JPH0653759B2 - Method for producing tumor cell growth inhibitory and tumoricidal cell factors - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a method for producing a novel component for inhibiting tumor cell growth and a novel tumor cell killing factor from human and animal platelets in a highly concentrated state. Regarding

(Prior Art) Currently, there is no complete drug treatment method for tumors, and despite the fact that many tumor therapeutic agents have been developed by many researchers all over the world, they still remain clinically surgical and radiological. The mainstream is the combined use of drugs with treatment methods. Tumor therapeutic agents are roughly classified into cancer chemotherapeutic agents and cancer immunotherapeutic agents.

On the other hand, recently, many studies have been published on physiologically active substances in platelets. The representative ones are as follows.

Wasteson et al. From platelets MW 28,000-34,000, PI9.
0-11.0 PDGF (Platelet-Derived Crowth Facto
r) was isolated and reported to have a cell growth promoting effect on smooth muscle cells, fibroblasts, etc. (Wasteson, Å. et a.
l: Biochem. J. 193 , 907 (1981), Kenneday, B.A. B
et al: J. B. C. 256 , 8896 (1981)). Lange et al. Obtained PBP (Platelet Basic Protein) from platelets and obtained MW 11,000 to 15,000 PI 9.0 to 11.0, Swiss
It is said to have a cell growth promoting action on 3T3 cells and the like (Lang
e. E. et al: Proc. Natl. Acad. Sci. USA, 77 , 5
914 (1980)). In addition, from platelets, casters C
TAP-III (Connective Tissue-activatng Peptide)
(Castep. CW et al: Proc. Natl. Acad. Sci.
USA, 80 , 765 (1983), Brown et al., PDECM (P
latelet-Derived Endoth-elial Cell Mitogen) (Bro
wn, T. et al = Proc. Natl. Acad. Sci. USA, 80 ,
1641 (1983)), and Spawn et al., TGF-β (Transf
orming Growth Factor β-type) (Sporn,
M. B. et al: J. B. C. 258 , 7155 (1983)), and all of them are found to have a growth promoting effect on various cells or an activity similar thereto.

Furthermore, the present inventors have called platelet-derived MW1 called JR-8403.
A novel antitumor iodide substance of 0,000-20,000 and pI 9.0-10 was discovered and a patent is pending (Japanese Patent Application No. 60-58789).

As described above, the presence of a component in the platelet extract that suppresses or kills the growth of human or animal tumor cells has not been reported except by JR-8403 by the present inventors.

(Problems to be Solved by the Invention) As described above, the currently used cancer chemotherapeutic agents are so-called cytotoxins, which exert an action by non-specifically suppressing the growth of cells, and therefore tumor cells Not only the normal cells but also leukopenia, infertility, hair loss, malformation,
Due to extremely serious side effects such as carcinogenesis, the amount used is strictly limited. Further, the cancer immunotherapeutic agent exerts a therapeutic effect not by directly suppressing tumor cells but by acting on a biological defense mechanism and indirectly suppressing tumor growth. Therefore, there are few serious side effects compared with cancer chemotherapeutic agents, but in many cases tumor patients do not have sufficient biological defense function, and their therapeutic effects are not always sufficient compared with cancer chemotherapeutic agents. It is expected that a therapeutic agent for a tumor that has less of these side effects and that selectively acts on tumor cells will be developed.

(Means for Solving the Problem) The present inventors have extensively investigated the effects of blood-derived substances on various animal cells, and as a result, they have a specific and direct effect on human and animal tumor cells in platelet extracts. , It was discovered that there is an active ingredient which exhibits a growth inhibitory effect or a cell killing effect, but does not give any change to normal cells. Furthermore, as a result of intensive studies on the present active ingredient derived from platelets, a novel tumor cell growth inhibitory and cytocidal factor (hereinafter referred to as JR-85) having physical properties such as molecular weight and isoelectric point which are different from those of JR-8403 described above.
Called 05. ) Succeeded in the manufacturing method probability. That is,
Platelets derived from various animals and humans are repeatedly frozen and thawed to destroy cells and granules, extracted with acidic water, and used as materials for gel filtration, ion exchange, reverse phase, isoelectric point separation, etc. It was discovered that the specific activity of JR-8505 can be increased several thousand times or more by combining the above, and the present invention was completed.

The present invention, after destroying platelets, is extracted with an acidic aqueous solution or a water-organic solvent mixed solvent solution, and if desired, a fraction having a tumor cell growth inhibitory activity of an estimated molecular weight of 50,000 to 150,000 is collected by gel permeation method, and then, , Chromatographic molecular weight 50,000 ~ 150,000, isoelectric point 4.5 ~
5.3, positive for ninhydrin reaction, inactivated by trypsin, but has a tumor cell growth inhibitory effect that is not inactivated by heating in physiological saline at 60 ° C for 30 minutes and contact with 0.2N hydrochloric acid for 60 minutes or 70% formic acid for 30 minutes at room temperature A method for producing a tumor cell growth-suppressing and tumor-killing cell factor, which comprises collecting a fraction.

Human platelets were used as the main raw material in the examples of the present invention, but nonhuman animals such as rodents, cats, dogs,
Similar active ingredients can be found and used in platelets of warm-blooded animals such as cattle. Further, the indicator cells in the examples include human, rat, mouse, etc.,
From the effects on them, JR-8505 is considered to be a tumor cell growth inhibitory and cell killing factor with little species specificity.

JR-8505 obtained by the present inventors differs from various substances conventionally extracted from platelets in physical properties and physiological activity and is considered to be a novel physiologically active substance.

In order to obtain JR-8505, for example, the extraction operation of platelets whose cells are disrupted by freezing, thawing or by a homogenizer is carried out with an aqueous solution of an inorganic or organic acid or a water-organic solvent mixed solvent solution. Preferable examples thereof include 1M-acetic acid, 1M-acetic acid-containing 75% ethanol, 0.1N-hydrochloric acid-containing 75% ethanol and the like. JR-8505
Can be extracted with a relatively strong acidic aqueous solution or an aqueous or hydrated organic solvent without deactivation, and contaminant proteins and the like can be easily removed. A clear extract can be obtained from the extract by combining it with centrifugation and filtering with a filter having pores of about several μm. When the extract contains an organic solvent such as alcohol, it is preferable to use a solvent precipitation method using alcohol or the like, and by combining precipitation with ether, ethanol, acetone, isopropyl alcohol, petroleum ether, ligroin, etc., JR-8505 Can be precipitated. A preferred example of the combination of solvents is ether and ethanol.

The precipitate thus obtained is taken to obtain an acidic aqueous solution.

If desired, the extract can be used as a carrier for biogel (BioG
el) P-100 (manufactured by Bio-Rad, USA), Sephadex G-150 (manufactured by Pharmacia Japan) and the like are used for gel filtration.

The activity of JR-8505 can be identified by the test method described below, and the extract or the active fraction collected by gelling the extract is subjected to at least one of ion exchange chromatography, reverse phase chromatography and chromatofocusing. The JR-8505 active ingredient is collected by operation.

In the ion exchange chromatography, both an anion and a cation exchanger can be used, and the base material can be either a natural or synthetic polymer. Examples thereof include QAE- and DEAE-Sephadex, SP- and CM-Sephadex, Sepharose, MonoS and MonoQ HR16 / 10 (Pharmacia Japan), Biorex 70 (USA, BioRad) and the like. .

The acidic extract that has passed or has not passed through the above gel filtration is added to the column of the ion exchanger after pH correction. At that time JR-85
05 The condition is such that the activity is adsorbed on the exchanger, and then the elution is carried out by gradient elution with an aqueous solution of a neutral salt in which the adsorbate is successively increased in concentration. Any neutral salt can be used as long as it is water-soluble and has no cytotoxicity, but sodium chloride is most preferably used.

Reversed-phase chromatography is performed using a carrier having a hydrophobic group. Examples of the carrier include silica gel modified with an alkyl group such as Micro Bondapak (μBondapak) series (Waters, USA) and Synchropak RP-P series (Synchropak, USA), MCI. -GEL CHP-20P series (manufactured by Mitsubishi Kasei), Amber light (Amberl
ite) XAD series (Rohm and Haas, USA)
Examples of such polystyrene-based high-porous adsorption resins include Particularly preferred among these are Syncropack RP-P (C ₁₈ ), MCI. CHP-20P and the like.

The acidic extract that has passed through the gel or not is added to a column containing a carrier to adsorb JR-8505. Next, the adsorbate is gradient eluted using an aqueous solution of a hydrophilic organic solvent whose concentration is gradually increased. Examples of hydrophilic organic solvents include methanol, lower aliphatic alcohols such as isopropyl alcohol, lower aliphatic ketones such as acetone, lower aliphatic nitriles such as acetonitrile, and preferred examples are acetonitrile and isopropyl alcohol. Is.

As an example of chromatofocusing, MonoP column is used for FPLC system (Pharmacia, Japan)
Done in. As an example of chromatofocusing, a sequential pH gradient system is formed in a MonoP column by Polybuffer 74 (Pharmacia, Japan) containing Pharmalyte 4-6.5 (all are manufactured by Pharmacia, Japan) and sequentially eluted according to the isoelectric point. It

The fraction of the eluate obtained by chromatography such as ion exchange, reverse phase chromatography or chromatofocusing is tested for JR-8505 activity by the test method described below, and an effective fraction is collected. JR by ion exchange, reverse phase chromatography or chromatofocusing
Any of the purification operations of -8505 may be used, and desirably, two or more of these operations may be combined.

The JR-8505 thus obtained is a white powder and has a positive ninhydrin reaction, a molecular weight of 50,000-150,000 (gel filtration method), and S
It is single in DS electrophoresis and has an isoelectric point of 4.5-5.3.

(Effect of the invention) The highly purified JR-8505 according to the present invention has a possibility of becoming an epoch-making drug in drug therapy for cancer patients and the like, and can enhance a biological defense mechanism for preventing degeneration of normal cells. You can also expect sex.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these.

Example (1) Platelets derived from 10 L of fresh human blood were frozen and thawed 3 times.
After repeating 120 times, 1M-acetic acid aqueous solution (120 m) was added and the mixture was stirred for 1 hour, and then centrifuged at 11,000 xg for 60 minutes to remove insoluble matter and freeze-dried (1.96 g). 20 to this dry powder
m 1M-acetic acid aqueous solution was added, the mixture was placed in a homogenizer at room temperature and frozen and thawed twice.
Centrifuge for 5 minutes to obtain the extraction supernatant
m 1M-acetic acid aqueous solution was added and re-extracted, and the supernatant was combined with the above extraction supernatant. Sephadex G-150 was packed in a column (volume: 7.2 × 100 cm) and equilibrated with a 1 M-acetic acid aqueous solution, and then the above extract was placed on the upper surface of the resin and eluted with a 1 M-acetic acid aqueous solution by a descending method (flow rate 60 m / hour, Temperature 4 ° C).

The elution curve is shown in FIG. In the figure, all markers (1), (2), (3), (4) and (5) with arrows are manufactured by Pharmacia and Japan (see the following table).

The elution position of the JR-8505 active component was at an estimated molecular weight of 50,000 or more and 150,000 or less, and an active fraction of 267 m was collected.
This was freeze-dried to obtain 206.5 mg of dry powder. In the JR-8505 activity test described later, the 10 μl fraction derived from this active elution fraction showed a significant growth inhibitory effect as compared to the control of the target _L929 cells.

Example (2) Platelets obtained from fresh fresh blood 5 were disrupted by freezing and thawing twice, and 75% ethanol water containing 0.1N-hydrochloric acid was added thereto for 200 m and stirred at 5 ° C. for 2 hours. After that, centrifuge at 10,000xg for 45 minutes,
1.0M-acetic acid buffer solution pH 5.5 (20 m) was added to further adjust the pH to a final pH of 5.5. After standing at 5 ° C. for 2 hours or more, centrifugation was performed again at 10,000 × g for 60 minutes at 5 ° C.
A 5-fold volume of an equal volume mixture of ether and ethanol was added to the supernatant, and the mixture was allowed to stand at -20 ° C for 2 days and nights. Next, centrifugation was performed at 12,000 xg for 60 minutes at 0 ° C, and the precipitation was carried out in Examples.
Extraction was performed with a 1 M acetic acid aqueous solution in the same manner as in (1) to obtain 18.1 m of an extract. . Sephadex G-150 was packed in a column (capacity: 2.4 × 100 cm), equilibrated with 1M-acetic acid aqueous solution, and the above extract was placed on the upper surface of the resin, followed by 1M- by descending method.
Elute with aqueous acetic acid (flow rate 15.0 m / hr, temperature 5
C). JR-8505 Active ingredient has an estimated molecular weight of 50,000-150,00
Elution at position 0 gave an active fraction of 180m,
When subjected to the JR-8505 activity test described below, the target tumor cells were significantly inhibited in growth as compared with the control.

Example (3) The JR-8505 active fraction 167m obtained in Example (1) was filtered with a membrane YM-10, which was a membrane with a molecular weight cut of 10,000.
(Amicon) was used to perform salt exchange, and finally a 20 mM-phosphate buffer pH 7.5 solution of 152 m was prepared. DE
AE-Sephadex A-25 on a column (2.4x50c
m), equilibrated with 20 mM-phosphate buffer pH 7.5,
The above-mentioned JR-8505 active ingredient was adsorbed on this, washed thoroughly with the same buffer, and then gradient elution was carried out with a 20 mM-750 mM sodium chloride solution (flow rate 25 m / hr, 5 ° C.). The elution curve is shown in FIG. JR-8505 activity is 400-650 mM
Elution was performed at the position of the sodium chloride solution, and an active fraction of 108 m was captured. This fraction was dialyzed against water and then freeze-dried to obtain 7.4 mg of dry powder. This active fraction 10μ
The origin showed a significant growth inhibitory effect on the target tumor cells in the JR-8505 activity test.

Example (4) To the powder (7.4 mg) obtained by freeze-drying the JR-8505 active fraction obtained in Example (3), 6 m of 0.025% trifluoroacetic acid aqueous solution was added and homogenized at room temperature. This was frozen, thawed, and centrifuged at 12,000 xg for 30 minutes to obtain an extract. The centrifugal precipitate was further washed with 2 m of 0.025% -trifluoroacetic acid aqueous solution, re-centrifuged, and the supernatant was combined with the above extract. Silica gel to which an alkyl group (C ₁₈ ) is bonded, a column of Syncropack RP-P (1
0.0mm x 250mm) high performance liquid chromatography system (manufactured by Gilson, USA), equilibrated with 0.025% -trifluoroacetic acid aqueous solution, and then adsorbing 3m of the above extract through a column to adsorb 0.025% -trifluoroacetic acid. After washing with the liquid, gradient elution was performed with the same solution of 0-80% acetonitrile. The elution curve is shown in FIG. JR-8505
The active ingredient was eluted at the position of acetonitrile concentration 45-65%. This column operation was repeated 3 times, and the active fractions obtained by treating all the extracts were combined and freeze-dried to obtain 1.8 mg of white dry powder. Dissolve this product in 10m physiological saline, and dilute it 100 times.
When the activity test was carried out, the target tumor cells were significantly suppressed in growth compared to the control.

Example (5) Chromatofocusing was performed using 2.0 m of 10 m of the JR-8505 active fraction obtained in Example (4). MonoP pre-pack HR5 / 20 column (Pharmacia) was set in the FPLC system (Pharmacia), the JR-8505 active ingredient was adsorbed, and Pharmalite 4-6.5 (Pharmacia) was used, pH 3.5- 6.5
PH gradient elution was carried out between. JR-8505 active ingredient has a pH of 4.5-5.7
Was eluted at the position of to obtain an active fraction of 3.0 m. The elution curve is shown in FIG. Molecular weight cut of this active fraction 3,500
Was dialyzed against physiological saline for 2 days and night, and freeze-dried. The above operation was repeated to treat the JR-8505 active fraction obtained in Example (4) to obtain 0.35 mg of a white powder. This product has a molecular weight of 50,0
00-150,000 (gel pass), isoelectric point 4.5-5.3, ninhydrin reaction positive, physiological saline solution 60 ℃ for 30 minutes, or 100 ℃ 1 minute heat treatment and 0.2 at room temperature
The tumor / cell growth inhibitory activity was not lost even by the acid treatment in which N-hydrochloric acid was contacted for 60 minutes or 70% -formic acid for 30 minutes.

This product is trypsin (derived from beef pancreas, manufactured by Difco)
When it was treated with a concentration of 0.1 mg / m at 37 ° C. for 24 hours, its activity was almost lost.

Example (6) Action of JR-8505 on tumor cells.

KB cells (human nasopharyngeal cancer) that are tumor cells, HEP-2
Cells (human laryngeal cancer), HeLa cells (human cervical cancer), G
-361 cells (human malignant melanoma), K-562 cells (human leukemia), L-1210 cells (mouse leukemia), and L-929 cells (mouse connective tissue cancer) were previously cultured for 48 hours,
1 × 10 ^{4 of} each cell was cultured for 5 days in a 5% carbon dioxide gas stream at 37 ° C. in 1 m of Eagle's medium containing 10% calf serum. Sample-free control group and examples
The test group to which JR-8505 obtained in (5) was added (100 ng / medium m) was simultaneously cultured, and the number of viable cells not stained with trypan blue was counted under a light microscope every 24 hours. Moreover, the dose-response curve was created by conducting experiments by changing the amount of the sample used. One example thereof is shown in FIGS. 5 and 6.

Example (7) Action of JR-8505 on normal cells.

Normal cells Flow7000 cells (human fetal foreskin), primary rat liver cells, primary calf kidney cells, primary chicken whole high cells were subcultured in advance for 2 or more generations, and 1x10 ^{4 of} each cell was 10%. In addition to 1m of Eagle's medium containing calf serum, JR-8505-free control group, Example
The test group was added to JR-8505 (4) and the group to which JR-8505 of Example (5) was added, and the cells were cultured for 5 days at 37 ° C. in a 5% carbon dioxide gas stream. The number of viable cells not stained with trypan blue was calculated by a light microscope on the 1st, 3rd, and 5th days. Based on the cells of the control group, the cell growth inhibitory effect of the sample was confirmed. In addition, JR-8505 (200 ng / m) obtained in Example (4) using primary cultured rat liver cells as target cells
Was used as the experimental group A, and the JR of Example (5) was used.
The results obtained for the experimental group B using −8505 (250 ng / m) as the sample are shown in FIG. Similarly, using JR-850 of Example (5), all primary high cells of chicken were used as target cells.
The results obtained with 5 (600 ng / m) as the sample are shown in FIG.

JR-8505 activity test method L-929 cells (mouse connective tissue cancer) are used as target cells. As the medium, an Eagle medium supplemented with 10% calf serum (0.1% supplemented with non-essential amino acid solution, manufactured by Dainippon Pharmaceutical Co., Ltd.) is used. To a 96-well multi-dish tray (Nunc, Denmark), 1 × 10 ⁴ target cells were added as a suspension of the above medium (150 μ), and a sample was added as a physiological saline solution (50 μ). To the control group, the same amount of physiological saline alone is added, and the cells are cultured in an open system in a 5% carbon dioxide gas stream at 37 ° C. and 100% humidity. After culturing for 2 days, trypsin treatment and trypan blue staining are performed, and the number of unstained viable cells is counted under a light microscope. The number of viable cells in the sample-added group relative to the control group is counted, and the 50% -growth inhibition value is calculated from the ratio. The reciprocal of the dilution ratio of the sample (50 μ) showing the 50% -growth inhibition value was calculated by JR-
8505 activity, 1 unit. (J. Biol. Chem. 259 , 686.
-691 (1984) Aggarwal, B .; B. et al. reference)

[Brief description of drawings]

FIG. 1 shows the elution curve of gel permeation (1M-acetic acid aqueous solution) using Sephadex G-150 of Example (1), and FIG. 2 uses DEAE-Sephadex A-25 of Example (3) as a carrier. Elution curve of gradient elution chromatography with 20 mM-750 mM sodium chloride aqueous solution, FIG.
Elution curve of gradient elution with 0-65% acetonitrile (in 0.025% -trifluoroacetic acid) in high performance liquid chromatography using silica gel bonded with an alkyl group (C ₁₈ ) of (4), Syncropack RP-P, Figure 4 shows the pH using Pharmalite 4-6.5 in Example (5).
Graded elution curves, Figures 5 and 6 are examples of
The results of the activity test (target cells: _L929 ) of the factor (100 ng / m) of the present invention obtained in (5) and the dose action curve are shown in FIG. 7. Experimental group A shown in Example (7) [Example Activity test for normal cells of factor (200 ng / m) of the present invention obtained in (4) and experimental group B [factor of the present invention (250 ng / m) obtained in Example (5)] (target cells: primary culture As a result of rat liver cells, FIG. 8 shows that in Example (7), normal cells (target cells: primary-cultured chicken fetus) using the factor (600 ng / m) of the present invention obtained in Example (5). The result of the activity test for cells is shown.

Claims (3)

[Claims] 1. After destroying platelets, extraction is performed with an acidic aqueous solution or a water-organic solvent mixed solvent solution, and if desired, a fraction having an estimated molecular weight of 50,000 to 150,000 and having a tumor cell growth inhibitory action is collected by gel filtration, and then the fraction is collected. , Chromatographic molecular weight 50,000 ~ 150,000, isoelectric point 4.5 ~
5.3, positive for ninhydrin reaction, inactivated by trypsin, but has a tumor cell growth inhibitory effect which is not inactivated by heating in physiological saline at 60 ° C for 30 minutes and contact with 0.2N hydrochloric acid for 60 minutes or 70% formic acid for 30 minutes at room temperature A method for producing a tumor cell growth-suppressing and tumor-killing factor, which comprises collecting a fraction. 2. The method according to claim 1, wherein the gel filtration is carried out under strongly acidic or neutral conditions. 3. Chromatography is (1) ion exchange chromatography in which an active ingredient is adsorbed on a cation exchanger, and then gradient elution is carried out using a concentration gradient aqueous solution of a neutral salt, (2) having a hydrophobic group. Adsorb the active ingredient on the carrier,
Next, reverse-phase chromatography with gradient elution using a hydrophilic neutral organic solvent, and (3) Chromatofocusing in which the active ingredient is adsorbed on a cation exchanger and then sequentially eluted in isoelectric point order with a pH gradient eluent. The method according to claim 1, wherein the method is any one of, or preferably, two or more of them are combined.