JPS5821621A - Remedy for malignant tumor containing tnf (tumor necrosis factor) - Google Patents

Abstract

PURPOSE:The titled remedy that is made by making a TNF-inducer act on the cells resulting from transplantation of human-originating cells, which can produce TNF and is established as a cultured strain, in a body of animals other than human, thus containing TNF. CONSTITUTION:Human-relating cells, which can produce TNF and are established as a cultured strain, such as Namalva cells are transplanted in the body of a warm- blood animal such as bird such as chicken or a mammarian such as dog or cat or inoculated in a diffusion chamber to proliferate, as receiving the supply of body fluid containing nutrients from the animal. Then, a TNF inducer is made to act on the resultant cells in virto or in vivo. Thus, the formation of TNF is induced in high activity and the TNF is separated and purified in a large amount. The TNF is used to given a remedy for malignant tumotors. The preparation is used in a form of capsules, tablets as an enteric coating, a suppository, injection solution, further collunarium, eye drops or ointment.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a therapeutic agent for malignant tumors containing Tumor Necrosis Factor.

Tumor Necrosis Factor
ROSISFactor (hereinafter abbreviated as TNF) is E, A, Carswellet al, Pr.
Nat, Acad, Sci, USA, Vol.
, 72, Nn 9 +3666-3670 (19
1975) and E, Pick (ed.) [TumorNecro
sis Factor Otori Lymphoki
ne Reports, J Vol, I I
.

For example, as described in Academic Press, 1980, etc., rabbits are exposed to Bacillus.
lus Calmette -Gu6rin (B C
G), Corynebacterium parvu
Meth A is a protein-like substance that is induced and produced in serum by parenteral administration of a TNF inducer such as dototoxin, and is the name given to a substance that has the ability to cause hemorrhagic necrosis in Meth A sarcoma. 1. It is known that it has a cytotoxic function, especially against tumor cells.

Because of these functions of TNF, TNF has been expected to be a therapeutic agent for malignant tumors since its discovery.

TNF is prepared from the serum of animals such as rabbits and rats, and is said to have no species specificity. However, in order to use it for human treatment, it must be derived from living human cells to avoid antigenicity that may arise during treatment. It is extremely safe and excellent in terms of side effects.

The present inventors have demonstrated that human T
We have investigated methods for producing NF and have conducted intensive research to determine whether TNF is useful as a therapeutic agent for malignant tumors.

As a result, the cultured human-derived cells were grown in vitro (i.
Instead of being inoculated and grown in a nutrient medium (n vitro), they are transplanted into a warm-blooded non-human animal, or inoculated into a diffusion chamber, while receiving a supply of nutrient-containing body fluids from that animal. It was discovered that TNF can be induced and produced with high activity by multiplying the cells and acting on the obtained cells with a TNF inducer in vivo or in vitro, and that TNF can be easily produced in large quantities by purifying and fractionating the TNF. The present invention was completed by confirming that TNF is an excellent therapeutic agent for malignant tumors.

Unlike when living cells are grown in vitro, the method for producing TNF used in the present invention not only eliminates or significantly saves on nutrient media containing expensive serum, but also It is extremely easy to maintain and manage during proliferation, and moreover, it has the characteristics of high induced TNF activity. That is, transplanting cultured human-derived cells into the body of a warm-blooded animal other than humans, or
If the animal is housed in a diffusion chamber that can receive body fluids, and this chamber is buried inside the animal's body and reared normally, the animal can utilize the body fluids containing nutrients supplied from the warm-blooded animal's body. This allows the cells to proliferate easily. Furthermore, the growth of these cells is stable compared to when grown in vitro;
Its major characteristics include a high proliferation rate, a large amount of cells obtained, and a marked increase in the yield of TNF per cell. The cultured human-derived cells used in the present invention may be any cell that can be transplanted into a warm-blooded animal other than humans and easily proliferate, and that can produce TNF. For example, [Journal of C11nical
Microbiology Vol, l J 115~
Namalv described on page 117 (1975)
a-cell, I, ``YOshi [Nature Vo
BALL-1 cells, TALL-1 cells, as described in I, 267 J pp. 843-844 (1977);
NALL-1 cells, l”Journal of Imm
unoigy Vol, 113J 1334-13
M-7002 cells, B-7, described on page 45 (1974)
Established cell lines such as 101 cells, normal mononuclear cells,
Cells that have been cultured by treating granular white blood cells with various viruses, drugs, radiation, etc. are freely used.
It is not limited to the established cell lines described herein. These cells can be freely used alone or in combination of two or more types in the process of inducing and producing TNF, which will be described later. If desired, it is also possible to use, for example, freshly prepared human leukocytes.

The warm-blooded animals used in the present invention may be those in which human-derived cells can proliferate, such as chickens, birds such as pigeons, dogs, cats, monkeys, rabbits, goats, pigs, horses, cows, Mammals such as guinea pigs, rats, hamsters, regular mice, and nude mice can be used.

Transplanting human-derived cells into these animals may cause an unfavorable immune reaction, so in order to suppress such reactions as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or newborns. Preferably from childhood.

In addition, these animals may be subjected to pretreatment such as irradiation with X-rays or gamma rays at a dose of 200 to 600 rem, or injection of antiserum or immunosuppressants, etc., to weaken the immune response before transplantation. .

When the animals used are nude mice, the immune response is weak even when they are grown, so cultured human-derived cells can be transplanted without the need for these pretreatments, and the cells can be rapidly grown. This is particularly advantageous since it can be propagated.

In addition, human-derived cells that have been cultured are first transplanted into hamsters and grown, and then these cells are then transplanted into nude mice. It is also possible to further stabilize the proliferation of the derived cells and further increase the amount of TNF induced and produced from them.

In this case, transplantation may be performed not only between the same species and the same genus, but also between the same class and phylum. The site within the animal body to which human-derived cells are transplanted may be any site where the transplanted cells can proliferate, such as the allantoic cavity, vein, abdominal cavity, or subcutaneous site.

In addition, porous filtration membranes capable of blocking the passage of animal cells without directly transplanting human-derived cells into the animal body, such as membrane filter-1 ultrafiltration membranes or follower fibers having a pore diameter of about 10 to 10 m, are also available. A well-known diffusion chamber of various shapes and sizes equipped with the above-mentioned diffusion chambers is buried in the animal's body, for example, in the abdominal cavity, and the above-mentioned culture strain is grown in the chamber while being supplied with body fluids containing nutrients from the animal body. Any human-derived cells that have been transformed can be grown.

In addition, if necessary, a chamber/bar that connects and perfuses the solution containing nutrients in the chamber with body fluids in the animal body is attached to the surface of the animal body, and the human-derived cells in the chamber are allowed to proliferate. It is possible to see through the animal's condition, and it is also possible to make only a portion of this chamber removable and replaceable, allowing cells to proliferate to the fullest lifespan without slaughtering the animal.
It is also possible to further increase the amount of cells produced per animal.

Methods using these diffusion chambers do not allow human-derived cells to come into direct contact with animal cells, so not only can only human-derived cells be easily collected, but there is also less risk of causing an immune reaction, which is preferable. There is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely.

The transplanted animal can be maintained and managed simply by continuing the normal care and management of the animal, and is convenient because no special handling is required even after transplantation.

The period for growing human-derived cells is usually 1 to 10
The goal can be achieved within a week.

It has also been found that the number of human-derived cells obtained in this manner reaches approximately 10 to 10 cells or more per animal.

In other words, the number of human-derived cells proliferated by the TNF production method used in the present invention reaches about 10 to 10 times or more than the number of cells transplanted per animal, and the number of human-derived cells grown by the method for producing TNF used in the present invention reaches about 10 to 10 times or more than the number of cells transplanted per animal, and is Approximately 10 when seeding and propagating rice
~10 times or more, which is extremely favorable for the production of TNF.

TNF from human-derived living cells grown in this way.
The method to induce and generate is free. It is also possible to cause the TNF inducer to act in the animal body in which it has grown. For example, a TNF-inducing agent is directly applied to human-derived cells grown in suspension in ascites in the peritoneal cavity, or tumor cells generated subcutaneously, to induce TNF production, and then TNF is extracted from the serum, ascites, or tumor. All you have to do is purify and separate it.

Furthermore, human-derived proliferating cells can be removed from an animal body and treated with a TNF-inducing agent outside the body to induce TNF production. For example, human-derived cells grown in ascites are collected, or subcutaneous tumors containing human-derived cells are excised and dispersed, and the resulting cells are heated at approximately 20 to 40°C.
The cells may be suspended in a nutrient medium maintained at a constant concentration of about 10 to 10 cells, treated with a KTNF inducer to induce TNF production, and then purified and fractionated.

Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells can be left in the chamber or removed from the chamber and treated with a TNF inducer to induce TNF production.

In addition, for example, TNF is first induced and produced in grown human-derived cells while still in the animal body, and then TNF is induced outside the animal body in human-derived cells collected from a specific part or the whole of the same animal. How to generate it, once again T
Animals used by methods such as using the cells used for induced production of NF twice or more for induced production of TNF, or increasing the number of cells obtained by implanting or replacing the chamber connected to the animal body, etc. T per individual
It is also possible to further increase the amount of NF produced.

TNF inducers typically include, for example, BCGlCory
nebacterium parvum 1 lipopolysaccharide, endotoxin polysaccharide?Which one or more types are used?Generally, first, 1, e.g., B C
After a certain period of parenteral administration of one or more of Corynebacterium and the like,
Human-derived cells are collected and treated with, for example, ribopolysaccharide,
TNF may be induced by one or more endotoxins, polysaccharides, etc. acting in vitro. The TNF induced in this way is
It can be easily purified and separated and collected by performing known purification and separation methods such as salting out, dialysis, filtration, centrifugation, concentration, and freeze drying. If a higher degree of purification is required, For example, by combining known methods such as adsorption-elution to an ion exchanger, gel filtration, isoelectric point fractionation, and electrophoresis, it is possible to collect TNF of the highest purity.

The activity of TNF has been described by E. Pick [Tumor Nec.
rosisFactor in Lymphokine
Reports, J Vol, I I, Acad
A known method was used to measure the number of surviving cells after culturing for a certain period of time using L-929 cells as reported in emicpress (1980).

TNF used in the present invention can be prepared, for example, by the following production example.

Human-derived mononuclear cells treated with 5V-40 and cultured were transplanted subcutaneously into newborn hamsters, and after raising them in the usual way for one week, live BCG cells were intraperitoneally injected for 10 minutes.
The animals were injected with 100% and kept for another 2 weeks. Approximately 1 that occurred under the skin
After 5 g of tumor was excised and cut into small pieces, the cells were suspended in trypsin-containing physiological saline to disperse and separate the cells. The cells were incubated with human serum 5 v/v Eagle containing gold at pH 7.2.
Cells were diluted to a culture medium with the same composition, washed with a minimum basic medium of 37°C and kept at 37°C, to a cell concentration of approximately 5×10/dK, and endotoxin from E. coli was added to the medium at a concentration of approximately 10 μfi/m2.
1 of the mixture was added and kept for 16 hours to induce TNF production. This is 4℃, about i,ooo,! Centrifugation at pH 9, remove the precipitate, and dilute the resulting supernatant to pH 7, 2, 0, 0.
Dialysis was performed for 21 hours against physiological saline containing 1M IJ phosphate buffer, and the filtrate obtained by further microfiltration was concentrated and lyophilized to obtain a powder containing TNF activity. The resulting powder was mixed with G, Bodo's report (8ymposium o
n preparation, 5 tandariza
tion and clinical use of
1nterferon, 11th International
onal Irrmu-nobioiological S
ymposium 8&9June 1977, Za
greb.

Interferon was removed by means such as adsorption/desorption on ion exchange, molecular weight fractionation by gel filtration, concentration, and microfiltration according to Yugoslavia), and further purified by ammonium sulfate salting out and ConA-Sepharose affinity chromatography, and Meth A sarcoma was purified. Approximately 1.000,000 units of highly purified TNF, which is characterized by having hemorrhagic necrotic ability and not having any adverse effects on normal cells, was obtained.

The TNF thus obtained had a specific activity of 350,000 units/da without being contaminated by the inducer used.

Below, the effectiveness, toxicity, and
Describe usage and dosage.

Experimental Example I A piece of human breast cancer tissue is subcutaneously transplanted into the back of a BALB/C-derived nude mouse. Table 1 shows the amount of TNF obtained in the above production example from the time when the tumor volume was about 200 mm.

As a control, TNF-free physiological saline was intravenously injected.

Table 1 Mine: There is a statistically significant difference compared to the control value when the risk rate is 5 or less.

Experimental Example 2 A group of 10 BDF+ male mice weighing around 25 g,
A Lewis lung cancer cut into 2 mm squares was subcutaneously transplanted into the back.

After transplantation, 1 oo and 1,000 units Z kg of TNF obtained in the above-mentioned production example were intravenously injected once daily from 8B1, and on day 21, the mice were sacrificed and tumor weights were measured.

The results are shown in Table 2. As a control, TNF-free physiological saline was intravenously injected.

*There is a statistically significant difference compared to the control value when the risk rate is 51 or less.

Experimental Example 3 Acute Toxicity When we conducted an acute toxicity test of TNF obtained in the above production example using mice 9 days after birth, the toxicity of TNF was extremely low, and the LDso when injected intraperitoneally was 200. 0
00 units/kg or more.

As is clear from the above experiments, the TNF of the present invention is
It is extremely safe due to its effective dosage and can be advantageously used in the treatment of various malignant tumors.

The malignant tumor in the present invention is prevented by TNF,
or a disease to be treated, such as a malignant tumor such as breast cancer, lung cancer, liver cancer, bladder cancer, uterine cancer, precancerous cancer, colon cancer, leukemia, lymphoma, skin cancer, etc.

Furthermore, when applied to malignant tumors, for example, a part of a patient's tumor is taken, treated with the TNF of the present invention in vitro to increase the immunogenicity of the tumor, and then returned to the tumor patient's body. This malignant tumor can also be treated.

The daily dose of TNF of the present invention for adults is 1 to 50.00.
0,000 units, preferably 1 to 1,000,000 units for local applications such as local injections and eye drops, 10 to 5,000,000 units for ointments, and for systemic injections such as intravenous and intramuscular injections. The dosage is 100 to 10,000,000 units, and 100 to 50,000,000 units for oral administration, but the dosage can be adjusted as appropriate depending on the usage or symptoms. If necessary, it can be prepared into a pharmaceutical preparation by a conventional method together with any conventional pharmaceutical carrier, base, or excipient.

The shape of the TNF-containing malignant tumor therapeutic agent of the present invention can be freely selected depending on the purpose.

Oral preparations include enteric preparations such as capsules, tablets, and powders; intrarectal preparations include rectal suppositories; injections include, for example, freeze-dried injections that are dissolved in distilled water for injection; It can also be used as nasal or eye drops, or as an ointment.

Examples of formulations are shown below, but the formulations are not limited thereto.

Example 1 Injection TNF prepared in the above manufacturing example 500,000
The unit is dissolved in 2001 saline and aseptically filtered using a membrane filter. The filtrate is filled in 2 ml portions into sterilized glass containers, freeze-dried, and sealed tightly to obtain a freeze-dried powder preparation.

This product is suitable for the treatment of breast cancer, lung cancer, liver cancer, leukemia, etc.

Example 2 Ointment The TNF prepared in the above production example was added to a small amount of liquid paraffin according to a conventional method, and then Vaseline was added to give 20,000 ml of ointment.
It was made into an ointment of unit/g.

This product is suitable for treating skin cancer, breast cancer, lymphoma, etc.

Example 3 Eye solution vapor 800 ml water 511 Il of β-phenylethyl alcohol and 20 ml of TNF prepared in the above production example.
ooo, oj.

Add salt to strengthen the unit and add distilled water to 1.000
ml and used as eye drops.

This product is suitable for the treatment of retinoblastoma, etc.

Example 4 Enteric-coated tablet When tabletting the TNF prepared in the above manufacturing example using a mixture of starch and maltose according to the conventional method, the amount of TNF was adjusted to 200,000 units per tablet (100 cm) of the product. Enteric-coated tablets were prepared by coating the tablets with methylcellulose phthalate.

This product is suitable for the treatment of colorectal cancer, colon cancer, liver cancer, etc.

Patent Applicant: Hayashibara Biochemical Research Institute Co., Ltd. Procedures Amendment Written on August 28, 1981 Commissioner of the Japan Patent Office Shima 1) Haruki Tono L Case Indication 1986 Patent Application No. 120459 2 Name of Invention Tsumor Necrosis Factor Malignant tumor therapeutic agent containing a Line 8 and page 11, No. 1
r E, Pick r 'l'umor Necro written in lines 8-15
sis Factor in Lymphokine R
eports, J VOl, II, Academ
ic press.

In 1980, the RG pickli 'pumor
NecrosisFactor in ” Lymph
okines” Vol, II, pp 285-
272゜Academic press, 1981,''.

(2) “Name name 1,” written on page 2, line 9 of the specification.
” is a [name, JK correction.

(3) Insert a codified sentence after “freely used” on page 5, line 8 of the specification.

[In addition, the genes capable of producing TNF in these cells can be modified by means of cell fusion using polyethylene glycol or Sendai virus, or by genetic recombination using enzymes such as DNAIJ gauze, restriction enzymes (nucleases), and DNA polymerases. It can be introduced into cultured lymphoblastoid cells that can be easily passage-cultured by other means, to increase the proliferation rate and to increase the TNF per cell.
(4) rL-929 cells described in page 11, line 15 of the specification should be corrected to rTNF-sensitive L-929 cells.

(5) r, 5V-40” written at the end of page 11 of the specification
sv-40 virus”.

(6) “Skin cancer, etc.” stated in the first line at the end of page 11 of the specification
will be corrected to "skin cancer, neuroblastoma, etc."

Claims (1)

[Claims] Tumor Necrosis Factor-producing cultured human-derived cells are transplanted into the body of a warm-blooded animal other than humans, and the cells obtained by growing while receiving body fluids from the warm-blooded animal are Alternatively, a malignant tumor therapeutic agent containing TSUMOA necrosis factor obtained by further purification and fractionation of TSUMOA necrosis factor produced by the action of a TSUMOA necrosis factor inducer in vitro.