JPS5959625A - Stabilization of tumor encrosis factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for stabilizing cancer necrosis factor (hereinafter abbreviated as i' N This paper relates to a method for stabilizing F.

In the present invention, TNF refers to [administration of one or more substances having a reticuloendothelial system activation effect to a mammal,
Tumor-bearing animals are then inoculated with bioactive agents induced by injection of endotoxin from Durham-negative bacteria or by adding endotoxin from Durham-negative bacteria to a tissue culture system containing activated macrophages of mammalian origin. It is a substance defined as "a factor that causes necrosis of certain types of cans by causing necrosis." 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 cancer in mice. In addition, TNF is a group in vitro
It has almost no harmful effect on normal cells, and some types of cancer cells (for example, L-M derived from mouse cancer)
It is known to have the ability to kill cells. As described above, TNF has an anticancer effect, is species-nonspecific, 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 the amount of F no Sato is extremely small. T
In order to widely and safely use NF as an anti-convulsant agent, it must be purified in a nail-like form, stored in a solution or frozen state for a long period of time, or freeze-dried.
This is an essential operation when producing i' NF industrially in large quantities.

However, the present inventors have found that when a high-purity I'NF solution is stored, frozen, or frozen + 2-dried, the activity decreases markedly3.
To date, no reports have been made regarding the stability of NF. 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 conducted intensive research on stabilization of TNF. By adding stuffed fruits, certain sugars and sugar-related compounds to 'rNF,
It has been found that the activity of TNF is maintained even after storage for a considerable period of time, or even after operations such as preparative purification and lyophilization, and the present invention was completed based on this finding.

The present invention is characterized in that at least one substance selected from D-glucose, D-caractose, D-quinlose, [1]-glucuronic acid, trehalose, dextran, and O.hydroxyethyl starch is added to TNF. The present invention relates to a method for stabilizing TNF.

The TN F raw material used in the present invention is produced by a known method. As such, for example, Matt
llews et al., Br, J. Cancer, 4
2 (1980) 416, Green et al., J.N.
a11. CancerInst, 59 (1977
) 1519 methods are mentioned. The method will be explained below.

That is, 2. One or more substances having a reticuloendothelial system activating effect are injected intravenously or intraperitoneally into a mammal (eg, mouse, rabbit, guinea pig, etc.). Durum-positive bacteria, protozoa, or yeast are usually used as substances that activate the reticuloendothelial system, and are administered in a live state, a dead state (for example, after heat treatment or formalin treatment), or as a bacterial cell extract component. 3. Durham-positive bacteria include, for example, 1'ropi
onibacterium acnes (Coryn
cbn+cl:eriumparvum),
Propionibacterium granu
losum(Corynel)acLerium gr
Propionibac such as anulosum)
Leria, Bacillus CalmeLL
e-Gu4rin (BCG), MycobacL
Mycoba like crium smegmatis
cteria, Nocardia erythrop
olis.

Noca like Nocardia gardneri
Examples include rdias. Examples of protozoa include malaria parasites and toxoplasma gondii. For yeast, usually Saccharomyces cercvi
Zymosan extracted from siae etc. is used. Moreover, synthetic polymer compounds such as pyran copolymers can also be used.

Endotoxin obtained from Durham-negative bacteria 7 to 14 days after administration of a substance that has a reticuloendothelial system activation effect, for example,
E. coli, green IIl'! 1. Polypolyzatucaride derived from typhoid fever is injected intravenously into the mammal. 1.5-2 hours after the injection, the mammal's body fluids (e.g.
Ascitic fluid, lymph fluid, etc.) and/or serum or plasma can be obtained, or one organ such as the liver, one tile, etc. of the animal can be homogeneously crushed and extracted with physiological saline.
Get p fee. .

The method for producing the TN F raw material used in the present invention is not limited to the above. That is, a TNF raw material production method such as a cell culture method can also be adopted.

The TNF raw material produced in this way is 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 anion exchange fat, gel 1 filtration, and electrophoresis. When these methods are combined to advance the separation and purification process to increase the degree of purification, TNF gradually becomes unstable. For example, specific activity (activity of TNF contained in 1 mg of total protein, unit of activity will be described later) 500,000 units/
TNF samples purified to mg are very unstable, as shown in the Examples. TNF samples 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 which has become unstable due to the degree of purification as described above, and may be either a solution or a powder, or a solution containing TNF.

1) of the solution containing TNF used in the present invention
It is preferable that H is maintained at 5 to 10, and more preferably adjusted with an appropriate buffer. Such buffers include, for example, phosphate FtU Ilj solution,
tris (hydroxy+ncl, hyl
) aminn1nethaneco-hydrochloric acid buffer and the like. Depending on the purpose, salt may be added. Salts used include sodium chloride and potassium chloride, and the concentration thereof is determined depending on the purpose. For example, when used for injection, sodium chloride is added to the solution to a concentration of 0.15M to form an equi-arcuous solution.

Substances used in the present invention (hereinafter referred to as substances of the present invention)
Among them, in the case of D-glucose, ■)-caractose, D-xylose, D-'/''lucuronic acid and trehalose, these phosphoric acid esters or salts thereof, acetic esters, methyl ethers, methyl glycosides, etc. Since derivatives of D-glucuronic acid also have a TNF stabilizing effect, the method of the present invention also includes a method of adding these derivatives. Also included are alkaline earth gold FA f4i such as alkali metal salts and calcium and magnesium salts.As hydroxyethyl starch, the average molecule @
300 of 1 to 400,000 or an average molecular weight of 20,000.

Preferably. The dextran preferably has an average molecular weight of 10,000 to 80,000, for example,
Dextran], 0, 40, 70 (average molecular weight 100 (10, 40000, 700 (10), respectively)
can be mentioned. Among these substances, trehalose is most preferred.

The substances of the present invention can be used alone or in an appropriate combination of two or more.

The additive concentration of the present invention is 10 mg or more, more preferably 10 mg per ml of solution containing TNF.
0 mg or more. The two limits of additive concentration are determined from common sense and economical viewpoints, and are 500 m/ml of the same solution.
It is g. The amount of the substance of the present invention to be added to powder containing FNF is as follows:
The solution concentration is chosen as above.

The method of addition is not particularly limited, but for example, the powder of the substance of the present invention may be directly added to the TNF-containing solution, the powder may be dissolved in water or an appropriate buffer solution beforehand, or the powder may be added to the TNF-containing solution. An example of this method is to mix it with a TNF-containing powder and add it. The timing of addition may be during the minute purification process or during the formulation process.

When two or more types of substances of the present invention are added, the total amount of the substances may be adjusted so that the concentration and amount of the substances added are as described above.

In this way, the solution containing 'I' NF to which the substance of the present invention has been added is stored at a temperature of 0 to 30"c %, preferably 0 to 10 T, or subjected to separation, purification, and formulation operations in its solution state. It is preferable to do so.

Furthermore, when the same solution is stored in a frozen state, it is desirable that the temperature be below O''CC, preferably below -20''C. In a solution containing TN F to which the substance of the present invention has been added,
storage in solution or frozen state or separation and purification;
The activity of TNF is maintained during the formulation process.

There are two ways to measure the activity of TNF: one is to measure the tumor necrosis 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) 3666. This method is suitable for transplanted 84eth A sar
This test measures the effect of TNF on necrosis of coma-induced tumors. That is, (BALB/c
C57BL/6) F l Mouse armpit region ironically 2×
10 MeLllA sarcom; + Ill cells are transplanted. After 7 days, the size of the transplanted tumor was 7 to 7 days later.
8+ nm, with good blood circulation without hemorrhagic necrosis, etc., were selected, and 0.5 ml of the TNF sample diluted with physiological saline was injected into the tail vein, and 24 hours later, the mice were evaluated according to the following criteria. Make a judgment.

(-): No change ±): Faint hemorrhagic necrosis (11): Moderate hemorrhagic necrosis (necrosis extending over 50% from the center of the graft surface) (m,): Significant hemorrhagic necrosis (graft The central part of the cancer is severely necrotic, with only a small amount of surrounding cancer tissue remaining) Group vi I: TNF active side standard by r O method, 1, for example, ゛, Rufr et al r I-ym
plo+ki++cs, Vol, 2. (td, by
E.

Pick, Δacademic Press, N, Y,
(1ri80) 2 :l 5 l, or Kull
L J, Innnunol,, I 2fi(]!
18+) 127] methods can be mentioned.

The method used by the present inventors is an improvement on these, in which TNF is LM, fl1+ cells (American Type Culture Collection).

cct, 1.2).

That is, i' NF sample 01m sequentially diluted with culture medium.
1 and Il, -M, ? with a concentration of 1 O/ml. Add 0.1 ml of culture medium suspension of IIl cells to a 96-well tissue culture microplate (Flow Laboratory). medium ha
Eagle's Minimum Essential Medium containing Ov/v% bovine fetal blood serum (its composition is, for example, 1-
Tissue Culture-1 Edited by Junnosuke Nakai et al. 1, Asakura Suriten, 1967
Incubate a 6° microplate for 48 hours at 37"C in air containing 5% carbon dioxide. After incubation, add glutaraldehyde 2 (17
Add zl and fix 411 cells. 3. After fixation, wash and dry the microplate, and add 0.1% 0.05% methylene blue solution. +nl to stain surviving cells. After rinsing away excess methylene blue and drying, the remaining methylene blue was extracted with a 3% hydrochloric acid solution and
Measure the absorbance at 1 in 5 minutes using a Quikuchik Multiscan (Flow Laboratories, Inc.) 1. This absorbance is proportional to the number of surviving cells fl 'I'
Determine by graph or calculation the dilution rate of the TNF sample that corresponds to 50% of the absorbance of the control without the addition of the NF sample, and calculate that dilution rate! 11th place (U) Defined as /inl. Hereinafter, in vitro analysis of T N F in the present invention will be described.
All tro activities are expressed in this unit.

According to the method of the present invention, the activity of TNF, which is expected to be an anticancer agent, is maintained even when it is stored in a solution, frozen, or lyophilized state, and during separation and purification and formulation operations. It becomes possible to efficiently and stably provide NF on an industrial scale. Furthermore, it is safe to administer to the human body, and is extremely advantageous when FNF is used as an anticancer agent.

Next, the present invention will be explained in more detail with reference to Examples 1.
,.

Example 1 T from Uzaki with a specific activity of 500,000 U/mg
Using NF, T with an activity of 100OU/ml
NF solution (0.1 containing 0.15 M sodium chloride)
M phosphate buffer pH 7.0) was prepared. This TNF
D-glucose, D-galactose, D-xylose, sodium D-glucuronate, trehalose, dextran 10, Textrumf 0 and hydroxyethyl starch (average molecular weight approximately 20,000(1) (HE) were added to the solution.
S, manufactured by Ajinomoto) at 100 mg/ml and: 30
It was added to a concentration of 0 mg/ml, stored at 4°C, and stored over time (2 days, 7 days.

In vitro, as described herein, on the 30th.

And residual activity was measured using an in vivo evaluation method.

In the case of the in vitro method, the residual activity rate (%) 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 treatment solution is
After concentrating 30 times using a superconcentration device, Mini Mojir NM-3 (manufactured by Asahi Kasei Kogyo, sold by Funakoshi Pharmaceutical), 0.5 ml per mouse was administered into the tail vein of 5 caged mice per group. Evaluation was made in accordance with the 24-hour time zone criteria. However, in the case of Textlan and hydroxyethyl starch addition, the residual activity was not measured because it was not possible to concentrate to a predetermined concentration.

Example 2 'f'N with active concentration similar to that used in Example 1
In the F solution, various substances of the present invention used in Example 1],
Add to OOmg/ml and 300 mg/ml and repeat freezing (-70°C) and thawing once.
3 times), and its residual activity was collected.

It was measured using an evaluation method. Table 1 shows the results in the form of residual activity rate (%).
Shown below.

Example 3 TN F with active concentration similar to that used in Example 1
The residual TNF activity after adding various concentrations of trehalose to the solution and storing it at 4-PaC for 7 days was determined by i.v.i.
It was measured by the t ro evaluation method, and the residual activity rate (%) was calculated based on the value. The results are shown in the drawing.

(Left below) Comparative Example D-glucose, sodium D-glucuronate, trehalose, and hydroxyethyl starch were used as the substances of the present invention, and as comparative additives, D-glucose, sodium D-glucuronate, trehalose, and hydroxyethyl starch were used. Various saccharides and saccharide-related compounds were used.

TN F with an active concentration similar to that used in Example 1
Add 10 mg/'ml or 20 mg/'ml of additives to the solution.
0 + ng/ml and stored at 4C for 7 days, the residual TNF activity was measured by an in vitro evaluation method, and the residual activity rate (%) was calculated based on the value. The results are shown in Table 2.

(must margin) Table 2 Comparative Examples As is clear from the above examples and comparative examples, according to the stabilization method of the present invention, during operations such as storage in a solution state, freezing, and thawing, It is possible to stably maintain the l6 character of TNF.

[Brief explanation of drawings]

The figure is a graph showing the relationship between the residual activity of 1"N F in viLro after storage for 7 days at 4°C with respect to various additive concentrations of trehalose. Yushibe Tsuboi Patent applicant Nippon Pharmaceutical Co., Ltd.

Claims (1)

[Claims] Can necrosis factors include D-glucose, D-galactose, and D
- A method for stabilizing cancer necrosis factor, which comprises adding at least one substance selected from xylose, D-1'' lucuronic acid, trehalose, dextran, and hydroxyethyl starch.