JPH0623108B2 - Tumor formation inhibiting composition - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to inhibiting tumor cell-associated tumor progression in vitro and actual tumor progression in vivo in warm-blooded animals.

The disease of cancer is caused by the progression of malignant tumors. Extensive medical research has been devoted to reducing and overcoming the causes of cancer. To date, no cure for cancer has been found. However, much has been learned about the mechanisms by which warm-blooded animals avoid the distress associated with cancer. Based on these findings, the present invention provides a substance that suppresses tumor progression and a method of suppressing the progression thereof.

Prior Art: Cells contained in mammalian body fluid include lymphocytes, monocytes,
There are macrophages and polymorphonuclear cells. These cells act as a natural monitoring system to combat tumor progression from lower mammals (eg rodents) to humans. Recently, it has been found that a small population of specific lymphocytes or lymphoid cells called "Natural Killer" or "NK" cells destroy tumor cells and thereby prevent the progression of cancer. According to the weight of NK cells
₂ O _2), or oxygen-containing radicals such as hydroxy anion ^(· OH) and superoxide ion _{(O 2} ^·)
It is suggested to have cytolytic activity associated with the development of Harbelman et al., "Science", Vol. 214, 2, 1981 for NK cells and active oxygen phenomena.
Mon, pages 24-30; Rodel et al., "Nature", 29.
8: 5, August 1982, pp. 569-572; Natan et al., "Journal of Immunology", 129,
No. 5, November 1982, pp. 2164-2171; and Mavier et al., "Journal of Immunology", Volume 132, No. 4, April 1984, 1980-.
See page 1986.

Of course, there are many compounds that release reactive oxygen species. However, this factor is the only N
Complements the function of K cells, or is introduced into a site where tumor formation has not sufficiently occurred to allow NK cells to function,
It does not mean that tumor progression is suppressed. Compounds that release reactive oxygen species generally do this very quickly, but their efficacy in combating tumor progression in warm-blooded animals such as humans appears to require at least a slower and more sustained release rate. Until the present invention, this has not been effectively achieved. If oxygen is released too quickly, both tumor and normal cells can be attacked.

The present inventors' U.S. Pat. No. 4,041,149 (issued Aug. 9, 1977) describes various forms (including dental tablets) in which the active ingredient is peroxodiphosphate, which suppresses the production of halitosis. Compositions are described. Peroxodiphosphate compounds differ from most oxygen-donating compounds in that they do not initially supply hydrogen peroxide rapidly. Rather, it gradually releases hydrogen peroxide, so
When compared to hydrogen peroxide at the same concentration, the amount of oxygen released by peroxodiphosphate is one-tenth of the amount of oxygen released and utilized by hydrogen peroxide. Furthermore, in the presence of alkaline phosphatase or acid phosphatase present in the body of warm-blooded animals (including mice, rats, humans, etc.), about 50% of active oxygen is released within 20 hours at 25 ° C. Only.

Problems to be Solved by the Invention: An advantage of the present invention is that tumor progression is suppressed against tumor cells in vitro and actual malignant tumor progression in vivo in warm blooded animals ranging from rodents to humans. Is to be done.

Another advantage of the present invention is that it provides a method of inhibiting tumorigenesis by introducing into the living host a substance that gradually releases oxygen.

Means for Solving the Problems: According to a particular aspect, the present invention provides a non-toxic, water-soluble pharmaceutically acceptable derivative of peroxodiphosphate dissolved or dispersed in a pharmaceutical carrier of about 0.1-10%. To a composition comprising

In another aspect, the invention provides about 0.1-6 per kg body weight.
g of a nontoxic dose of a nontoxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate, dissolved or dispersed in a carrier for formulation, per day per kg body weight of a warm-blooded animal. The present invention relates to a method for suppressing the formation of tumors containing malignant tumor cells, which comprises administering to a warm-blooded animal host by ingesting in a manner to supply about 0.1 to 6 g per.

According to another particular aspect, the present invention provides a non-toxic, water-soluble pharmaceutically acceptable formulation of peroxodiphosphate at a non-toxic dose of about 0.1 to 2 g / Kg body weight of a warm-blooded animal having malignant tumor cells. Method for inhibiting tumor formation, which comprises systemically administering a composition comprising a derivative capable of being dissolved or dispersed in a carrier for preparation to a warm-blooded animal host in a manner to supply about 0.1 to 2 g per 1 kg of body weight of the warm-blooded animal. Regarding

Peroxodiphosphate compounds (PDPs, peroxodiphosphate derivatives) are pharmaceutically acceptable non-toxic compounds, which exceed the salts set forth in the aforementioned US Pat. No. 4,041,149. These compounds include alkali metal (eg lithium, sodium and potassium) salts, alkaline earth metal (eg magnesium, calcium and strontium) salts, zinc salts and tin salts, and the organic peroxodiphosphate C.
Also included are salts such as _1-12 alkyl, adenylyl, guanylyl, cytosilyl and thymilyl esters, and quaternary ammonium salts. Alkali metal salts, especially potassium salts, are preferred among the inorganic cationic salts. Tetrapotassium peroxodiphosphate has a molecular weight of 346.35 and active oxygen content
Stable, odorless, fine free-flowing, white, non-hygroscopic crystalline solid with 4.6%. Tetrapotassium peroxodiphosphate is 47-51% water-soluble at 0-61 ° C, but is insoluble in common solvents such as acetonitrile, alcohols, ethers, ketones, dimethylformamide, dimethylsulfoxide and the like. A 2% aqueous solution has a pH of about 9.6
And its saturated solution has a pH of about 10.9. 1 at 25 ° C
The 0% aqueous solution showed no loss of active oxygen even after 4 months, and 5
The 10% solution at 0 ° C. showed 3% active oxygen loss over 6 months.

Organic salts appear to be particularly suitable for administration against malignant tumors. Among the organic esters, those that impart hydrophobicity, such as those having a C _1-12 alkyl group, and those that allow for rapid uptake of the pexoxodiphosphate moiety by cells, such as adenylyl, guanylyl, cytosilyl and thienyl. Myryl esters are preferred.

Formulations suitable for oral ingestion are tablets coated with a substance that resists degradation by gastric acid at a gastric pH (about 1-3).
This is because pequoxodiphosphate is inactivated by such gastric acid. Carriers containing tableted granules of solid peroxodiphosphate are higher than this
It is dissolved by intestinal fluid having a pH (approximately 5.5 to 10), does not inactivate peroxodiphosphate, and keeps it in a state of being enzymatically acted upon by phosphatase existing in humans and other warm-blooded animals. A preferred tablet coating solution is a fatty acid ester such as N-butyl stearate (typically about 40
To 50 parts by weight, preferably about 45 parts by weight); wax, such as carnauba wax (generally about 15 to 25 parts by weight, preferably about 20 parts by weight); fatty acid, such as stearic acid (generally about 20 to 30 parts by weight, preferably Is 25 parts by weight); and a cellulose ester such as cellulose acetate phthalate (generally about 5 to 15 parts by weight, preferably about 10 parts by weight); and an organic solvent (generally about 400 to 90 parts by weight).
0 parts). Other desirable coating materials include ceramics, and copolymers of maleic anhydride and ethylenic compounds such as polyvinyl methyl ether. This type of coating is different from orally disintegrating tablets, where the tablet material generally comprises about 80-90 parts by weight mannitol and about 30-40 parts by weight magnesium stearate.

Tableted peroxodiphosphate granules are prepared by blending about 30-50 parts by weight of peroxodiphosphate with about 45-65 parts by weight of solid polyhydroxy sugar (eg, mannitol) to form a solution of polyhydroxy sugar compound (eg, sorbitol). Wetting with about 20-35 parts by weight, sieving to constant size, blending with about 20-35 parts by weight of a binder (eg magnesium stearate) and compressing the granules in a tablet machine into tablets. Manufactured by.
The tableted granules are coated by spraying them with a foam of a solution of the coating material and drying to remove the solvent. Tablets of this kind generally differ from compressed dental tablets without special protective coating.

If the administration is by oral gavage, the effective dose of peroxodiphosphate in the indicated manner is about 0.1.
~ 6 g / Kg (body weight) / day; administration is systemic,
For example, by intramuscular, intraperitoneal or intravenous injection, the dose is about 0.1-2 g / Kg body weight / day.

Physiologically acceptable pyrogen-free solvents are suitable carriers for use in the art-recognized manner for systemic administration. Saline buffered with phosphate to a physiological pH of about 7-7.4 is a preferred carrier for systemic administration. This type of solvent differs from the humectant vehicle commonly used in dentifrices. Liquids of this type are generally sterilized by deionized distilled water, and Tsuji et al.
October 984, pp. 35-41) and a test to confirm that it is pyrogen-free by the Limulus amebocyte lysate (LAL) test, and then prepared in sterile pyrogen-free water. Prepared phosphate buffer (eg, pH about 8.5-10) and about 1-100 mg of the peroxodiphosphate derivative, and sodium chloride to a concentration of about 0.5-1.5% by weight. The solution is re-sterilized by passing through a microporous filter and then filled into vials for use.
As an alternative, sodium chloride 0.86% by weight, potassium chloride 0.
Other solutions such as Ringer's solution containing 03 wt% and calcium chloride 0.033 wt% may be used.

The peroxodiphosphate compound (PDP) gradually releases hydrogen peroxide in the presence of a phosphatase-type enzyme according to the following formula.

In the above formula, X is a non-toxic, pharmaceutically acceptable cation or completes the organic ester moiety. The phosphatase that decomposes peroxodiphosphate is saliva,
And in plasma, intestinal fluid and white blood cells. Slow oxygen release is particularly effective in complementing the efficacy of NK cells against malignant tumor cells associated with peroxodiphosphate therapy. When warm-blooded animals are treated with PDP according to the present invention, it is desirable to employ a mode in which the treatment is continued at least until the time when the tumor recurs.

The invention is illustrated by the following example. All amounts are by weight unless otherwise noted.

Example 1 In vitro test for tumor cytotoxicity of PDP In this test the effect of PDP on the proliferation of murine myeloma (SP ₂ line) cells was investigated at various concentrations (Table 1). Human gingival fibroblasts were used as control normal cells (Table 2). Cells were grown in Dworbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, MEM vitamin 1x, L-glutamine 1x, NEAA ₃ 1x, gentamicin 1x. This was incubated at 37 ° C. in a humidified CO ₂ atmosphere. Approximately 1 to 3 × 10 ^{5 in} each well of a 24-well microtiter plate containing 2 ml of medium.
Cells were placed and various concentrations of PDP (potassium salt) were added.

After the incubation, the viability of the cells was judged by removing a certain portion from the hole at the time indicated in Table 1.
Viability was assessed by trypan blue exclusion test. Fresh medium was added daily to each well to maintain the required growth conditions. The inhibition rate was calculated by comparing the% of cells surviving in phosphate buffered saline (PBS) with that of PDP. The data are summarized in Table 1.

These results indicate that the potassium salt of PDP is highly cytotoxic and murine to murine myeloma (cancer) cells as compared to the control buffer.

Table 2 describes the effect on normal cells (human gingival fibroblasts).

The data in Table 2 show that PDP 100-500 mcg / ml had no significant effect on cell proliferation, but 1000 and 2500 mcg / ml.
It shows that even in normal cells, the viability is reduced in ml. It is worth noting that the effect on myeloma cells (Table 1) is more pronounced than the effect on normal cells (Table 2) even at high concentrations.

Lithium salt, sodium salt, magnesium salt of PDP,
Similar results were obtained with calcium salts, strontium salts, zinc salts and stannous salts, as well as C _1-12 alkyl, adenylyl, guanylyl, cytosilyl, thymilyl esters and tetramethylammonium salts of organic peroxodiphosphates or PDPs. Was obtained.

Example 2 Effect of PDP, Potassium Pyrophosphate (KPP) and PBS (Phosphate Buffered Saline) on Tumor Progression In Vivo Mean Body Weight 20 g ± 3 g, Genetically Equal Valve (Balb) of 25 Groups Each 75 / C mice were treated with (a) control phosphate buffer (PBS), (b) potassium peroxodiphosphate (PDP) and PBS (pH 7.0), and (c) pyrroline. Potassium acid (KPP) and P
Treated with BS (as a phosphate control). Animals were prepared for inoculation of malignant SP ₂ cells (murine myeloma carcinoma cells) by intraperitoneal administration of 0.2 ml of pristane to each animal. After 3 weeks, the animals were treated by ingestion in the following manner. Group (a) 0.2 ml PBS administered intraperitoneally; group
(b) Administration of 2.0 mg of PDP suspended in 0.2 ml of PBS; and Group (c) Administration of 2.0 mg of KPP in 0.2 ml of PBS (3 consecutive days). SP ₂ for each animal 48 hours after the third injection
Cells (mouse tumor cells, murine myeloma) 2-3 × 10 ⁶
Individuals were inoculated (intraperitoneal). The animals were then dosed with each of these substances once daily for 5 days / week. That is (a)
PBS, (b) PDP or (c) KPP. Animals were scored weekly for tumor progression and death. The data are based on the Mantel-Hentzel method (statistical viewpoint of analysis of data obtained from a retrospective survey of diseases, Journal of National Canon Institute, Volume 3, 719-748.
Pp. 1959). The data in Tables 3, 4 and 5 indicate that PDP is significantly more effective in suppressing tumor progression in mice as compared to PBS or KPP, thereby inhibiting tumor progression in reactive oxygen species. It is proved that it is not supplied by phosphate but by phosphate.

PBS, KPP and PDP were intramuscularly and intravenously administered in PBS at the same concentration, or orally, 45 parts of N-butyl stearate, carnauba wax 2
Similar results were obtained when administered at a concentration of 1 mg / ml (0.1%) in a stable carrier of 0 part, 25 parts stearic acid and 10 parts cellulose acetate phthalate.

Other inorganic salts of PDP, especially lithium salt, sodium salt,
Similar results were obtained with magnesium, calcium, strontium, zinc and stannous salts. Organic compounds of PDP, especially C _1-12 alkyl, adenylyl, guanylyl, cytosilyl, thymilyl ester, and tetramethylammonium salts are also effective in combating the growth of murine myeloma malignant tumor cells.

Example 3 500 parts of potassium peroxodiphosphate and 641 parts of mannitol were blended together and a 10% sorbitol solution 32.5
Part wetted to form wet granules, dried at 49 ° C. and sieved through a 12 mesh US sieve (sieve opening 1.68 mm). Then, 35 parts of magnesium stearate was added as a binder, and the composition was compressed by a tableting machine to produce tableting granules.

These tablets were coated with the enteric coating solution described below.

Cellulose acetate phthalate 120 parts Carnauba wax 30 parts Stearic acid 10 parts 95% ethanol 450 parts Acetone 1000 parts Coating was performed by injection treatment in a common coating pan.

When the tablets thus produced were ingested, they passed through the stomach undegraded and the coating was then dissolved by the intestinal fluid.

Example 4 Distilled deionized water in an autoclave at atmospheric pressure 2
Sterilized for 0 minutes. After cooling, Tsuji et al.
Pyrogen-free was tested using the Limulus amebocyte lysin (LAL) described in October 984, pp. 35-41). 0.1% containing 50 parts potassium peroxodiphosphate, sodium chloride in an amount corresponding to 0.9% of the solution, and KH ₂ PO ₄ and Na ₂ HPO _4.
M phosphate buffer (pH 9.4) was added to pyrogen-free sterile water. The solution was then sterilized by passing through a 0.5 microporous filter, then filled into a sterile file.

Although the present invention has been described in relation to particular examples, it will be apparent to those skilled in the art that various modifications can be made therein, and these are also included in the scope of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location A61K 33/42 8314-4C 47/02 G 7433-4C 47/12 D 7433-4C 47/14 D 7433-4C 47/38 D 7433-4C

Claims (10)

[Claims] 1. A tumor formation-suppressing composition comprising a dose of 0.1 to 10% of a nontoxic, water-soluble pharmaceutically acceptable derivative of peroxodiphosphate dissolved or dispersed in a carrier for the preparation. The tumor carrier is a phosphate buffered saline solution having a pH of 7.0-7.4, or a coating tablet material resistant to degradation by gastric acid but degraded by intestinal fluid of pH 5.5-10. Composition for suppressing formation. 2. A composition according to claim 1, wherein the pharmaceutical carrier is a phosphate buffered saline solution. 3. A composition according to claim 1 wherein the pharmaceutical carrier is a coating tablet material which resists degradation by gastric acid but is degraded by intestinal fluid at pH 5.5-10. 4. A coating of tablets with a carrier for formulation comprising 40 to 50 parts by weight of fatty acid ester, 15 to 25 parts by weight of wax, 20 to 30 parts by weight of fatty acid, and 5 to 15 parts by weight of cellulose ester. The composition according to item 3. 5. The method according to claim 4, wherein the fatty acid ester is N-butyl stearate, the wax is carnauba wax, the fatty acid is stearic acid, and the cellulose ester is cellulose acetate phthalate. Composition. 6. A non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate is a salt selected from the group consisting of alkali metals, alkaline earth metals, zinc and tin. The composition according to item 1. 7. The salt is potassium peroxodiphosphate,
The composition according to claim 6. 8. The composition according to claim 1, wherein the derivative is a C _1-12 alkyl ester of peroxodiphosphoric acid. 9. A non-toxic, water-soluble pharmaceutically acceptable derivative of peroxodiphosphate is blended with a solid polyhydroxy sugar, the blend is moistened with a solution of the polyhydroxy sugar compound and sieved to size. The content of the tablet was kept constant, and this was blended with a binder and compressed into a tabletted granule.
Coating by spraying a film of coating liquid which is dissolved by intestinal fluid of
A process for producing tabletted granules having a coating which is not decomposed when passing through the stomach and is dissolved by intestinal fluid having a pH of 5.5 to 10. 10. Deionized distilled water is sterilized to make it pyrogen-free, and then a phosphate buffer and a peroxodiphosphate derivative and sodium chloride are added thereto. A method for producing a liquid preparation suitable for intragastric administration of a non-toxic, water-soluble pharmaceutically acceptable derivative.