JPS5939829A - Method for stabilizing tumor necrotic factor - Google Patents

Abstract

PURPOSE:To prevent the deterioration in activity of a tumor necrotic factor useful as a carcinostatic agent in the step of separating, purifying, freeze-drying the tumor nectotic factor, etc. and stabilize the factor, by adding a nonionic surfactant to the above-mentioned tumor necrotic factor. CONSTITUTION:A nonionic surfactant is added to stabilize a tumor necrotic factor (hereinafter abbreviated to TNF). The TNF is specific for species, and unreactive with normal cells. Therefore, the TNF is expected as a carcinostatic agent, but the activity thereof is remarkably deteriorated by storing or freezing or freeze-drying the solution of the TNF in high purity. Thus, it is found that the nonionic surfactant having preferably 9-20HLB value from the viewpoint of the dispersibility and solubility is added thereto to suppress the deterioration in activity thereof. A polyoxyethylene ester of a fatty acid, may be cited as the above-mentioned surfactant, and the concentration thereof to be added is 1mug- 1mg, preferably 5-100mug, based on one ml solution containing the TNF.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing cancer necrosis factor (hereinafter abbreviated as TNF), and more particularly, to a method for stabilizing TNF during storage, separation and purification, freeze-drying, and other operations. It is something.

In the present invention, TNF is defined as [a method by which one or more substances having a reticuloendothelial system activating effect is administered to a mammal, and then an endotoxin derived from Durham-negative bacteria is injected, or an endotoxin derived from a mammal-derived A physiologically active substance that is induced by adding endotoxin derived from Durham's bacteria to a tissue culture system containing macrophages, and is defined as a "factor that causes necrosis of certain Yuzu cancers when inoculated into animals with bile cancer." It is. It is known that TNF not only causes necrosis in certain cancers, but also that its action is not species-specific.

For example, TNF obtained from rabbits can kill cancers in mice. Furthermore, TNF is in v
It is known that it has almost no harmful effect on normal cells in vitro and has the ability to kill certain types of cancer cells (for example, LM cells derived from mouse cancer).

As described above, 1' NF has an anticancer effect, is nonspecific to yuzu, and does not act on normal cells, so it is expected to be an anticancer agent.

Traditionally, TN induced in animals or tissue culture systems
It is known that a very small amount of FO accumulates. TN
In order to widely and safely use F'i as an anticancer agent, it is important to make it with 11 glazes, and long-term storage in a solution or frozen state, or freeze-drying, is important.
This is an essential operation when industrially manufacturing NF into large gongs.

However, the Ministry of the Invention and others found that when a TNF solution with a high purity is stored, frozen, or freeze-dried, the activity is significantly reduced. °Also, 1'N of purity
No report examining the stability of F has been published to date. Under these circumstances, despite its anticancer effect, it is impossible to efficiently and stably provide high-purity TNF on an industrial scale.

In view of these current circumstances, the present inventors have conducted extensive research into stabilizing TNF, and as a result, we have found that by adding a small amount of nonionic surfactant to TNF, which is inexpensive and easy to obtain, we have found that it can be stored for a long period of time. We discovered that the activity of TNF is maintained even after separation, purification, freeze-drying, and other operations.

The present invention relates to a method for stabilizing TNF, which is characterized by adding a nonionic surfactant to TNF.

Nonionic surfactants used in the method of the present invention
Since it does not have any adverse effect on the purity level assay of 1'NF and can accurately measure purified pure anti-filtrate, the present EiJJ method can be used to detect TNF during purification operations such as column chromatography and concentration, or during solution storage. The stabilization of 1/C is particularly suitable.

The T N p raw material used in the present invention is produced by a known method. As such, for example, Matt
hews et al., Br, J. Cancer, 42
(1980) 416, Green et al., J. Natl.
+Cancer In5t, 59 (1977) 1
There are 519 methods. The method will be explained below.

That is, an infusion of a substance that has a reticuloendothelial system activating effect on mammals (e.g. mice, rabbits, guinea pigs, etc.)
Or inject two or more transplants intravenously or intraperitoneally. Durham-positive bacteria, protozoa, or 40-mouth yeast are usually used as substances that have a reticuloendothelial system activating effect, and they can be used in live or dead conditions (for example, after TJ, heat treatment or formalin treatment), or bacterial cell extraction. Administered as a component.

Here, examples of Durham-positive bacteria include, for example, Propio
nibaeterium acnes (Coryne
bacterium parvutn), Propio
nibacterium granulosum (Co
rynel) acterium granulosum
) like AlPropionibacteria,
Bacillus Calmette-Guer
in (BCG), Mycobacterium srn
Mycobacteria, such as egmatis
Nocardia erythropolis.

NocarcNa gardneri like Noca
Examples include rdias. Examples of protozoa include 1, such as malaria parasites and Toxoplasma gondii. For yeast, usually Saccbaromyces cerevi
Zymosan extracted from S. siae etc. is used. Furthermore, synthetic polymer compounds such as bilan copolymers can also be used.

Endotogycin obtained from Durham-negative bacteria 7 to 14 days after administration of a substance having a reticuloendothelial system activation effect, for example,
Big Ill! -, Pseudomonas aeruginosa, and typhoid fever 1-derived polysaccharide are injected intravenously into the mammal's body. , the suckling M on the injection ridge! Contain body fluids (e.g., ascites, lymph fluid, etc.) and/or serum or plasma of JJ;
In addition, organs such as the liver and lungs of the animal are uniformly crushed.
, and extracted with physiological saline to obtain TNF raw material.

The method for producing the TNF raw material used in the present invention is not limited to the above. In other words, T
NF raw material production method can also be adopted.

The TNF raw material produced in this manner is separated and purified using a combination of conventional biochemical separation and purification methods. Examples of such methods include salting out using ammonium sulfate, ion exchange chromatography using a single cloud ion exchange resin, gel filtration, and electrophoresis.

When these methods are combined and the separation and purification process is carried out to reach all levels of purification, TNF gradually becomes unstable. For example, TNF contained in total protein with specific activity (1uv)
The activity and unit of activity will be described later) The TNF sample purified to 500,000 units/ly.
It is unstable. TNF test phases with specific activities lower than this also have their activity reduced to varying degrees by storage, freezing, lyophilization, and other operations. The object of the present invention is TNF that has become unstable due to the -L degree of purification.
and, in particular, a solution containing TN F.

I)J( of the TNF-containing solution used in the present invention is preferably maintained at 5 to 10, and more preferably adjusted with an appropriate buffer solution.

Such buffers include, for example, phosphate buffers,
tris (hydroxymethyl)
Examples include arninomethane)-hydrochloric acid buffer.

Depending on the purpose, salt may be added. Examples of the salts used include thorium chloride and potassium chloride, and the concentration thereof is determined depending on the purpose.

For example, when used for injection, thorium chloride i is added to a concentration of 0.15 M to make an isotonic solution.

Examples of the nonionic surfactant used in the present invention include polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene solpitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, Examples include hardened polyoxyethylene mustard oil, polyoxyethylene castor oil, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene polyoxypropylene block polymer, sorbitan aliphatic ester, sucrose fatty acid ester, and glycerin fatty acid ester. From the point of view of dispersibility and solubility, HL
B (Hydrophile LipophileBa
lance ) values of 9 to 20 are preferred, and more preferably those with an HLB value of 13 to 18 are used.

These can be used alone or in an appropriate combination of two or more.

Polyoxyethylene fatty acid ester has 8 to 2 carbon atoms.
It has a saturated or unsaturated fatty acid residue of 0, and the number of moles of ethylene oxide (hereinafter abbreviated as E, Ol) added is about 4 to
50 is preferred, and examples thereof include polyoxyethylene stearate, polyoxyethylene laurate, and polyoxyethylene oleate.

Polyoxyethylene alkyl ether has 8 or more carbon atoms
20 saturated or unsaturated alkyl groups, E, 0. Those having an added mole number of about 5 to 55 are preferred, and examples thereof include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, and polyoxyethylene oleyl ether.

Pholioxyethylene alkylphenyl ether has a saturated or unsaturated alkyl group having 5 to 10 carbon atoms, and E
,0. Those with a valence mole number of about 5 to 55 are preferable, such as polyoxyethylene p-octylphenyl ether and polyoxyethylene p-nonylphenyl ether.
'Can be done.'

Polyoxyethylene rubitan fatty acid ester has a residual saturated or unsaturated fatty acid having 8 to 20 carbon atoms, and has E
, O, the number of added moles is preferably about 5 to 50, for example, polyoxyethylene rubitan monolaurate,
Examples include polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan tristearate.

Polyoxyethylene glycerin fatty acid ester has a saturated or unsaturated fatty acid residue having 8 to 20 carbon atoms, and has E
,0. Those having an added mole number of about 5 to 50 are preferred, and examples thereof include polyoxyethylene glycerin monostearate and polyoxyethylene glycerin/monooleate.

Polyoxyethylene hydrogenated castor oil and polyoxyethylene castor oil u, E, O, number of added moles is about 20 to 150
Preferably.

The polyoxyethylene polyoxypropylene alkyl ether has a saturated or unsaturated alkyl group having 12 to 18 carbon atoms, and preferably has an E, O content of about 65 to 85, for example, polyoxyethylene polyoxypropylene. Cetyl ether is mentioned.

The polyoxyethylene polyoxypropylene block polymer has an average molecular weight of about 3,000 to 15,000 and an E, 0
．． A content of about 65 to 85 inches is preferred.

The sorbitan fatty acid ester is preferably a mono- or sesquiester of a saturated or unsaturated fatty acid having 8 to 20 carbon atoms, such as sorbitan monolaurate, sorbitan monopalmitate, and sorbitan sesquioleate.

The sucrose fatty acid ester is preferably a monoester of a saturated or unsaturated fatty acid having 8 to 20 carbon atoms, for example,
Examples include sucrose monopalmitate and sucrose monostearate.

The glycerin fatty acid ester is preferably a monoester of a saturated or unsaturated fatty acid having 8 to 20 carbon atoms, such as glycerin monostearate and glycerin heptanooleate.

Among the above nonionic surfactants, relatively low toxicity polyoxyethylene rubitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene castor oil, and polyoxyethylene polyoxypropylene block polymer are particularly preferred. Among others, polyoxyethylene (20) sorbitan monooleate, E, 0. Polyethylene hydrogenated castor oil with an added mole number of 60 and an average molecular weight of about 8350, E, 0. Preference is given to polyoxyethylene polyoxypropylene block polymers with a content of 80%.

The concentration of the surfactant added is 1 μt to 1 μt per 1 solution containing TNF, preferably 5 μV to 10 μ2
, more preferably 10μ2 to 100μ2. The method of addition is not particularly limited; for example, the surfactant may be added directly to the TNF-containing solution, or the surfactant may be dissolved in water, an appropriate buffer, or an organic solvent such as ethanol beforehand. Can be mentioned.

The addition time may be during the separation and purification process or during the formulation process.

A method of adding two or more surfactants is also included in the method of the present invention. In that case, the total amount of surfactants may be adjusted to the above-mentioned addition number.

It is preferable that the TNF-containing solution to which a nonionic surfactant is added is stored, separated and purified, and formulated at 0 to 30°C, more preferably 0 to 10°C, in its solution state. Furthermore, when storing the same solution in a frozen state, it is desirable to keep it at 0°C or lower, more preferably -20°C or lower.

In the TNF-containing solution to which a surfactant is added in the present invention, the TNF activity is maintained even during storage in a solution state or frozen state, or during separation and purification and formulation operations. On the other hand, anionic surfactants have a strong denaturing effect on TNFK, and cationic surfactants themselves have strong cell-modifying effects, making it impossible to accurately measure TNF activity. It cannot be used as a stabilizer.

There are two ways to measure the activity of TNF: one is to measure the tumor necrotic effect in vivo, and the other is to measure the cancer cell killing effect in vitro.

In vivo methods include, for example, Carswel
The method of l et al., Proc, Nat, Acad, S
ci, USA, 72 (1975) 5666. This method uses transplanted MetlIA sa
This test measures the effect of TNF on necrosis of rcoma tumors. That is, (BALB/cXC57B
L/6) 2 x 10S Meth A sarcoma cells were transplanted into the axillary region of F, -r mice. After 7 days, select a mouse with a transplanted tumor size of 7 to 81111 mm in diameter, no hemorrhagic necrosis, and good blood circulation, and inject 0 or 5 d of TN diluted with physiological saline from the tail vein.
Sample F is injected, and 24 hours later, judgment is made according to the following criteria.

←): No change - H = Faint hemorrhagic necrosis (@: Moderate hemorrhagic necrosis (5 minutes from the middle of the transplanted cancer surface)
0-ml or more necrosis) ml: Significant hemorrhagic necrosis (severe necrosis in the central part of the transplanted cancer, with only a small amount of surrounding cancer tissue remaining) TNF activity measurement using an in vitro method is described, for example, by Ruff et al. Lymphokine Reports
Vol, [, ed, byE, Pick, Acad
emic Press, N.Y. (1980)2
35] or Kull et al. (J. Immunol.
, 126 (1981) 1279).

The method used by the present inventors is an improved version of these methods and measures the effect of killing 6!0, TNF-, -M cells (American Type Culture Collection, CCL1, 2) i. That is, L of TNF samples diluted in successive medium at concentrations of 0.1- and 105 kg/wt.
- Add the medium suspension of M cells to a 196-sized tissue culture microplate (Flow Labora, IJ-Company). The medium was Eagle's Minimum Essential Medium containing 10 v/v% bovine fetal 91 serum (its composition was
For example, "Tissue Culture" edited by Junnosuke Nakai et al., Asakura Shoten,
(described in 1967). The microplate is incubated at 67° C. for 48 hours in air containing 500 g of carbon dioxide. After culturing, add 20 μtf of glutaraldehyde.
Add to fix the cells. After fixation, microplate was washed, dried, and washed with 5% methylene blue solution.
d and stain the surviving cells.

After washing away excess methylene blue and drying, extract with the remaining methylene blue'fr:596 hydrochloric acid solution,
The absorbance at 65 nm is measured with a Titertic Multiscan (Flow Laboratory). This absorbance is proportional to the number of surviving cells. The dilution of the TNF sample corresponding to 50% of the absorbance of the control without the addition of the TNF sample is determined by graph or calculation, and the dilution rate is defined as the unit (U)/d. Hereinafter, all in vitro activities of TNF in the present invention are expressed in this unit.

According to the method of the present invention, the activity of TN・F +MW, which is expected to be an anticancer agent, is maintained even during storage in a frozen or lyophilized state, separation and purification, and formulation operations, so that high purity can be obtained. T N F f, (It becomes possible to provide efficiently and stably on an industrial scale.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 A TNF' solution (0.1M phosphorus containing 0.15M sodium chloride Acid buffer pH 7.0) was prepared. Add 10μr/g of various nonionic surfactants to this TNF solution.
/, store at 4℃, and store over time (2 days,
7th.

30 days) in vitro as described herein.

The total residual activity was then measured using an in vivo evaluation method.

In the case of the in vitro method, the residual activity rate (Q) was calculated from the obtained measured values. The results are shown in Table 1 together with the results of Example 2.

In addition, in the case of the in vivo method, the treated solution is passed through an ultrafiltration concentrator, mini module NM-5 (manufactured by Asahi Kasei Corporation,
After concentrating 38 times with Funakoshi Pharmaceutical Sales), one mouse was injected into the tail vein of 1 #5 mice with bile cancer. l O,5- administered;
24 hours after administration, evaluation was performed according to the criteria.

Example 2 Various nonionic surfactants were added to a TNF solution having the same active concentration as that used in Example 1 at a concentration of 10 μf/me, and the mixture was frozen (-70'C) and thawed 9 times. 1 time, 3 times), and its residual activity was determined in vitro.
It was measured by the f value method. Table 1 shows the results in the form of residual activity rate (ch).
Shown below.

Example 3 Various concentrations of polyoxyethylene (20) sorbitan monooleate (Tween 80) were added to a TNF solution with an active concentration similar to that used in Example 1.

Wako Tsumugi Pharmaceutical Co., Ltd.), polyoxyethylene (6) stearate and ssannonion 5-61 NOF Co., Ltd.), polyoxyethylene (25) lauryl ether (Brij35. Wako Tsumugi M), polyoxyethylene (60) hydrogenated castor oil (
NIKKOL HCO-60, manufactured by Nikko Chemicals) and polyoxyethylene polyoxybrobylene block polymer (average molecular weight approximately 8550, E, O content 80
%) (Pluronic F68.

(manufactured by Asahi Denka) and stored at 4°C for 7 days, and then the residual TNF activity was measured using an in vitro evaluation method.
The residual activity rate (%) was calculated based on that value. The results are shown in Figures 1 to 5, respectively.

Comparative Example As a nonionic surfactant of the present invention, Tween80
, NIKKOL HCO-60 and Pluroni
c F 68 f was used, and as comparative additives, sodium dodecyl sulfate, which is an anionic surfactant, and cetyltrimethylammonium bromide, which is a cationic surfactant, were used.
Tests were also conducted using various alcohols and gold, which are well known as usual solution stabilizers for physiologically active substances.

1) Stabilizing effect In the case of a surfactant, o, oos and 0.05
In the case of alcohols, 1 is added to 0.5% for ethanol and 10% for other 7A/coles. Activity was measured.

(() Cytotoxicity To test the cytotoxicity of the additive itself, a solution of the above concentration was made into a sea bream, diluted 100 times with the medium, and L- Ma
Toxicity to cells: investigated. At that time, those that showed lethal toxicity of 50 or more to LM cells were determined to have cytotoxicity.

Table 2 shows the results of 1) and i+).

As is clear from the above Examples and Comparative Examples, according to the stabilization method of the present invention, it is possible to stably maintain the activity of TNF during operations such as storage in a solution state, freezing, and thawing. This not only applies to the purification process and formulation process during TNF production, but also guarantees product stability when commercialized.

[Brief explanation of the drawing]

Figures 1 to 5 show the TNF in vitres after storage at 4°C for 7 days with respect to the added concentration of various nonionic surfactants.
. It is a graph showing the relationship between residual activity rates. '-') f>/°, t> s-g sum... 5 (i
I 4 corners NIKKO Hco-to Reiyto$iL (sl/
J) play with

Claims (1)

[Claims] A method for stabilizing cancer necrosis factor, which comprises adding a nonionic surfactant to cancer necrosis factor.