Application hederagenine 3-o- α -l-rhamnopyranosyle (1→2)-[ β -d-glucopyranosyle (1→4)]- α -l-arabinopyranosyde or containing extract of pulsatillae radix as a therapeutic agent for solid tumours

FIELD: medicine; pharmacology.

SUBSTANCE: application of hederagenine 3-O- α -L-rhamnopyranosyle(1→2)-[b-D-glucopyranosyle(1→4)]-α -L-arabinopyranosyde or containing extract Pulsatillae radix and application of water-soluble fraction of Pulsatillae radix extract containing hederagenine 3-O- α -L- rhamnopyranosyle(1→2)-[b-D- glucopyranosyle(1→4)]- α -L-arabinopyranosyde as a therapeutic agent for solid tumours.

EFFECT: large-scale inhibition of tumour increase on solid tumours cells; relatively low toxicity; decrease of side effects of well-known antitumour agents.

8 cl, 3 ex, 3 tbl

 

This invention relates to the use of hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside, which is represented by the following formula (I):

or containing extract of Pulsatillae radix as a therapeutic agent for solid tumors.

PROTOTYPES

Pulsatillae radix is the dried root of a species of Pulsatilla, which belongs to the family Ranunculaceae (Ki Hwan Bae, a Korean Medicinal Herbs, 1999). According to Chinese medicine it is known that Pulsatillae radix has the effect of removing the elevated temperature and blood detoxification. It is also used as anti-inflammatory, astringent, hemostatic agent and agent against diarrhea and to treat bloody stools, malaria, nosebleeds and bleeding from the teeth. Its flower is called Pulsatillae Flos and is used to treat malaria and smallpox. Its leaves are called Pulsatillae Folium and used to treat pain in the waist, swelling or pain in the heart. In addition, it is reported that a decoction of Pulsatillae radix has antibacterial action against amoebic dysentery and pesticide action against Trichomonas.

Pulsatillae radix contains about 9% saponins and currently selected ingredients such as protoanemonin, anemonin, ranunculin, hederagenin, betulin acid, derivatives ol analboy acids and their glycosides, which is represented by the following formula (II):

The pharmacological effects of the above ingredients is still not widely understood, but it is reported that protoanemonin has mitotoxicity (Vonderbank, F., Pharmazie 5, 210, 1950). Li and others (Li, R. Z. and others, Yao Hsueh Hsueh Pao, 28, 326 31, 1993) also reported that ranunculin has cytotoxicity against KV-cells by inhibiting DNA polymerase.

Hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside isolated from Pulsatilla cerna and P. koreana researchers Shimozu, etc. (Chem. Pharm. Bull., 26, 1666, 1978); from P. chinensis researchers Yoshihiro and others (J. Nat. Prod., 62, 1279, 1999) and from Serjania salzmanniana Schlecht researchers Ekabo and others (J. Nat. Prod., 59, 431, 1996). Kang and others (Arch. Pharm. Res., 12(1), 42-47, 1989) also distinguished it from P. koreana and confirmed its structure. Yoshihiro and others in the above article reported that hederagenin and derivatives of oleanolic acid have shown cytotoxicity against cells HL-60 human leukaemia. They report that hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside isolated from Chinese Pulsatillae radix (Pulsatilla chinensis) has low toxicity, namely to 3.8 µg/ml ED50against cells HL-60. However, most of the saponins and many of the IDA natural products usually demonstrate the same level of cytotoxicity, and therefore, we cannot say that the above connection is based on antitumor activity. Thus, it is not known that hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside and antitumor activity, in particular against solid tumors.

The applicants of the present invention have identified deoxyadenylate of medical plants, including Anthriscus sylvestris Hoffman, Pulsatillae radix, etc. and found that this substance inhibits the cell growth of solid tumors by inhibiting angiogenesis, and received a patent of Korea (Korea patent No. 315200). The applicants of the present invention have conducted extensive studies on preparation of antineoplastic agents from medicinal plants. As a result, they received from Pulsatillae radix fraction, which is poorly soluble in an organic solvent, but is easily soluble in water, and separated from this fraction antitumor connection by thus the present invention.

Thus, the aim of the present invention is to provide a therapeutic agent for solid tumors containing anticancer compound isolated from Pulsatillae radix, or fraction of Pulsatillae radix containing such a compound as an active ingredient.

One aspect of the brew is its invention provides a therapeutic agent for solid tumors, extract of Pulsatillae radix containing hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside as the active ingredient.

Used herein, the expression "solid tumor" refers to any tumor with the exception of blood cancers, typical example of which is a tumor of the lung.

In the present invention the extract of Pulsatillae radix containing hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside, can be obtained by extraction of Pulsatillae radix with an aqueous solution of ethanol and sludge formation when adding there acetone to obtain water-soluble fractions (WT). Or it can be obtained by extraction of Pulsatillae radix with an aqueous solution of ethanol, the formation of precipitation adding there acetone to obtain water-soluble fraction and passing the fraction through a column of Sephadex LH20 with obtaining fractions (SPX3)with Rfof 0.48-0.50 and giving red staining, and then blue staining after spraying with sulfuric acid followed by heating.

Another aspect of the present invention provides a therapeutic agent for solid tumors, including hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside as the active ingredient.

Hereafter the present invented the e will be explained in detail.

According to the present invention the extract of Pulsatillae radix is extracted with 50% ethanol to obtain the fraction of WT, slightly soluble in acetone, and then this fraction is purified on Sephadex LH20, receiving a fraction SPX3, and from the faction SPX3 finally get clean SB365. This connection is hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside and has higher antitumor activity against solid tumors formed by cancer cells mouse lung cells LLC (Lewis lung carcinoma), or tumor cells of the human lung, cells NCI-H23 than clinical medicine adriamycin.

In particular, this process of obtaining antitumor fraction of Pulsatillae radix and discharge from her antitumor substance is as follows.

(1) Receiving antitumor WT fraction of Pulsatillae radix

Powder Pulsatillae radix is extracted with 50% aqueous ethanol and dried under reduced pressure. To the obtained dried substance add acetone 5-10 times the amount. The mixture was shaken, centrifuged at 3000 rpm and the supernatant decanted, receiving insoluble part. The above process was repeated twice. The remaining undissolved part is readily soluble in water, it is denoted "fraction WT". This fraction is displayed on the t relatively high antitumor activity in BDF1 mice transplanted with cells LLC and naked mice transplanted with cells NCI-H23.

(2) Obtaining fractions SPX3 from the faction WT

This fraction WT dissolved in a given quantity of an aqueous solution of methanol with different concentrations and then fractionary on a column of Sephadex LH20, stabilized in the same solvent. In this case, the best allocation is achieved by the use of 80% aqueous methanol, and the appropriate size of the filled column is 60×4 cm to 500 mg fraction WT. The result is the fraction SPX1 (non research tubes 26-66), SPX2 (non research tubes 66-91), SPX3 (non research tubes 91-111) and SPX4 (non research tubes 111-138). After spraying with sulfuric acid fractions obtained on the plate with silica gel, and heating plates fraction SPX3 network over time first red staining, and then blue staining and contains as a main ingredient manifested connection with Rfof 0.48-0.50 in. It is shown that it has high antitumor activity in BDF1 mice transplanted with cells LLC and naked mice transplanted with cells NCI-H23.

(3) the Allocation of SB365 faction SPX3

To highlight the antitumor substance from the fraction SPX3, demonstrating antitumor activity, conduct HPLC, getting a net connection SB365. To identify patterns SB365 conduct the reaction Liberman-Burcard, research IR,1H-NMR,13C-NMR and hydrolysis of a mixture of ethanol/sulfuric acid. The results confirm that SB365 is hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside, which is saponify ingredient, which was isolated from Pulsatillae radix.

Extracts of Pulsatillae radix of the present invention or isolated from pure connection SB365 have a weak cytotoxicity against cells of solid tumors, but unexpectedly exhibit excellent antitumor activity in animal experiments. Thus, they can solve the problems caused by previous anticancer agents in clinical use, such as decreased immune reactions due to the decrease of blood cells. Expect also that they demonstrate low toxicity relatively rapidly dividing cells, such as hematopoietic cells and other

Fraction of Pulsatillae radix and SB365 according to the present invention can be combined with commonly used pharmaceutically acceptable carriers and produce in the form of various preparations, conventional in the pharmaceutical field, for example, preparations for oral administration such as solutions, suspensions, etc.; preparations for injection, such as solutions or suspensions for injection, ready to use dry powder and injection, and others; preparations for local application, such as ointments, creams and solutions. In particular, the active ingredient of the present invention is soluble in water and can be dissolved in various solutions, such as saline, ringer's solution, nutrient solution and other Such pharmaceutical preparations can be administered intravenously, subcutaneously, intraperitoneally or locally.

The recommended dosage of the active ingredient of the present invention to a person is 3.5-8.0 mg/kg of body weight in the case of SB365, 20-40 mg/kg body weight in the case of fractions SPX3 or 200-300 mg/kg in the case of fraction WT. The optimal dosage is 6.5 mg/kg of body weight in the case of SB365, 25 mg/kg body weight in the case of fractions SPX3 or 250 mg/kg body weight in the case of fraction WT. However, this dosage can be adjusted appropriately depending on the age, body weight, health, severity of illness of patients.

Figure 1 shows sample TLC on silica gel fraction WT.

Figure 2 shows samples TLC on silica gel fractions SPX3, purified on a column of Sephadex LH20.

3 shows the HPLC chromatogram of fraction SPX3.

4 shows HPLC chromatogram SB365.

BEST mode for carrying out the present INVENTION

The invention will be better understood from the following examples. The person skilled in the art will readily appreciate that the described specific substance and the result is you are only illustrative, not intended to be limiting and should not limit the invention, which is described in more detail in the next following claims.

Example 1. Getting faction WT

50 g of the powder Pulsatillae radix is shaken out three times with 500 ml of 50% aqueous ethanol and dried extract under reduced pressure, obtaining 22 g of dried substance. To the obtained dried substance add 300 ml of acetone and the mixture is shaken and centrifuged at 3000 rpm, the Supernatant removed, receiving sediment. This precipitate twice repeat the treatment with acetone. The acetone layer is cast and dried undissolved portion, receiving 17.8 g of the dried substance (WT fraction). The obtained fraction WT subjected to TLC on silica gel (manifesting solvent mixture of butanol:acetic acid:water in the ratio 4:1:1, the color reaction: spraying with sulfuric acid followed by heating). The result is shown in figure 1. Figure 1 is a blue spot with Rfin the range from 0.48 to 0.50, corresponds to the active ingredient of the present invention, which is described above. As shown in experimental example 1, fraction WT demonstrates relatively high antitumor activity (the degree of inhibition of tumor growth: 57%) in BDF1 mice transplanted with cells LLC.

Example 2. Obtaining fractions SPX

560 mg of fraction WT fractionary then on to the column, Sephadex LH20 (200 g, 60×4 cm), using a mixed solution of methanol and water (80:20) with a flow rate of 1 ml/min and the volume fractions of 0.5 ml/tube. These fractions is applied on a thin layer of silica gel in order and are getting faction (the solvent mixture of butanol:acetic acid:water in the ratio 4:1:1, the color reaction: spraying with sulfuric acid followed by heating). The result is shown in figure 2. Figure 2 SPX1 (139 mg, 24.8 per cent) obtained by collecting research test tubes with numbers from 26 to 66 and consists of 4 main spots, the lower of which gives a yellow coloration after interaction with sulfuric acid. SPX2 (344 mg, 61.4 per cent) obtained by collecting research test tubes with numbers from 66 to 91 and consists of 2 main spots. SPX3 (61 mg, 10.9 per cent) obtained by collecting research test tubes with numbers from 91 to 111 and network over time first red staining, and then blue staining after spraying with sulfuric acid and subsequent heating. Fraction SPX3 contains spot with Rfin the range from 0.48 to 0.50, as the main ingredient. SPX4 (15.7 mg, 2.8 per cent) obtained by collecting research test tubes with numbers from 111 to 138. Faction SPX3 and SPX4 have a relatively high degree of purity, showing a single spot on thin layer.

As shown in experimental example 1, fraction SPX3 demonstrates a 60% degree of ingibirovany the tumor growth after 15 days after injection. In contrast, SPX1, SPX2 and SPX4 not demonstrate any action, and, thus, it can be assumed that the substance which gives a blue coloration with sulphuric acid, is an ingredient with anti-tumor activity. This fraction SPX3 can be used as an antitumor agent by itself.

Example 3. The allocation of SB365

To highlight the pure substance from the fraction SPX3 spend HPLC as follows.

As stationary phase used reversed-phase silica gel (RP-C18, 250×10 mm production Metachem), and as the mobile phase used a mixed solution of methanol and water (80:20). The wavelength of detection is 210 nm, flow rate 1 ml/min. and the Result is shown in figure 3. As shown in figure 3, SPX3 consists of 3 main substances. From the obtained fractioned quantities peaks at Rf8.5 minutes and 10.4 min contain small amounts of ingredients, and the peak at Rf23,3 min contains the main ingredient. Thus, assume that the latter has antitumor activity. As described above, collecting the substance with Rf23,3 min, which gives a blue coloration with sulphuric acid, and is an active ingredient, to obtain 2.8 mg SB365 of 31 mg SPX3. The collected fraction at Rf23,3 min, dried and subjected to HPLC under the above conditions to determine the pure is s. The result is shown in figure 4. Figure 4 confirms that SB365 is a pure substance. Received SB365 directly used for structural identification and investigation of antitumor activity below.

As shown in experimental examples 1 and 2, SB365 shows 81% and 82.1% of the degree of inhibition of tumor growth in BDF1 mice transplanted with cells LLC and naked mice transplanted with cells NCI-H23, respectively, we can say that this is an excellent antitumor activity.

Example 4. Identification and confirmation of the structure of the active compounds SB365

Highlighted above connection SB365 is a white amorphous substance with TPL 239-241°and [α]D+23,6° (c, 0,2, MeOH) and positive reaction Libermann-Buchard and, therefore, it is confirmed that it is a glycoside. In addition, the IR spectrum (cm-1) see peaks at 3400 (broad, OH), 2940 (broad, C-H), 1695 (C=O), 1455, and 1040 (C-O). The substance is considered a glycoside, on the basis of absorption peaks in the range of 1000-1100 and 3000-3400.

According to1H-NMR substance has an NMR spectrum typical of saponins. Six groups-CH3see if 0,91, 0,92, and 0.98, and 1.00, 1.07 and to 1.21 ppm and another group-CH3see in the form of a doublet at 1,64 ppm Of the spectrum can be seen that the compound contains one group ramnose in their sugar groups is H. Anomeric protons see when and 6.25 (broad), 5,11 (1H, J=7,80 Hz) and equal to 4.97 ppm (1H, J=6,66 Hz). Thus, SB365 confirmed as a glycoside having three sugar group. According to13C-NMR hydroxymethylene group see when 65,4 ppm (C-23) and three signals of anomeric carbons see if 104,2 (C-1'), 106,7 (C-1"') and 101.7 ppm (C-1'). Two olefinic carbon see if 122,5 ppm (C-12) and 144,8 ppm (C-13) and one carboxyl carbon see if 180,2 ppm (C-28). In General, if sugar is connected at 28 to the position (180,2 ppm → 176,2 ppm), is high field shift of the glycosylation of about 4 Hz. In this connection, the above phenomenon is not observed and, therefore, confirmed that the compound has no sugar group in the 28th position.

Then the connection hydrolyzing in a mixture of ethanol/sulfuric acid for the identification of its sugar groups and the structure of the aglycone. SB365 confirm as hederagenin after comparing physico-chemical data of the hydrolysis product of the aglycone, data13C-NMR and1H-NMR. In addition, comparative TLC confirmed that the hydrolyzed sugars are rhamnose, arabinose and glucose.

On the basis of the above results and data published in the literature confirm that SB365 is hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside.

1H-NMR and13C-NMR of compound SB365 shown in the following table 1.

Table 1
Position1N (ppm)J (Hz)13(Ppm)Position1N (ppm)J (Hz)13(Ppm)
S-138,9Arabinose
C-226,1C-1'equal to 4.97 d6,66104,2
C-33.28 d10,981,0C-2'80,4
C-443,5C-3'75,4
C-548,1C-4'76,2
S-618,1C-5'63,9
32,8The rhamnose
S-839,7C-1"6,25 cm.101,7
S-947,8C-2"72,3
C-1036,9C-3"72,4
S-1123,9C-4"74,1
C-12the 5.45122,5C-5"69,6
S-13144,8C-6"1,645,9418,6
C-1442,1Glucose
S-1528,3C-1"'5,11 d7,80106,7
S-1623,8C-2"'75,0
C-1746,2C-3"'78,5
S-1841,9C-4"'71,2
S-1946,4C-5"'78,8
S-2030,9C-6"'62,5
S-2134,2
S-2233,2
S-234,36, to 3.67Overlap65,4
S-241,0714,0
S-250,9116,0
S-260,9817,4
S-271,2126,3
S-28-180,2
S-290,9232,8
S-301,0023,7

Example 5: the antitumor activity BDF1 mice transplanted with cells LLC

In this experiment used mice BDF1, healthy male mice with body weight 18-25, these animals provide food and water without restrictions in place with the temperature controlled in the range of 23-24°and fed food for mice without antibiotics. The LLC cells grown subcutaneously in C57BL/6 mice for 14 days. Select the fabric content is relevant cells LLC, and add cold sterilized saline (5 ml/g tissue), receiving the cell suspension. 0.2 ml of a suspension of cells transplanted subcutaneously in the groin area BDF1 mice.

At 24 h after transplantation of the above mice are divided into several groups consisting of 5 mice. Then the samples of WT fraction, fractions SPX and SB365 was dissolved in saline and injected intraperitoneally at a concentration of 280 mg/kg (WT), 70 mg/kg (SPX1), 171 mg/kg (SPX2), to 30.5 mg/kg (SPX3), 8,1 mg/kg (SPX4) and 6.4 mg/kg (SB365). Negative control group injected only with saline, and the positive control group injected with adriamycin (0.5 mg/kg). Injection designate, starting from 24 h after transplantation of the tumor injected samples consistently once a day for 7 days, stop the introduction for one day and then continue for 6 consecutive days.

To assess the toxicity of SB365 on mice experimental mice are weighed twice a week. Antitumor activity calculated after measuring the volume of tumors in the control and intervention groups at the 14-th and 15-th day after injection of the sample as follows:

Tumor volume (mm3) = length (mm) × width2(mm2)/2.

The degree of inhibition of tumor growth (%) = (s-T) × 100/S.

(From: mean tumor volume in the control group; T: average tumor volume in issled the emnd group). The results are presented in the following table 2.

Table 2

The degree of inhibition of tumor growth (IR, %) fractions Pulsatillae radix and SB365 on BDF1 mice transplanted with cells LLC
The fraction of connectionsThe number of miceThe degree of inhibition of tumor growth (%)
14-daya)15-daya)
WT55655
SPX151012
SPX252530
SPX355760
SPX45810
S36558279
Adriamycin56064
a)Days after the transplantation of tumor cells

As shown above in table 2, on the 15th day after the transplantation of tumor cells fractions of WT and SPX3 demonstrate the degree of inhibition of tumor growth 55% and 60%, respectively, and SB365 shows degree of inhibition of tumor growth of 79%, higher than adriamycin - 64%.

P the emer 6. Antitumor activity on naked mice transplanted with cells NCI-H23

In this experiment, used as experimental animals female Nude mice, 5 weeks of age weighing 16-25 g, obtained from Harlan Co. (USA). Mice used after aklimatyzacji for 1 week in an aseptic room for animals. In this room for the animals maintain a temperature of 22±2°C, humidity 55±5% and 12-hour light / dark cycle, which is regulated automatically. Solid feed for experimental animals, radio-sterilized, and drinking water is sterilized in an autoclave. Animals provide food and drinking water without restrictions. Use of cell line human tumor from the National Cancer Institute (NCI), USA and stored in Korean Research Institute of Bioscience and Biotechnology (KRIBB), Korea. Of human tumor cells, mice transplanted tumor cells lung cells NCI-H23. Tumor cells with a concentration of 3×107cells/ml transplanted mice subcutaneously at a volume of 0.3 ml/20 g body weight. Samples injected mice intraperitoneally every day for 13 days, namely, from the 1st to the 14th day, excluding the 8th day after the transplantation of tumor cells. Determine the size of the formed during injection of the tumor in each animal and also determine any changes in body weight. On the 16th day after the transplantation of tumor cells naked we shall kill her, and the tumor is isolated and weighed. Animals of the positive control group injected intraperitoneally adriamycin 0.5 mg/kg of body weight on the 1st, 5th, 9th and 14th day. The result is shown in the following table 3.

Table 3

The degree of inhibition of tumor growth (IR, %) by SB365 in Nude mice transplanted with cells NCI-H23
GroupThe degree of inhibition of tumor growth (%) NCI-H23
A negative. controlAdriamycin (0.5 mg/kg)SB365 (1.6 mg/kg)SB365 (3.2 mg/kg)SB365 (6.4 mg/kg)
The 16-daya)-61,540,152,382,1
a)Days after the transplantation of tumor cells

As shown above in table 3, SB365 in the amount of 6.4 mg/kg shows a high degree of inhibition of tumor growth 82,1% on the 16th day after the transplantation of tumor cells.

Example 7. The study of cytotoxicity

Tumor cells A549, SK-MEL-2 and MCF-7 receive from KRIBB and used in this experiment. A nutrient medium is prepared by adding sterilized distilled water for injection, one package of L-glutamine-containing RPMI1640 medium, 100 ml of serum PL is Yes cows (FBS), inactivated by heating on a water bath with a temperature of 50°C for 30 min, 2 g NaHCO3, 100,000 units of penicillin and 100 mg streptomycin, adjusting the pH of the mixture by means of HCl 0.1 N., bringing the total volume to 1 l and disinfecting mixture by filtration, and stored until use at a temperature of 4°C. Cells remain through breeding once in three days and to separate the cells from the walls using a solution containing 0.5% trypsin and 2% EDTA in physiological buffer solution (PBS).

Cytotoxicity of tumor cells are determined according to the method using sulforhodamine (SRB), NCI developed in 1989 to determine the in vitro antitumor activity of the drugs.

In particular, the cells are separated from the walls by using a 0.5% solution of trypsin-EDTA and then prepare a cell suspension 3-5×104cells/ml of the cell suspension is added to cells in 96-cell tablet (180 μl/cell) and incubated tablet in the incubator at 37aboutC, 5% CO2within 24 hours

The sample was dissolved in dimethyl sulfoxide (DMSO) and diluted with nutrient medium or three times with distilled water, and receiving appropriate for the experiment, the concentration, and make serial dilution to final concentration of DMSO is 0.2% or less. For each cell in 96-cell tablet add 20 ál of serially diluted solutions of samples and C is the incubated tablet in the incubator at 37° C, 5% CO2within 48 h At time Tz(zero time) adding the sample solution collected cells on the tablet. Remove the medium from Tztablet each tablet upon completion of incubation and added to the tablets of 10% trichloroacetic acid (TCA) (50 μl/cell). The obtained tablets leave to stand for 1 h at 4°for immobilization of cells on the bottom of the tablet. Upon completion of cell immobilization tablets washed 5-6 times with water to remove the remaining solution TCA and the resulting plate is dried at room temperature so that they do not contain water.

To completely dry the tablets add 50 ál of dye solution with 0.4% SRB in 1% acetic acid, staining the cells for 30 minutes the tablet Then washed 5-6 times with 1% acetic acid solution to completely remove SRB, not associated with cells. The tablets are dried at room temperature. Add 100 ál of 10 mm Tris solution to dissolve the dye. Then measure the OD (optical density) using a microplate reader at a wavelength of 520 nm.

The value of the ED50sample tumor cells [50% effective dose (ng/ml): concentration at which the growth of tumor cells is inhibited by 50%] calculated in the following way. The value of Tzdefined as the OD value at the start of the incubation after addition of the were acquired, the value of control (UAC) is defined as the OD of the cell not treated with the sample, and the value of T (test) is defined as the OD of the cell-treated sample. From the values of Tz, S and T determine the cytotoxicity of the agent according to the following formula:

in the case of Tz≥T, (T-Tz)/(C-Tz)×100,

in the case of Tz<T, (T-Tz)/Tz×100.

From the values calculated above are set ED50sample using the regression function of the Lotus.

As a result, the value of the ED50for SB365 on tumor cells of the human lung A, human melanoma cells SK-MEL2 and tumor cells of human breast MCF7 is >20 μg/ml >10 μg/ml and >10 μg/ml, respectively. Therefore, SB365 has low cytotoxicity on cells of solid tumors.

Example 8. Obtaining solution for injection containing fraction WT

250 mg of WT fraction obtained in example 1 dissolved in 10 ml of physiological solution, receiving the injection.

Example 9. Obtaining a dry powder for injection containing fraction SPX3

25 mg faction SPX3 obtained in example 2, dissolved in 10 ml of ringer's solution, sterilized and then dried by freezing, getting ready to use dry powder for injection. Before applying this powder to restore in distilled water for injection./p>

Example 10. Obtaining solution for injection containing S365

6,5 mg S365 obtained in example 3, dissolved in 10 ml of ringer's solution and sterilized, receiving the injection.

INDUSTRIAL APPLICABILITY

Fractions of WT and SPX3 Pulsatillae radix and SB365, hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside isolated from fractions according to the present invention not only possess a high degree of inhibition of growth of tumor cells of solid tumors, but can also be used in the usual manner by dissolving in a variety of solutions, including saline, ringer's solution or nutrient the solution, as they are easily soluble in water and have relatively low toxicity, reducing side effects of the previously developed anticancer agents. Thus, it is expected that they are very useful as therapeutic agents for solid tumors.

1. The use of water-soluble fraction of the extract of Pulsatillae radix containing hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside, as a means for the treatment of solid tumors.

2. The use according to claim 1, where the daily dose funds, based on active substance ranges from 200 to 300 mg/kg body weight.

3. The use according to claim 1, where the extract Pulsatillaeradix represents the fraction with R fin the range from 0.48 to 0.50 and gives a red coloration, and then blue staining after spraying with sulfuric acid followed by heating, and the specified fraction is obtained by extracting Pulsatillae radix with an aqueous solution of ethanol, with subsequent sedimentation of the precipitate with acetone to obtain water-soluble fraction and passing the fraction through a column of Sephadex LH20.

4. The use according to claim 3, where the water-soluble fraction of the extract of Pulsatillae radix is administered in a daily dose of from 20 to 40 mg/kg body weight.

5. The use according to any one of claims 1 to 4, wherein the water-soluble fraction of the extract of Pulsatillae radix, intended for the introduction, is dissolved in a solution selected from the group consisting of physiological saline, ringer's solution and the nutrient solution.

6. Application hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside as the active substance of the medicinal product for the treatment of solid tumors.

7. The use according to claim 6, where the daily dose of hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside ranges from 3.5 to 8 mg/kg body weight.

8. The use according to claim 6, where hederagenin 3-O-α-L-rhamnopyranose(1→2)-[β-D-glyukopiranozil(1→4)]-α-L-arabinopyranoside intended for the introduction, is dissolved in a solution selected from the group consisting of saline, the ringer's solution and the nutrient solution.



 

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2 cl, 6 tbl

FIELD: medicine.

SUBSTANCE: invention describes the use of 9-oxoacrydin-10-acetic acid, its pharmaceutically acceptable salts and compound ethers to induce or increase susceptibility of the prostate cancer to hormonal therapy, and the method of treatment of the prostate cancer where a patient is treated with sufficient injections of 9-oxoacrydin-10-acetic acid its pharmaceutically acceptable salts and compound ethers and with hormones aimed to reduce the impact of androgens. Also there is a method to induce or increase susceptibility of the prostate cancer to hormonal therapy aimed to reduce the impact of androgens by 9-oxoacrydin-10-acetic acid, its pharmaceutically acceptable salts and compound ethers.

EFFECT: prevents fast development of hormonal refraction which is the reason for unsuccessful treatment of prostate cancer.

26 cl, 3 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: this invention is related to treatment of diseases characterized by failures of signal transmission along the MAP kinase path, and the patient is treated with injections of isohinilin-based compounds. The use of isohilinin-based compounds is similarly described as a method of melanoma treatment.

EFFECT: method of this invention is primarily used to treat cancer types related to RAF kinase mutations, especially of melanoma.

4 tbl, 76 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention is a covalent conjugant or endoperoxyde related to artemizinin and ferriferous protein.

EFFECT: more efficient treatment of malignant tumor infections caused by pathogens.

23 cl, 2 ex, 1 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: novel micellar preparation is invented, which contains the anticancer agent with increased water-solubility, and which provides its high concentration in blood after the intravenous injection. The micellar preparation is produced from block copolymer with the general formula (1): where. R1 identifies the atom-methyl group; R2 identifies the tri-methyl group; R3 identifies the methylen group; R4 identifies the acetyl group; R5 identifies the hydroxyl group, optionally substituted by aryl C2-8alcoxyl group, substituted C1-4alkylamino group or amino group, containing the residue of amino acid or peptide derivative; n has integral values from 5 to 1000, m has integral values from 2 to 300, and x has integral values from 1 to 300; provided, percentage of hydroxyl groups in R5 is from 0 to 99%, and x has value not more than m; and taxane anticancer agent at concentration from 0.1 to 50 weight% per total mass of the agent and block copolymer.

EFFECT: preparation has high medical activity and reduced side effects.

7 cl, 4 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention presents several polymorphous forms and an amorphous form of {2-fluorine-5-[3-((E)-2-pyridine-2-ilvinyl)-1N-indazole-6-ilamine]phenyl}amide 2, 5-dimethyl-2N-pyrazole-3-carbonic acid, a pharmaceutical formula containing such polymorphous or amorphous forms, as well as ways of using such pharmaceutical formulas for therapy of ill conditions mediated by protein kinases such as cancer and other pathological conditions associated with undesirable angiogenesis and/or cell proliferation, as well as the mode of modulating protein kinase receptor activities on the basis of the polymorphous forms.

EFFECT: obtained polymorphous forms possess improved solubility and biological accessibility when taken per os.

22 cl, 40 dwg, 18 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention relates to the benzo-iso-selenium zolonyl derivatives with the general formula (I) or (II) , where R - C1-C6-alkylen, phenyliden, biphenyliden, R' - polysaccharide residue or residue , where M - Pt or Pd.

EFFECT: compounds possess anti-inflammatory, antiviral and antithrombotic activities.

12 cl, 7 ex, 8 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: invention relates to the pyrimidine derivatives with general formula I and their pharmaceutically acceptable salts, which possess tyrosine kinase ZAP-70, FAK and Syk-inhibiting activity, and to their application. The compounds can be used for treatment or prevention of diseases or conditions, where the activation of tyrosine kinase ZAP-70, FAK and/or Syk is involved, e.g., heart failure, chronic obstructive pulmonary disease, Alzheimer's disease et al. In general formula (I) , X is for CR0; R0, R1, R2, R3 and R4 - each identifies independently hydrogen; C1-C8alkyl; hydroxyC1-C8 alkyl; or R3 and R4 together with the adjoined nitrogen and carbon atoms create the 5-10-term heterocyclic ring and, besides, contain 1, 2 or 3 heteroatoms, chosen from N, O and S; or R1 , R2 and R3 each identifies independently the halogen; C1-C8alcoxy; hydroxyC1-C8 alcoxy; C1-C8 alcoxy -C1-C8 alcoxy; phenyl; 5-6-term heterocyclic ring; containing 1-4 heteroatoms, chosen from N and O; nitro; carboxy; -N(C1-C8alkyl)C(O)-C1-C8 alkyl; -CONR10R11; -SO2N(R10)R11; where R10 and R11 each identifies independently the hydrogen; hydroxy; C1-C8 alkyl; C2-C8alkenyl; C3-C8cycloalkyl; C3-C8 cycloalkyl -C1-C8 alkyl; C2-C8 alcoxy -C1-C8 alkyl; hydroxy C1-C8 alcoxy -C1-C8 alkyl; hydroxyC1-C8 alkyl; or 5-10-term heterocyclic ring, containing up to two heteroatoms, chosen from N and S; or R1 and R2 together with the adjoined carbon atoms create the aryl or 5-10-term heteroaryl radical, containing one or two heteroatoms, chosen from N, O and S; or R5 and R6 identifies independently from each other the hydrogen; halogen; cyano; C1-C8 alkyl; haloC1-C8alkyl; R7, R8 and R9 identifies independently from each other the hydrogen; hydroxy; C1-C8alkyl; haloC1-C8alkyl; C1-C8alcoxy; -Y-R12, where Y means the simple link or O and R12 means C1-C4alkyl, substituted or unsubstituted 5-, 6- or 7-term heterocyclic ring, containing 1, 2 or 3 heteroatoms, chosen from N, O and S; carboxy; -N(C1-C8 alkyl)-CO-NR10R11; - -N(R10)(R11); R7 and R8 or R8 and R9, correspondingly, together with the adjoined carbon atoms create the 5- or 6-term heteroaryl containing 1, 2 or 3 heteroatoms, chosen from N; or 5- or 6- term carbocyclic ring; or R7 means hydrogen, hydroxyl, C1-C4alkyl, unsubstituted or terminally substituted hydroxyl group; C1-C8alcoxy group, unsubstituted or terminally substituted by hydroxyl, C1-C4 alcoxy group or unsubstituted or substituted C1-C4alkyl 5-6-term heterocyclic ring, containing 1-3 heteroatoms, chosen from N and O; Provided, one R1, R2 or R3 means -CON(R10)R11; or -SO2N(R10)R11.

EFFECT: therapeutic efficiency is increased.

7 cl, 14 tbl, 184 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention can be used in medicine and pharmaceutical industry and relates to the cell proliferation inhibitor and to the anti-tumor agent, which contains the 3-phenylcinnolin analogue as an active component, and has the formulas , where J presents the A-C-B, where C is carbon, A is alcoxy, OH, acyloxy or amino acid residue, B is hydrogen, alkyl group, carbonyl group or = N-NH2 together with A, K - (CH2)q; L - N-W, where N -nitrogen atom, or W-C-W′, where C - carbon atom; W - alkyl group or hydrogen atom; W′ - alkyl, phenyl, carboxyl or alcoxycarbonyl group or hydrogen; J-K-L-M corresponds to C(O-Y)=CH-C(W)=CH or M - group (CH2)m; Z - oxygen; X - alcoxyl group, halogenated alkyl group, nitrogroup, cyanogroup or halogen; X' - halogen or hydrogen; m and q = 1, n and n' = 0 or 1, and (n + n') less than 2.

EFFECT: improved anti-tumor agent is available based on described compound.

82 ex

FIELD: medicine.

SUBSTANCE: composition containing the population of complexes of one or more heat-shock proteins (HSP) and antigenic peptides (AgP) are administered to the patient; in addition, at least one therapeutic agent, which doesn't contain the HSP is administered, to prevent or treat cancer. The population of complexes is received by use of the method, which provides the protein hydrolysis by one or more proteases and its decomposition by one or more non-enzyme methods, with obtaining the AgP population and formation of HSP with AgP complex. The protein agent is obtained from the cells with the same cancer antigenicity that should be treated or prevented in subject, and contains at least 50% or 50 different proteins of original antigen cells or cell fractions. The kit includes the containers with the described composition and treatment agent.

EFFECT: invention provides increased immune response in cancer and improved of therapeutic effect with reduced side action of treatment agent when used in combination with composition.

30 cl, 1 tbl

FIELD: medicine.

SUBSTANCE: vegetable raw is extracted, extract is mixed with vaseline and anhydrous lanoline. As a preliminary vegetable raw - white bryony roots - are crushed for fractions not more than 3 mm, defatted with mixture chloroform - ethanol (2:1) during 7-10 hours with further removal of dissolvent. Dried sovent cake is extracted with 70% ethanol at temperature 18-20°C, with proportions: extractor 1:2.0 during 144 hours in six diffusers; then at temperature 50-55°C vaseline and anhydrous lanoline are melted proportionally 85:10, mixture is cooled up to temperature 25-30°C; this mixture is combined with 5 portions of white bryony roots liquid extract.

EFFECT: drug without irritant odor.

2 tbl, 4 dwg, 8 ex

FIELD: medicine; medicine industry.

SUBSTANCE: laminaria powder with higher iodine content 0.2-0.35% is extracted from water at 89-90°C, mass proportion of laminaria powder-water is 1:3-1:5 during 120-180 minutes of mixing, mixture filtrating and combining received filtrate with food laminaria powder for pasty mass. This mass is dried until water content reaches 7-10%, dried product is crushed for fractions not more 120 mc.

EFFECT: iodine content product.

2 cl, 4 ex

FIELD: medicine; pharmacology.

SUBSTANCE: ointment for burns, dermatoses treatment and wound healing contains turpentine, beeswax, sea-buckthorn oil, cow's butter, pig's fat and biologically active substance selected from: aloe extract, plantain extract, beggars-ticks extract, rosehip extract, St. John's wort oil, linseed oil, calendula oil, bur oil, camomile oil, or propolis.

EFFECT: stimulating effect on regenerative processes in connective tissues and epithelium.

19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to new compounds of the formula (1a) or its pharmaceutically acceptable salt, esters or imides where A is a thiophenyl group containing, probably, substitution, the thiophenyl group A containing, probably, substitution with one or several groups as follows: alkyl, halo or arylalkyl, Y is O, S or NR2 where R2 is hydrogen or alkyl group containing 1 to 6 carbon atoms, and R1 is an non-ramified alkyl group containing 6 to 25 carbon atoms, ramified alkyl group containing 6 to 25 carbon atoms, aryl alkyl group where the alkyl group contains 2 to 25 carbon atoms or phenyl group containing substitution with one or several groups as follows: phenyloxy, phenylthio, SO2-phenyl, alkylphenyl, CO-phenyl, CONR16- phenyl, NR16CO-phenyl or NR16 -phenyl containing, probably, substitution where R16 is hydrogen or alkyl group containing 1 to 4 carbon atoms, the groups phenyloxy, phenylthio, SO2-phenyl, alkylphenyl, CO-phenyl, CONR-phenyl or NR-phenyl containing, probably, substitution with one or several groups as follows: halo, alkyl, alkylhalo or phenyl group containing substitution with one or several groups or alkyl groups provided the above compound is not 5-methyl-2-(4-metoxyphenyl)amino-4H-thieno[2,3-d][1,3]oxazine-4-on, 6-amyl-2-(4-chlorophenyl)amino-4H-thieno[2,3-d][1,3]oxazine-4-on or 6-amyl-2-(4-metoxyphenyl)amino-4H-thieno[2,3-d][1,3]oxazine-4-on Invention also relates to method of obtaining compounds of the formula (Ia) or (IIa), to pharmaceutical compound and application, as well as cosmetic technique.

EFFECT: obtaining of new biologically active compounds and pharmaceutical compounds based on them.

27 cl, 4 ex, 1 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: extract of the birch bark containing the specific amount of betulin is used in prevention and treatment of Parkinson's disease.

EFFECT: extract of birch bark is efficient for prevention and treatment of Parkinson's disease.

19 dwg, 7 tbl

Antiallergic agent // 2324491

FIELD: medicine; pharmacology.

SUBSTANCE: antiallergic agent is produced by toluene extraction of the birch bark and contains the active components such as betulin, lupeol and O-caffeate taken in particular quantities.

EFFECT: agent is efficient against allergy and doesn't reveal side effects.

11 tbl, 9 ex

FIELD: medicine; veterinary.

SUBSTANCE: homeopathic drug Lyarsin is used as a pharmaceutical agent. 0.7 mL of Lyarsin per head is administered intramuscularly to the animals suffering from hepatosis, at the first trimester of pregnancy, twice in 3 days.

EFFECT: method reduces number of stillborn puppies and increases brood per pupped dam.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: film dosage form sticking to mucous, containing at least on active substance from hemp. The form contains the polymeric matrix, which is used as a reservoir for the active substance and sticking to mucous. The form can have the multilayer structure. It can also contain the non-active ingredients, odorants or aromatisors. The film form is used in treatment of pain in such diseases as carcinomatosis, AIDS, trauma and other disorders.

EFFECT: invention provides better tolerance to administered substances and rapid onset of administered drug, simplifies administration procedures.

19 cl

FIELD: medicine.

SUBSTANCE: therapeutic-cosmetic agent for the external use contains pantohematogen, water, polymeric gel-forming component, as well as dimethyl sulfoxide and water. The components are taken in particular quantitative ratio.

EFFECT: agent shows stable efficiency in treatment of osteoarthrosis and spinal osteochondrosis.

7 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: therapeutic-cosmetic agent for the external use contains pantohematogen, water, polymeric gel-forming component, as well as dimethyl sulfoxide and water. The components are taken in particular quantitative ratio.

EFFECT: agent shows stable efficiency in treatment of osteoarthrosis and spinal osteochondrosis.

7 cl, 2 dwg

FIELD: medicine, oncology, amino acids.

SUBSTANCE: invention relates, in particular, to the development of an antitumor preparation based on natural substances. Invention relates to an amino acid preparation comprising at least one modified essential amino acid obtained by treatment of amino acid by ultraviolet radiation (UV) at wavelength 250-350 nm for 12-80 h at temperature 15-30oC or with ozone at temperature 15-25oC. The modified amino acid has no toxicity for health cells. Also, invention relates to a method for preparing such preparation. Invention provides the development of an antitumor preparation based on modified amino acids and expanded assortment of antitumor preparations being without cytotoxicity for normal cells.

EFFECT: valuable medicinal antitumor properties of preparation.

8 cl, 4 tbl, 2 dwg, 4 ex

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