JPS6016926A - Antineoplastic agent - Google Patents

Abstract

PURPOSE:To provide an antineoplastic agent containing homocarnosine or its salt as active component, having increased tumor immune reaction, exhibiting antineoplastic effect, and having high safety. CONSTITUTION:The objective agent contains the homocarnosine of formula or its salt as an active component. Homocarnosine is a dipeptide extracted from the bovine brains. The antineoplastic activity of homocarnosine promoting the nonspecific active immune reaction (immune promoting agent) or promoting the tumor immune reaction different from the direct action to the transplanted cancer cell or from the action of the conventional immune promoting agent, has been established by the use of two experimental systems of the allogeneic DDY- S-180 and the isogeneic BALB/C-METH-A. The administered homocarnosine is hydrolyzed in the body into L-histidine and GABA. The effective dose of homocarnosine, i.e. 50mg/kg (by subcutaneous injection) (2.5g for adult) corresponds to about 1.1g of GABA, and it can be said from the permissible dose of GABA, that the homocarnosine has high safety.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antitumor agent containing homocarnosine or a salt thereof as an active ingredient.

The development of tumor therapeutic agents is currently being carried out based on two broad concepts. One of these is based on the idea that cancer can be suppressed by inhibiting active nucleic acid biosynthesis in tumor tissues.

In Japan, for example, pleomycin (Nippon Kayaku Co., Ltd.), mitomycin C (MMC (Kyowa Hakko Co., Ltd.)
], 5-F'U (Kyowa Hakko Co., Ltd.) and the like are therapeutic agents created based on this idea. The second is non-specific active immunotherapy that utilizes the host's immunity, which is based on the idea of treatment with so-called immune stimulants. Zeria Pharmaceutical Co., Ltd.)], Crestin (
”PSK (Kureha Chemical Industry Co., Ltd., Sankyo Pharmaceutical Co., Ltd.)
] belong to this category.

However, both therapies have deficiencies that prevent them from achieving their full clinical purpose. The former has the disadvantage that its inhibitory effect on nucleic acid synthesis is not cancer-specific and therefore inhibits nucleic acid synthesis in tissues other than liver cancer tissues, resulting in unavoidable side effects.

In the latter therapy using immunostimulants, the immune response is not cancer-specific, and regardless of the quantity and quality of the immunostimulant used, the fundamental problem is that the amount of immune response produced is unique to the reticuloendothelial system organ. is limited by the response function of In order to suppress aggressively proliferating tumors, some means must be devised to increase the efficiency of the immune response.

Homocarnosine is /q67 bisano (pl5ano)
A dipeptide extracted from bovine brain by et al., namely L-
Histidinyl-γ-aminobutyric acid, approximately 0.00% in the brain
Contains 1%. Since its discovery, its physiological significance and pharmacological usefulness have remained unknown.

Hayashi et al. (/qA, S-year) focused on the inhibitory effect on the central motor system because homocarnosine is localized in the brain spinal cord and contains γ-aminobutyric acid (hereinafter abbreviated as GABA) in its structure, and found that homocarnosine is associated with epilepsy. An attempt was made to treat epilepsy by intrathecally injecting the patient's brain. Fujii and Elton Nis-Tuck et al. (7977
2007) is a cow brain extract that is derived from mouse Staphylococcus aureus (St, aphylococcus aureus).
discovered that it has an anti-infective effect against infections caused by
The active ingredient was determined to be homocarnosine.

The fact that homocarnosine does not show antibacterial properties in vitro but shows antibacterial properties when administered to animals suggests that homocarnosine has a sensitizing effect on the immune response, but this research has not been developed into immunological research since then, and the positive immune response To date, there have been no publications regarding the antitumor effect of enhancement. We present allogeneic pDy=s-/zaθ and allogeneic syngeneic BALB/C-M
Using two ETH-A experimental systems, we investigated the effects of non-specific active immune reactions (immune promoters) or tumor immunity on transplanted cancers, which do not have a direct effect on cancer cells and are different from the effects of conventional immune promoters. The present invention was completed by confirming the anti-tumor effect of homocarnosine, which enhances the response, on transplanted cancers.

Homocarnosine has the following chemical structural formula L-
Histidinyl GABA is a detide that is contained in approximately 0.000% of brain tissue.

(2) It is a white crystalline powder with a melting point of 0 to 3.0, and its 70% aqueous solution is colorless and transparent and slightly alkaline, with a weak alkaline taste.

The administered homocarnosine is hydrolyzed into L-histidine and GABA. L-histidine is an amino acid that is ingested in large quantities as a nutrient from daily foods, and is also a GA
BA has been commercialized as a brain stimulation agent [Daiichi Pharmaceutical Co., Ltd., Gammaron (trademark)], and is a substance whose safety has been confirmed. The effective amount of homocarnosine 3θ/kt (subcutaneous injection) (adult equivalent λ1.9g) is approximately GABA equivalent/7g.
It is. The amount of GABA allowed as a drug is 0.7S~/l/300~goo-glucose solution/, 2
~3 hours//day/~3 times. Therefore, from both of the above points, the Sθmy/ky of the antitumor effective amount of homocarnosine
(subcutaneous injection) is less than half the amount of intravenous injection, and its safety can be fully estimated.

Various methods are known for the synthesis of homocarnosine, and for example, it is synthesized as follows (Jou
RNA of Biological Chemls
try・, :18゜Δ(,2, da de de ~ S
0.2, /9A/).

Cal adds triethylamine to a suspension of benzyloxy-gamma-aminobutyric acid in methylene chloride. After the resulting solution has been cooled to -5° C., ethyl chloroformate is added and the mixture is kept at this temperature for 70 minutes. To this solution, diluted in methylene chloride cooled to θ℃
L made by adding triethylamine to a suspension of histidine methyl ester dihydrochloride
- Rapidly add the solution of histidine methyl ester. The resulting mixture was heated overnight A,! ;Leave it on oC.

It is then washed with water and /N-NaHCOg, dried over Na28OA and concentrated to a slag.

Dissolve the product in methanol and add /N-NaOH. After 3 hours at Kota DEG C., the solution is pH adjusted to the left with dilute sulfuric acid and concentrated to dryness under reduced pressure. The residue is extracted twice with hot ethanol and water is added to this extract. After adding 10% palladium-charcoal, C
In order to absorb 02, a caroxite tube (Caroxi
This mixture is hydrogenated in an apparatus equipped with a tube. After hydrogenation, the solution is filtered once and concentrated under reduced pressure. Dissolve the obtained residual Shiratuzo in water,
Adjust to pn 3 with dilute sulfuric acid. When ethanol is gradually added, the sulfate salt of zotakuputide separates as granular crystals. The product is filtered and recrystallized from water-ethanol in the same manner as above. Homocarnosine sulfate with a melting point of 2 θ'C (decomposition point) is obtained.

After filling a column with Dowex !rO and treating with /N-HC/, washing with water with neutral thymol blue, passing homocarnosine sulfate as /θ thi solution, and treating with thymol blue neutral. After washing with water until the sulfate group is removed, homocarnosine is eluted with /N-NH4O'H. After concentration, ethanol is added and the mixture is left in a cold room to obtain crystals of free homocarnosine.

The present invention includes a therapeutic agent consisting of a salt of homocarnosine, and the salt of homocarnosine includes a salt based on a caldenic acid group and an acid addition salt based on an amino group with a pharmacologically acceptable acid. There are also salts based on both caldenic acid and amino groups.

Salts based on caldenic acid groups include salts with metals such as sodium, potassium, calcium, magnesium, zinc and aluminum, ammonium salts and substituted ammonium salts, salts with trialkylamines and other amines such as triethylamine; Salts based on amine groups include hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid,
There are salts with inorganic acids and organic acids such as propionic acid, lactic acid, tartaric acid, citric acid, succinic acid, maleic acid, benzenesulfonic acid, and toluenesulfonic acid, and these can be converted into free homocarnosine by methods known per se. can be prepared by reacting with a stoichiometrically calculated amount of the selected acid or base.

Next, we will explain the excellent anti-lung cancer effect of homocarnosine by giving an experimental example.

Experimental example/Materials + DDY mouse, left week old male (obtained from Shizuoka Laboratory Animal Agricultural Cooperative Association) Tumor cells; Sarco-r/KO (Sarcoma
/go) (Komiyama stock, Kitasato Institute Cancer Research Department) 10 transplants I were transplanted subcutaneously in the midline scapula region.

Administration: From J+ time (1st day) after Wt tumor transplantation, the drug was administered subcutaneously on the back every other day for a total of 1S times (until day 29) and once every day for 10 consecutive days. Control animals Approximately 2 clnM L of physiological saline θ,/- was subcutaneously administered to the back of the transplanted site, and experimental animals received 0.0 mL of physiological saline θ,/- to the experimental animals.
θ/, 0, θS1 θ, 2, /Tn9 homocarnosine was administered subcutaneously to the back at a distance of about 2 meters from the transplant site.

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Ω Gyaku KōP Introduction -- 10 A Hemorrhoid 3 Experimental results: As shown in Table 1, administration of homocarnosine to three groups of mice had an antitumor effect on average survival days, mortality rate, and tumor volume. Indicated.

Experiment example Antitumor effect of combination use Material: Same as Experimental Example 1.

Tumor cells: Same as Experimental Example 1.

Transplantation: 705 pieces were transplanted subcutaneously to the midscapular region.

Administration: The drug was administered subcutaneously on the back, about 2 meters caudally from the implantation site, every day for 10 consecutive days from 7 days after tumor implantation. Control animals received saline 0. /d was administered subcutaneously on the back at a distance of approximately Jam from the transplanted site, and the experimental animals were given 0.0% in O6/1 physiological saline. / , 0.
,! ? ,/da of homocarnosine was administered subcutaneously to the back at a distance of about 2 cIL from the transplant site. Also, when using 3 OK-das alone, 0. /- 0°s KE in physiological saline intraperitoneally every other day/j times in total, 0. /ytt θ・, in physiological saline
3-KE 0K-1I 3 times intraperitoneally once every other day for a total of 3 times.
A solution containing homocarnosine/l was administered subcutaneously to the back at a distance of approximately -cm from the transplant site, 10 times daily.

Experimental results: homocarnosi when used in combination with 0K-4<32.

The effect of the button was enhanced. Especially in cases of extinction It is noteworthy.

Experimental example 3 Material family: Same as Experimental Example 1.

Tumor cells: Same as Experimental Example 1.

05 transplants were transplanted subcutaneously in the midscapular region.

Administration: Homocarnosine lQ10. from 21 hours (first day) after tumor implantation. /d Physiological saline daily/times continuous IO
The drug was administered subcutaneously to the back about 1 m from the transplant site.

Measurement: 73 days after transplantation, the animals were killed, the tumor was removed and weighed,
The average value of each 5 cases was calculated.

Experimental results: As shown in the average value of each 5 cases in Table 3,
The inhibitory effect of homocarnosine on tumor electrical storage is striking.

Experimental example: Antitumor activity of homocarnosine against Gumous sarcoma Materials:
BALB/C mouse, male S week old (obtained from Shizuoka Laboratory Animal Agricultural Cooperative Association) Tumor cells: METH-A (?-A-/-, 2) (Hokkaido University stock, stored at Daiichi Pharmaceutical Central Research Institute) Transferred Plant: Ascites type! 70 cells (/ in Table 7) and 10 cells (C in Table 7) were transplanted subcutaneously in the midscapular area.

Administration: The drug was continuously administered subcutaneously to the back of the patient about a distance caudal from the implantation site for 10 consecutive days from the time (first day) after tumor implantation.

For control animals, saline O3/4 was applied approximately 2 cm from the transplant site.
Homocarnosine in o, i y physiological saline/, 09 (/ in Table 1) and O, S da (co in Table DA) was administered to experimental animals subcutaneously on the dorsal part of the transplanted area. 1 mL was administered subcutaneously to the back.

Effect 1! ×105 Control 317. / /θ/10 /θOθH,C,/
Da 112. / 10/10 100 0.2. Ko ×
105 Control 3'1. A 9/q/DOO H, C, 0-3; W, 3, g 10/// 9/
/ Experimental results: Antitumor effects of homocarnosine were observed in BALB/C-METH-A as well as in the case of DI)Y-8-/gO. In addition, one case of coxIO transplantation was observed to disappear.

Summary (11D DY-S-/gO system 7 times every other day from day 1 to day 30 after tumor implantation, total 1!r)
7 consecutive doses and between days 1 and 1θ after transplantation.
Fosocarnosine showed an antitumor effect in prolonging the average survival time and reducing tumor volume compared to the control when administered 70 times per day.
The measurement results on day 3 showed that the tumor weight was significantly suppressed compared to the control. In particular, the Komiyama strain preserved in the Kitasato Research Institute's Department of Cancer Control is said to be a cancer strain that has an extremely strong proliferative ability than any Sarcoma/gO preserved in other laboratories in Japan, and is said to be a cancer strain that is more active than the conventional immune-stimulating cancer strain. Since this is a strain that cannot be easily suppressed with curative agents, the effect of homocarnosine shown in the Komiyama strain (S-/ZAO) fully demonstrates its effectiveness against human cancer.

(2) Combined administration of homocarnosine and 0K-4t3.1, which was used as a representative example of an immunostimulant, shows a more pronounced effect on prolonging the average survival period and eradicating the disease than either administration alone. No.-
Table 3 shows the experimental results shown in Table 3 when calculated according to the standards of the National Cancer Institute of the United States for determining the effectiveness of anticancer chemical substances.

Even in the T/C evaluation method, homocarnosine is 0K-lI3
It showed a better effect than 2. Furthermore, the combination of homocarnosine and 0K-1134 was more effective than homocarnosine alone.
Also, 0K-11, which was used by Acteip Zorase as -,
It is more effective than 3 drugs, and the effect is 33% when both are used together.
% increased.

Table S Treatment TiC Chi H, C・/■ /34 OK-II3.2θ, 5KE / / ? The final day of evaluation method determination is the 60th day. Median survival days (M,
S, T-median aurylval time)
is expressed as C×J M, S, T.=L □.

fM (where L is the lower limit of the death date of the middle mouse, C is one division of the death date, so usually /, fM is the total number of mice that died on the middle mouse death date, Ja is the middle mouse death date (This is the number of mice required to reach the intermediate level among mice.) In screening for anti-cancer chemical substances, TiC%L of 0 or more is evaluated as effective.

Administration of homocarnosine to group a of the f31 BALB/C-METH-A system prolonged the survival period, and the cases that disappeared showed an antitumor effect.

Since the BALB/CMETHA system is allogeneic, its antitumor effect on allogeneic syngenes without transplant immunity further ensures its effectiveness against human cancer.

The antitumor effects of homocarnosine as described above have shown that homocarnosine is effective against various organ cancers, such as gastric cancer, rectal cancer, breast cancer,
Uterine cancer, oral cancer, esophageal cancer.

Bile cancer, bile duct cancer, biliary tract cancer, pancreatic cancer, prostate cancer,
malignant thyroid tumor, lung cancer, brain tumor, liver cancer, tongue cancer,
When used alone or in combination with an immunostimulant such as 0K-13- to enhance its action, excellent therapeutic effects can be expected for the treatment of thymoma, skin cancer, etc.

The antitumor agent of the present invention may be in any dosage form as long as it is convenient for oral or parenteral administration of homocarnosine for diseases, such as injection solution, powder, granule, tablet, or capsule. There are a variety of dosage forms available, including enteric-coated tablets, ointments, suppositories, enemas, and lozenges, which can be formulated depending on the patient and the type of tumor.
They can be used alone or in combination depending on the symptoms. The clinical dose for adults estimated from basic efficacy experiments is generally O-5 to 311 (oral) per 7 days, and it is preferable to administer the drug in divided doses at appropriate time intervals depending on the symptoms.

Since homocarnosine is easily soluble in water, a 3%, 3% or IO aqueous solution of homocarnosine can be easily prepared under aseptic operation. This is sealed in an ampoule under a stream of inert gas and injected using an ordinary syringe. Immediately before injection, dissolve homocarnosine powder, which has been lyophilized and sealed in an ampoule or vial using aseptic procedures, in sterile distilled water, and immediately use it for injection as a 3%, 3%, or IO aqueous solution. Good too.

Powders, granules, tablets or capsules for oral administration may be prepared with binders such as syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone,
Excipients such as lactose, corn starch, calcium phosphate, sorbitol or glycine; lubricants such as magnesium stearate, talc, polyethylene glycol, hydroxypropylmethylcellulose or silica; disintegrants such as potato starch; or wetting agents such as sodium lauryl sulfate. and formulated by methods conventional in the art. Tablets may be coated by methods well known in the art.

To manufacture the ointment, according to techniques known in the pharmaceutical industry, an amount of finely powdered homocarnosine to provide an ointment of the desired concentration is added to an ointment base such as beeswax, spermaceti, dehydrated lanolin, white petrolatum, or higher alcohol. , mixed with macrogols or zolastic base (Hydrocarnol ointment base manufactured by Taisho Pharmaceutical K.K-), hydrophilic ointments listed in the Japanese Pharmacopoeia, water-absorbing ointments, or mixtures thereof, and added as necessary. Add oils such as Co97 oil, peanut oil, and olive oil, resins, glycerin, propylene glycol, surfactants, bactericides, fungicides, antioxidants, etc., and mix thoroughly until homogeneous. Let's practice together.

Suppositories are made in much the same way as ointments; for example, a preservative and homocarnosine are added to a dissolved suppository base, mixed uniformly, poured into a mold, allowed to solidify, and then taken out.

Next, examples of formulations of the antitumor agent of the present invention will be given.

Formulation example/(injection) Under aseptic operation, synthesize 3 pieces of homocarnosine into 1 bottle.
．． ! (both as homocarnosine) were filled into ampoules as an aqueous solution.

Formulation example (I111I granules) The following formulation using the synthesized homocarnosine Homocarnosine 0.2 1 Lactose 063 g Corn starch o, lI! ry granules /, 0
Granules were manufactured using 011.

Formulation Example 3 (Ointment) Using the synthesized homocarnosine, the following formulation was made using hydrocarboxylic ointment as a base: homocarnosine O0Ω
I Hydrocarbon Ointment Base99. g I00 g at 0-. A 1% ointment was prepared.

Formulation Example 1I (Layering agent) Using synthesized homocarnosine and using Fosco s-, rt (Maruishi Seiyaku KK) as a base, the following formulation (layering agent/part) Homocarnosine O20co I, ethyl 4-hydroxybenzoate θ, Oθθgs A layer agent was prepared using an appropriate amount of 1 Phosco 5-tt.

Homocal 7-nosine and ethyl 79-hydroxybenzoate are sieved through a 100-mesh sieve. Phosco S dissolved at r0°C
-j'j was added in multiple tones at a time to make it uniform. The mixture was poured into the mold at 0C for 3 times, allowed to cool to solidify at room temperature, and then cooled in a refrigerator. This was removed from the mold and wrapped in Nozera-Fin paper.

Claims (1)

[Claims] An antitumor agent containing homocarnosine or its salt as an active ingredient.