH-ED15 or pBSH-ED16. Cells are cultivated in cultural medium up to dense level of 0.4-0.6 OD6-00, then 0.1-0.3 mM of isopropyltiogalactoside is added into medium and cultivation is carried out for 2-3 h up to product accumulation. Further cells is exposed with ultrasound for 50-70 s at 0°C in presence of 0.1 % sodium deoxycholate. Bodies are dissolved in presence of 1 % sodium N-lauryl sarcosine. Suspension is multiply washed and centrifuged at 5000 g. In refolding process oxidation with air oxygen is carried out for 35 h, and target product is purified using chromatography depending on used incorporated plasmide.

EFFECT: apyretic product of high purity.

6 cl, 9 ex, 7 dwg

FIELD: medicine.

SUBSTANCE: preparation belongs to decapeptides considered to be an analog of receptor-binding fragment of TGFα from 22 to 31 amino acids in which the eighth amino acid residue Lys is substituted with Ser residue. The analog is bindable to wide range of cytotoxic agents and operate as vector in directed delivery of antitumor agents to tumor cells. The preparation also comprises decapeptide conjugate with a cytotoxic agent showing selective action with respect to tumors and capable of reducing tumor cells resistivity to cytotoxic agents. Conjugated parts are bound by a bond scissile with respect to acid hydrolysis. Another embodiment of the invention is related to pharmaceutical composition containing effective quantity of conjugate and carrier applicable as intravenous injections.

EFFECT: enhanced effectiveness of treatment; high antitumor action selectivity.

6 cl, 2 dwg, 2 tbl

FIELD: organic chemistry, polypeptides.

SUBSTANCE: invention relates to new antagonists of urotensin-II. Invention represents group of cyclic polypeptides of the general formula: (R1)a-AA1-cyclo-[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 wherein AA1 means L-isomer of aromatic amino acid; AA2 means L- or D-isomer of Cys; AA3 means L-isomer of aromatic amino acid; AA4 means L- or D-isomer of Trp; AA5 means L- or D-isomer of Lys, N-Me-Lys or Orn; AA6 means L- or D-isomer of Val, Thr, Leu, Ile, Tle, Nle or aromatic amino acid; AA7 means L- or D-isomer of Val, Thr, Leu, Ile, Tle, Abu, Nle or aromatic amino acid; R1 means hydrogen atom (H), lower alkyl, lower alkanoyl or lower acyl; a = 1 or 2; R2 means hydroxyl; group (OH), OR3, N(R3)2 or NHR3 wherein R3 means H, lower alkyl or arylalkyl. These cyclic polypeptides are used as antagonists of urotensin-II.

EFFECT: valuable biological properties of compounds.

24 cl, 1 tbl, 2 ex

FIELD: organic chemistry, amino acids.

SUBSTANCE: invention proposes agonists of somatostatin of the formula (I): X-A1-cyclo-(D-Cys-A3-A4-Lys-A6-A7)-A8-Y or its pharmaceutically acceptable salt wherein X represents hydrogen atom (H); A1 represents L-Cpa, L-Phe, L-Trp or L-Nal; A3 represents L-3-Pal or L-4-Pal; A4 represents D-Trp; A6 represents -NH-(CHR1)n-CO- wherein n = 2, 3 or 4; A7 represents L- or D-Cys; A8 represents D- or L-isomer of amino acid taken among the group consisting of Nal, Phe, Cpa and Trp; Y represents NH2; R1 represents hydrogen atom (H), and Cys in A3 is bound by disulfide bong in A wherein this disulfide bond is formed by thiol groups of each Cys residue.

EFFECT: valuable biological properties of compounds.

9 cl, 2 ex

FIELD: chemico-pharmaceutical industry.

SUBSTANCE: the present innovation deals with new stabilized pharmaceutical composition in its lyophilized form including the compound of formula I

as an active ingredient and lactose disaccharide as a stabilizing agent. The present pharmaceutical compositions are of high stability at storage. As for active ingredient it is not destroyed in the course of time.

EFFECT: higher efficiency.

10 cl, 15 ex, 6 tbl

FIELD: medicine.

SUBSTANCE: preparation comprises echinocandine substance of formula I or its pharmaceutically permissible salt, pharmaceutically permissible micelle-forming surface-active agent and non-toxic aqueous solvent and stabilizing agent.

EFFECT: improved stability and bioaccessibility properties.

48 cl, 4 tbl

FIELD: biochemistry.

SUBSTANCE: method relates to new cyclopeptides of general formula cyclo(R1-Arg-Ile-Lys-Pro-His-R2) selected from group containing: P11: cyclo(DPhe-Pro-Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly-Glu) (SEQ ID NO:5), P16: cyclo(Arg-Ile-Lys-Pro-His-Gln-Gly (SEQ ID NO:8), P17: cyclo(Pro-Arg-Ile-Lys-Pro-His-Gln-Gly) (SEQ ID NO:9), P19: cyclo(Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly-Glu) (SEQ ID NO:10), P20: cyclo(Dphe-Pro-Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly) (SEQ ID NO:11), P23: cyclo(DPhe-Pro-Arg-Ile-Lys-Pro-His-Gln) (SEQ ID NO:13), P24: cyclo(Gly-Arg-Ile-Lys-Pro-His) (SEQ ID NO:25), as well as P11, P20 and P23 with DPhe substituted by DTyr. Cyclopeptides are useful in systems for angiogenesis inhibition. System includes substrate with cyclopeptides attached by organic spacer arm optionally containing group cleavable by any fermentation system.

EFFECT: angiogenesis inhibiting cyclopeptides.

23 cl, 4 dwg, 2 tbl, 2 ex

FIELD: medicine, pharmacology, biochemistry.

SUBSTANCE: invention relates to the highly purified enzyme α-galactosidase A (α-Gal A) and to different methods for its purification. Invention relates to the development of α-Gal A preparation with changed charge and to methods for preparing such preparations. Also, invention relates to α-Gal A preparations with prolonged half-time life in blood current of mammal-host, to methods for their preparing, to methods and doses in administration of α-Gal A preparations in patient. Invention provides expanding assortment used for treatment of patients suffering with α-galactosidase A insufficiency.

EFFECT: improved method for treatment, valuable medicinal properties of preparations.

15 cl, 9 tbl, 10 dwg, 5 ex

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to macrocyclic peptides of the general formula (I): wherein W means nitrogen atom (N); R21 means hydrogen atom (H), (C1-C6)-alkoxy-, hydroxy-group or N-(C1-C6-alkyl)2; R22 means hydrogen atom (H), (C1-C6)-alkyl, CF3, (C1-C6)-alkoxy-group, (C2-C7)-alkoxyalkyl, C6-aryl or Het wherein het means five- or six-membered saturated or unsaturated heterocycle comprising two heteroatoms taken among nitrogen, oxygen or sulfur atom and wherein indicated Het is substituted with radical R24 wherein R23 means hydrogen atom (H), -NH-C(O)-R26, OR26, -NHC(O)-NH-R26, -NHC(O)-OR26 wherein R26 means hydrogen atom, (C1-C6)-alkyl; R3 means hydroxy-group or group of the formula -NH-R31 wherein R31 means -C(O)-R32, -C(O)-NHR32 or -C(O)-OR32 wherein R32 means (C1-C6)-alkyl or (C3-C6)-cycloalkyl; D means a saturated or unsaturated alkylene chain comprising of 5-10 carbon atoms and comprising optionally one-three heteroatoms taken independently of one another among oxygen (O), sulfur (S) atom, or N-R41 wherein R41 means hydrogen atom (H), -C(O)-R42 wherein R42 means (C1-C6)-alkyl, C6-aryl; R4 means hydrogen atom (H) or one-three substitutes at any carbon atom in chain D wherein substitutes are taken independently of one another from group comprising (C1-C6)-alkyl, hydroxyl; A means carboxylic acid or its alkyl esters or their derivatives. Invention relates to pharmaceutical compositions containing indicated compounds and eliciting activity with respect to hepatitis C virus and these peptides inhibit activity of NS3-protease specifically but don't elicit significant inhibitory activity with respect to other serine proteases.

EFFECT: valuable biochemical and medicinal properties of peptides.

106 cl, 9 tbl, 61 ex

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