GB2161813A - Anti-tumour glycoprotein - Google Patents

Abstract

An anti-tumour glycoprotein having a molecular weight of 30-100,000 and an isoelectric point of 3.5-5.5, which may be obtained from Human T Cell Leukemia Virus infected Human Cell and Murine Leukemia Virus infected Rat Cell.

Description

SPECIFICATION Anti-tumor agent Background of the invention The present invention relates to anti-tumor agent, containing the sugar protein as the principal ingredisnt that causes the cell functions to tumor cells particularly. Cells arising from Rat Fetal Lung causes the fusion from outside and the fusion from inside by Murine Leukemia Virus. It is consided that the fusion protein which causes the cell fusion is the sugar protein which exists Rat Fetal Lung Cell be infected by Leukemia Virus.

Pharma activity of the sugar protein was zealously examined in the present invention, it was confirmed that the sugar protein particularly causes the cell fusion to the tumor cells, further causes tumor cells growth disability, and then the present invention have reached to the present invention.

Summary ofthe invention It is an object of the present invention to provide anti-tumor agent having particular cell fusion faclties to the tumor cells as the principal ingredient and excellent the tumor cell growth disability acts.

It is an another object of the present invention to provide anti tumor agent which is not in the venom of that oth of acuter and chronie in administrating. An object of the above-mentioned present invention is the sugar protein which is consisted of 30,000 100,000 of molecular weight and 3.5-5.5 of the isoelectric point, such the sugar protein is combined N-acetyl-D-Galactosamine, and is attained with the matter mainly consisting or Rectine which causes the cell fusion.

Brief description ofthe drawings Figure 'shows a grrph, presenting the relationship of the growth inhibition to the protein concentration of anti-tumor agent in the present invention, Figure 2 shows a graph, presenting the relationship of the grwoth inhibition to the temperature of said anti-tumor agent, Figure 3 shows a graph, presenting the relation ofthe growth inhibiton to pH of said anti-tumor agent, Figure 4 shows a graph, presenting the results of the cell disturbance facluties test which is shown in the experiment 7, and FigureS shows a graph, presenting the resu Its of the cell disturbance faculties test which is shown in the example 15.

Detailed description ofpreferred embodiments It is possible to get the sugar protein which is effective components of anti-tumor agent in the present invention. That is to say, one of the producing methods is to be acquired Human Lymphoma or the culture cell. Another one is to be acquired Murine Leukemia Virus to Rat Fetal Lung(RFL) Cell. In the either case of them, the guiding principle of these producing methods is the fusion faculties for Rat Fetal Lung (RFL) cell.

And a piece having high fusion faculties and low Leukemia Virus producing faculties should be screened.

Cells obtained by screenting is cultured, and that cultured cells are freezed, melted and broken. Broken cells are given the salting out by ammonium sulfate, the precipitated protein which is said salting out object is centrifuged, and is made up to the soution agent, and then ammonium sulfate is removed by dialyzing, and further components such as broken cells film etc. are removed by centrifuging. And then the supernatant luquid is obtained. Such obtained supernatant liquid is purified by using CBH cephalos, ConCanavaline A Cephalos, DEAE Cephalos, and Gel electrophoresis, and purified supernatant liquid is separated, and then the sugar protein is obtained.

It is identified in the present invention that the sugar protein which is effective components for anti-tumor agent in the present inventon has 30,000-100,000 of molecular weight in gel electrophoresis analysis. Each characteristics of the sugar protein which is the principal ingredient of anti-tumor in the present invention, are shown as follows.

{Concervative property) Figure 1 shows the changes of the growth inhibition rate in the case of that the concentration of the protein was chaged variously and the concervative temperature was chaged to 40C and 37"C. And P388 was used for the protain in the case. As demonstrated in Figure 1, the lost-activity was not almost observed in a high concentration showing 95~100% of the growth inhibition (0.001 mg/ml, 0.0004 mg/ml) if it was within 1 day, even in the case of 4"C or 37"C. However the lost-activity was increased with getting low concentration (0.001 mg/ml, 0.00004 mg/ml).And also, even inder the condition of high concentration, about 80-90 have lost the activity in the case of 1 month concervative condition below 4 C.

(Thermal Stability) 0.00004 mg/ml pointing 50% of the growth inhibition of the protein in the present invention was examined, and the chages of the grwoth inhibition in the case of that each protein were heated at each temperatures for 10 minutes, is shown in Figure 2. As demonstrated in Figure 2, about 30~40% of the activity was lost under 4060'C heating, and about 70% of the activity was lost over 70"C heating.

zpHstabilityJ 0.00004 mg/ml pointing 50% of hyperplasia depression of the protein in the present invention was examined, and the changes of the growth inhibition in the case of that pH of the protein was chaged variously at 37"C, and the protein were cultured for 1 hour, is shown in figure 3.

As demonstrated in Figure 3, examined protein was the most stable in pH 7, and were lost 30~40% of the activity in sides of the acidity and alkaline. However, in the case of 24 hours culturing at 4"C, about 60% of the lost activity was observed at the side of acidity (pH 2, pH 5) and about 85% of activity was retained in the side of aikaline (pH 9), therefore, the lost-activity in the side of acidity was remarkable.

(Iso electric point) Measurements of isoelectric point were done by electrophoresis isoelectric point fraction process. The column of 110 ml capacity was used for this measuring. An electric shock is applied below about 1 00C, 300V, for 24 hours, by using Ampholyte (pH 3.5-10), and was fractionated per 1 ml. After measurements of pH and the protein, we have examined hyperplasia that isoelectric rate (pl rate) is aout 3.5-5.5.

According to the above-mentioned electrophoresis, the molecular weight was from 20,000 to 150,000. In the elvate, Murine Fetal Lung Cell is used as indication cell, the cell dusion act is examined as a guide to multi-core cells, and we have confirmed the cell fusion faculties. It was also observed that the suger protein particularly causes the cell fusion to tumor cells in vitro a2d also the cell growing disturbance from the result of examining the cell fusion faculties of obtained sugar protein in detail. And also we have tried to cause cancer to Murine Tigh Muscle by 3-Methyl Cholanthrene, and the intravenous injection of anti-tumor agent having the sugar protein in the present invention have been given to Murine Tigh Muscle.

The dates in which tumor -volume reaches its three times was 4.5 days in average, in non-injected group of the controlled. Furthermore, the the virulence of acute and chronic have not be observed in injecting anti-tumor agent in the present invention to normal murine. Anti-tumor agent in the present invention is possible to give in either orally or non-orally. In the case of orally giving, soft capsule, hard capsule, oral tablets, granular, minute granular, powder and liquid were used, and injection, drip infusion and various kind of administrations were used for non oral giving.

Anti-tumor agemt in the present invention is possible to be preparaed by using interface activity. And also anti-tumor agent in the present invention depends upon by desired treatment and treatment period, however it is considered that anti-tumor agent per 1 day in a full-grown person is usually 0.01 1000mg.

The present invention is discribed in the following experiments.

(Experiment 1) In this experiment, Human Leukemia Virus was acquired to Human Peripheral Blood Lymphocyte by the usual process. This Lymphocyte was devided, cells having cell fusion function was prepared as the guiding principle of fusion factor for cancer cells, and was chosen by the mixing cultivation. Hereafter called HR-1 as to the chosen cells.HR-1 cells were kept on a dedium containing Demethyl sulfoxide, at -80 C. Hr-1 cells 2x 10% pieces were suspended on 100 ml of a dedium containing 5% of Cow Fetal Body Serum added RPM 1640, and was made up to cell floating liquid. 2ml of cell floating liquid obtained in the above were inoculated to 100 ml of a dedium containing 5% of Cow Fetal Body Serum added RPM1640, and then were cultured for 4 days, at 37"C under the condition of 5% Carbonic Acid Gas mixed moistened air, in 175cmZ of slastic cultivation flask. After culturing, cells were gathered, rinsed and suspended by Phosphoric acid buffere solution (pH7.0) mixing physiological salt solution. And after that, the suspention was rapidly freezed and then was melted, cells were set to pieces.Secondly, Ammonium Sulfate was added until be saturated in the suspention of destroyed cells at a low temperature below 4"C, and the protein was precipitated. This precipitated protein was centrifuged by 20,000G for 30 minutes, obtained precipitates were suspended to PBS. And then a small amount of Ammonium Sulfate existing solution were removed by the dialysate, and again was centrifuged by 20,000G for 30 minutes, cell films, etc. were removed and the supernatant liquid was obtained.

Cell fusion act in this obtained supernatant liquid were checked, and it was confirmed that this supernatant liquid has the active matters. Accordingly, RFL cells were used for the indicated cells, obtained by a phase-contrast microscope, and appearance of multi-core cells were confirmed as the guiding principle.

{Experiment2) In this experiment, the supernatant liquid obtained in Experiment 1 was throughout RCA, Cephalos 4B Column which was prepared by RCA purified by Riunus Communis and Cephalos 4B, the non-adsorbed fracton was eluted by PBS, and then the adsorbed fraction was eluted by 0.2M Galactose PBS.

Such eluate was dialyzed through with PBS, Galactose was removed, and then the eluate was throughout Con-A Cephalos 4B Column, the non-adsorbed fraction was eluted by PBS. And after that, the adsorbed fraction was eluted by 0.2M Mannose PBS liquid.

The eluate was dialyzed through with PBS, and Mannose was removed fom the eluate. And then the sugar protein fraction was obtained. Cell fusion act of the sugar protein fraction was obtained as same as Experiment land it was confirmed that the sugar protein fraction has cell fusion fractions.

(Experiment 3) In this experiment, the supernatant liquid obtained in Experiment 2 was throughout Con-A Cepholos 4B colum, the non-adsorbed fraction was eluted by PBS, and then the adsorbed fraction was eluted by 0.2M Mannose PBS liquid. Such eluate was dialyzed through with PBS, Man nose was removed, and then the eluate was throughout RCA, Cephalos 4B Colum, the non-adsorbed fraction was eluted by PBS. And after that, the adsorbed fraction was eluted by 0.2M Galastose PBS. The eluate was dialyzed through with PBS, and Galactose was removed fom the eluate. And then the sugar protein fractions were obtained Cell fusion acts of the sugar protein fractions were observed as same as Experiment 1, and it was confirmed that the sugar protein has cell fusion faculties.

{Experiment 4) In this experiment, the sugar protein fraction obtained in Experiment 3 were throughout DEAE Cellulose Column, and the absorbed fraction was eluted by 2M NaCI.

The elute was dialyzed through with PBS, and the sugar protein fraction was obtained. Cell fusion acts of the sugar protein fraction was observed as same as Experiment 1, and it was confirmed that the sugar protein fraction has cell fusion faculties.

{Experiment 5) In this experiment, the sugar fraction obtained in Experiment 4 was throughout N-Acetyl-D Galactosamine Colum, and the adsorbed fraction was sluted by N-Acetyl-D Galactosamine. The eluate was dialyzed through by PBS, and the suger protein fraction was obtained. Cell fusion acts of the suger protein fraction was observed as same as Experiment a, and it was confirmed that the suger protein fraction has cell fusion faculties.

(Experiment 6J In this experiment, the sugar was detected by Shiff test from the sugar fraction obtained in Experiment 2,3,4 and 5, and protein was also detected by Coomassie brilliant blue. And also 12% of Acrylic Amide Gel (contains SDS) electrophoresis were done for 5 hours at 30mA. As that result, molecular weight was between 30,000 and 100,000.

{Experiment 7) In this experiment, constant values of cells were examined, these cells were cultured for 48 days under the condition of various kinds of concentrations of cell fusion factors, and then amounts of active cells were measured. Cells were cultured in lml/well of the cultured liquid, 2 wells per each dose were used in this Experiment. The cell growth inhibition rate is shown as follow.

Growth Inhibition % = cell ~ cell values in each testgroups x 100 cell values in the controlled groups Cells for experiment: (1) Normal Human Peripheral Blood Lymphocyte (2) JM Human Leakemia (3) Daudi (4) Balm 3 (5) CEM Figure 4 shows the results of tests in this experiment. Four kinds of used tumor cells have shown a higher susceptibity than normal lymphocyte.

(Experiment 8) In this experiment, RFL cell arising from WKA Rat Fetal Lung (reference GANN, 64,321--322(1973)) was acquired Moloney Murine Leukemia Virus by the usuai process. The concentration of the virus was 1,000-10,000 MOI for one RFL ceil. From this experiment, a lot of RFL cells have got the producing acts of the fusion factors.

These cells having cell fusion faculties were prpared as the guiding principle of fusion factor for cancer cells, and were chosen by the cell piling process. Cancer cells used for the guiding principle were XC, KB, FL, KC, RSb, HeLa, etc. Hereafter called RM-4 as to the chosen cells. RM-4 cells were kept on a dedium containing Dimethyl Sulfoxide, at -80 C. RM-4 cells, 2x107 pieces were suspended on 10ml of a dedim containing 10% of Cow Fetal Body Serum added Dulbecco Revolutionized Eagle, and was made up tp cell floating liquid.

Cytodexwas added to 10% of Cow Fetal Body Serum added Dulbeccorevolutionized Eagle, in Further8 10 ml ofthe above-mentioned cell floating liquid (5 x 105 1 x 106 cell/ml) were inoculated, and then RM-4 cells were cultured for 4 days, at 37"C, under the condition of 5% Carbonic Acid Gas mixed moistened air, in a plate having 1 Ocm of circumference. After culturing, cells were gathered, rinsed and suspended by Phosphoric Acid buffer solution (pH 7.0) mixing physiological salt solution (hereafter called PBS).

And after that the suspention was rapudly freezed and then was melted, cells were set to pieces.

Secondly, Ammonium Sulfate was added until be saturated in the suspention of destroyed cells at a low temperature below 4"C, and protein was precipitated. This precipitated protein was centrifuged by 12,000G for 30 minutes, obtained precipitates were suspended to PBS. And then a small amount of Ammonium Sulfate existing solution were removed by the dialysate, and again was centrifuged by 12,000G for 30 minutes, cell films etc. were removed and the supernatant liquid was obtained. Cell fusion acts in this obtained supernatant liquid were checked, and it was confirmed that this supernatant liquid has the active matters.

Accordingly, RFL cells were used for the indicated cells, observed by a phasepcontrast microscope, and appearance of the multi-cores cells were confirmed as the guiding principle.

(Experiment 9) In this experiment, the supernatant liquid obtained in Experiment 8, was throughout RCA, Cephalos 4B Column which was prepared by RCA purified by Ricious Communis and Cephalos 4B, the non-adsorbed fraction was eluted by PBS, and then the adsorbed fraction was eluted by 0.2M galactose PBS. Such eluate was dialyzed through with PBS, Galactose was removed, and then the eluate was throughout con-A Cephalos 4B Column, the non-adsorbed fraction was eluted by PBS. And after that, the adsorbed fraction was eluted by 0.2M Mannose PBS Liquid. The eluate was dialyzed through with PBS, and Mannose was removed from the eluate. And then the sugar protein fraction was obtained (hereafter called F-RM2). Cell fusion acts of F-RM2 was observed as same as Experiment 8, and it was conformed that F-RM2 has cell fusion faculties.

{Experiment 10) In this experiment, the supernatant liquid obtained in Experiment 8 was throughout Con-A Cephalos 4B Column, the non-adsorbed fraction was eluted by PBS, and then the adsorbed fraction was eluted by 0.2M Mannose PBS liquid. Such eluate was dialyzed through with PBS, Mannose was removed, and then the eluate was throughout RCA, Cephalos 4B Column, the non-adsorbed fraction was eluted by PBS, And after that, the adsorbed fraction was eluted by 0.2M galactose PBS. The eluate was dialyzed through with PBS, and Galactose was removed from the eluate. And then the sugar protein fraction was obtained (hereafter called F-MM3). Cell fusion acts of F-RM3 was observed as same as Experiment 1, and it was confirmed that F-RM3 has cell fusion faculties.

(Experiment 11J In this experiment, F-RM2 fraction was throughout DEAE Column, and the adsorbed fraction was eluted by 2M NaCI. The eluate was dialyzed through with PBS, and suger protein fraction was obtained (hereafter called F-RM4). Cell fusion acts of F-RM4 was observed as same as Experiment 8, and it was confirmed that F-RM4 has cell fusion faulties.

(Experiment 12J In this experiment, the sugar was detected by Shifftestfrom F-RM2, F-RM3 and F-RM4 obtained in Experiments 9, 10 and 11, and the protein was also detected by Coomassie brilliant blue. And also 10% of Acrylic Amide Gel electrophoresis were done for 2 hours at 3mA. As that result, molecular weight was between 20,000 and 150,000.

(Experiment 13J In this experiment, 5ml of F-RM2, F-RM3 and F-RM4 obtained in Experiments 9, 10 and 11, were suspended with 20ml of the physiological salt solution was controlled to OD230 = 0.4, and then injectable solutions were obtained. These injectable solutions were named injectable solution 9, 10 and 11.

(Experiment 14) In this experiment, the intramuscular injection of 0.2 mg of 3 methyl coranthren was given to the thigh muscle of 4 weeks birth BALTIC mouse, tumors of which cancer was caused chemically, were caused on the time of onset.

After the intramuscular injection, tumors were grown up to a little piece, and got possible to touch.

Therefore, after caused on the time of onset, four groups were devided by turns in 20 mice per one group.

The first one group was not injected as the control. In the second to fourth groups, 0.1 ml of injections 9, 10 and 11 obtained in Experiment 13, was given to tails as intravenous injections. Tumors inhibition effects were rated by three times of days of tumors. Table 1 shows that results, and also shows survival values of 10 days after tumors touching. Accordingly, it was confirmed by inspecting that both survival and death examples of tumors were maligrant fribrous Histiocytoma.

TABLE 1 Group Tumors inhibition effects (day) Survival values (60th day) First 4.5 0/20 Second 38 3/20 Third 41 3/20 Fourth 28 2/20 (Experiment 15J In this experiment, constant values of cells were examined, these cells were cultured for 48 days under the condition of various kinds of concentrations, and then amounts of active cells were measured. Cells were cultured in 1 ml/well of the cultured liquid, 2 wells per each dose were used in this experiment. The cell growth inhibition rate is shown as follow.

Growth inhibition % = Cell ~ Cell values in each test groups x 100 Cell values in the controlled groups Cells for experiment: (1) P388 Lymphoma DBA/2groups Murine same group transplaned tumors (2) L1210 Lymphoma DBA/2groups Murine same group transplaned tumors (3) L5178Y Lymphoma DBA/2groups Murine same group transplaned tumors (4) P815 Mastcytana DBA/2groups Murine same group transpianed tumors (5) B-16 Melanoma C57 BU6groups Murine same group transplaned tumors (6) Golden Hamster Fetal Cell (7) Normal Human Peripheral Blood Lymphocyte (8) Balm 3 Lymphoma Human root cells (9) Daudi Lymphoma Human root cells Figure 5 shows the results of tests in this experiment. Seven kinds of used tumor cells have shown a higher susceptibility than normal lymphocyte and normal Hamster Fetal Cells.

(Experiment 16J The effect of F RM4 which have exerted to the purified cell membranes and Lyso zone, wes observed in this experiment. Various kinds of cells were destroyed, cell membrances and Lyso zone were purified, F RM4 was added, and then the liberation into the cultured liquid of 5' mucleotidase acid phosphatase was continuously observed. The fusion factors as the present invention have caused a melting of the purified cell and Lyso zone. This reacts were caused very fast for 30 minutes by acting the fusion factors. And also in this reacts, the cell membramces and Lyso zone of the tumors cells have shown higher susceptibility than normal cells.

(Experiment 17J This has been practiced on the basis of the screening procedure of NCI. Female BDF, Murine (weight:12 t 1g) was used for hits experiment. Six murines units for one group and ascites type for tumors were used for this experiment. P388: 1 x 1 cells / mouse, L1 210:1 > c 1 105 cells/mouse. The treatment was started from Day 1 as measured by tumors explants day set Day 0. The route for administration ws in abdominal cavity.

An amount for administration was 0.1 ml/mouse (0.04 gum and 0.004W9). Two kinds of treatments which are the route of once and the route of every day, were done in this experiment.

TIC (survival duration on the average of the treated group/survival duration on the average of the contralled group) '125% I have evaluated effective when the above-mentioned results were obtained. As shown in Table 1, F RM4 is frequently used for NCI in vitro and in viro, and/or the first screening of minimizing cancer in Japan. And also F RM4 have shown remarkable effect for P388 and/or L1 210.

TABLE 1 Effects of F-RM4 on survival of tumor bearing murine.

Tumot Sample Dose No. ofmice mean TIC reroute) {route) OD280 mibody survival x day days control - 6 7.67 1.00 P388 F-RM4 0.04 x 1 6 12.50 1.63 p. (i.p.) 0.004x 1 6 10.17 1.33 0.04 x5 6 15.17 1.98 0.004 x 7 6 10.67 1.39 control - 6 8.00 1.00 L1210 F-RM4 0.04 x 1 6 10.50 1.31 i.p. (i.p.) 0.004 x 1 6 10.00 1.25 0.04 x5 6 9.00 1.13 0.004 x 7 6 8.83 1.10

Claims (4)

1. Anti-tumor agent consists of; 30,000-100,0000 of molecular weight by gei electrophoresis analysis, the sugar protein having 3.5 - 5.5 of isoelectric point and particular cell fusion faculties in tumor-cells which is effectively consisted of rectine combining with N-Acetyl-D-Galactosamine.

2. Anti-tumor agent according to claim 1, wherein said sugar protein is obtained from the cell films of the animal cells.

3. Anti-tumor agent according to claim 2, wherein said animal cells are the Human cell which acquired the Human T Cell Leukemia Virus.

4. Anti-tumor agent according to calim 2, wherein said animal cells are the Rat cells which acquired Murine Leukemia Virus.