JPS6140217A - Control of cancer growth - 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 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.

Cancer is a disease caused by the progression of malignant tumors.

A huge amount of 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 suffering associated with cancer.

Based on these findings, the present invention provides substances that inhibit tumor progression and methods for inhibiting tumor progression.

Prior art: Cells contained in mammalian body fluids include lymphocytes, monocytes,
There are macrophages and polymorphonuclear cells.

These cells act as a natural surveillance system against tumor progression from lower mammals (eg, fungi) to humans. In recent years, 'Natural Killer' (Natural Killer)
According to 0 proof weights that a small population of specific lymphocytes or lymphoid cells called ral K11ler# or ``NK# cells'' have been shown to destroy tumor cells, thereby halting the progression of cancer. , it has been suggested that NK cells have cytolytic activity associated with the generation of reactive oxygen species such as hydrogen peroxide (H2O2) or oxygen-containing radicals such as hydroxy anion ('OH) and superoxide anion (02°). Regarding NK cells and active oxygen phenomena, see No\-Pelman et al. 1'Science'', Vol. 214, 2. October 1981, pp. 24-30↓
Rodel et al., Nature, vol. 298, 5.1982
August, pp. 569-572; Nathan et al., 6 Journal Φ
Opken Immunology”, vol. 129, Ya 5. 1982 1
January, pp. 2164-2171 and Maviel et al., 6
Journal of Immunology'', vol. 132, 4.
There are, of course, many compounds that release active oxygen species.

However, this factor is the only compound that is introduced into the body to supplement the function of NK cells, or introduced into areas where tumor formation is not sufficiently occurring to activate NK cells and suppress tumor progression. It doesn't mean that you do. Compounds that release reactive oxygen species generally do so very rapidly, but efficacy in combating lung cancer progression in warm-blooded animals such as humans appears to require at least a slower and more sustained rate of release. Until the present invention, this had not been effectively accomplished. If oxygen is released too quickly, both tumor cells and normal cells can be attacked.

No. 4,041,149 (197
The specification describes compositions in various forms (including dental tablets) for suppressing the production of bad breath, in which the active ingredient is Belokundi phosphate. Diphosphate compounds differ from most oxygen supply compounds in that they do not initially supply hydrogen peroxide abruptly. Rather, it releases hydrogen peroxide gradually, so when comparing equivalent concentrations of hydrogen peroxide, the amount of oxygen released by beroxodiphosphate is not released and utilized by hydrogen peroxide. This is one tenth of the amount of oxygen in the Furthermore, in the presence of alkaline phosphatase or acid phosphatase present in the body of warm-blooded animals (including rats, humans, etc.), 2
Only about 50% of the active oxygen is released within 0 hours.

Problems to be Solved by the Invention: An advantage of the present invention is that tumor progression is inhibited in warm-blooded animals ranging from fungi to humans, relative to the progression of tumor cells in vitro and actual malignant tumors in vivo. Is Rukoto.

Another advantage of the present invention is that it provides a method of inhibiting tumor formation by introducing slowly oxygen-releasing substances into a living host.

SUMMARY OF THE INVENTION According to a particular aspect, the present invention provides a non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphoric acid dissolved or dispersed in a pharmaceutical carrier. Concerning a composition consisting of a dose of 10%.

According to another aspect, the present invention provides about 0.1 kg of body weight per kg of body weight.
Non-toxic dose of peroxodiphosphate of ~6g,
A composition comprising a water-soluble pharmaceutically acceptable derivative dissolved or dispersed in a pharmaceutical carrier is administered orally in a manner that provides about 0.1 to 6 g per IKg of body weight per warm-blooded animal per day. The present invention relates to a method for inhibiting the formation of tumors containing malignant tumor cells, which comprises administering to a warm-blooded animal host by ingestion.

According to another particular aspect, the present invention provides a non-toxic, water-soluble formulation of verocunnilic acid at a non-toxic dose of about 0.1 to 2 y/kg body weight of a warm-blooded animal having malignant tumor cells. A composition comprising a pharmaceutically acceptable derivative dissolved or dispersed in a pharmaceutical carrier is administered to a warm-blooded animal host in such a manner that about 0.1 to 2 g per body weight of the warm-blooded animal is supplied. This invention relates to a method for inhibiting tumor formation by systemic administration.

Peroxodiphosphate compounds (FDP, peroxodiphosphate derivatives) are pharmaceutically acceptable, non-toxic compounds that exceed the salts shown in the aforementioned U.S. Pat. No. 4,041,149. It is something. These compounds include alkali metal (e.g. lithium, sodium and potassium) salts, alkaline earth metal (e.g. magnesium, calcium and strontium) salts, zinc and tin salts, and the organoberoxodiphosphates Cl-12 alkyl, adenylyl and Also included are salts such as guanylyl, cytosilyl and thymylyl esters, and quaternary ammonium salts. Alkali metal salts, particularly potassium salts, are preferred among the inorganic cationic ones.

Tetrapotassium peroxodiphosphate is a stable, odorless, fine, free-flowing, white, non-hygroscopic crystalline solid with a molecular weight of 346.35 and an active oxygen content of 4.6%. Tetrapotassium peroxodiphosphate is 47-51% at 0-61℃
Although soluble in water, K is insoluble in common solvents such as acetonitrile, alcohols, ethers, ketones, dimethylformamide, dimethyl sulfoxide, and the like. An aqueous solution of 2T has a pH of about 9.6, and its saturated solution has a pH of about 10.
．． It has a pH of 9. At 25°C, the lO2 aqueous solution showed no loss of active oxygen at the edge of the blade, and at 50°C, the 10% solution showed 3% loss of active oxygen over 6 months.

Organic salts appear to be particularly suitable for administration against malignant tumors. Among the organic esters, those that confer hydrophobicity, such as those with a Cl-12 alkyl group, and those that allow rapid uptake of the bexoxodiphosphate moiety by cells, such as adenylyl, guanylyl, cytosylyl and thymyl. esters are preferred.

Formulations suitable for oral ingestion are tablets coated with a substance that resists degradation by gastric acid at intragastric pH (approximately 1-3). This is because beroxodiphosphate is cineactivated by such gastric acid. A carrier containing compressed granules of solid bexodiphosphate is soluble in intestinal fluids with a much higher pH (approximately 5.5-10) and does not inactivate the bereoxodiphosphate. , maintained in a state where it is subjected to enzymatic action by phosphatases present in humans and other warm-blooded animals. Desirable tablet coating liquids include fatty acid esters, such as N-butyl stearate (generally about 40-50 parts by weight, preferably about 45 parts by weight); waxes, such as carnauba wax (generally about 15-25 parts by weight, preferably about 20 parts by weight); fatty acids such as stearic acid (generally about 20-30 parts by weight, preferably 25 parts by weight); and cellulose esters such as cellulose acetate phthalate (generally about 5-15 parts by weight, preferably about 10 parts by weight); and organic solvents (generally about 400-9
00 part) Yosi Naru. Other desirable coating materials include shellac and copolymers of maleic anhydride and ethylene-based compounds, such as polyvinyl methyl ether. The coating of rice seeds differs from tablets that disintegrate in the mouth, where the tablet material generally comprises about 80-90 parts by weight of mannitol and about 30-40 parts by weight of magnesium stearate.

Compressed Bell Oxodiphosphate Granules About 30 to 50 parts by weight of Havel Oxodiphosphate are blended with about 45 to 65 parts by weight of a solid polyhuman hydroxysaccharide (e.g., mannitol) and a polyhydroxy sugar compound solution (e.g., sorbitol) is prepared. It is moistened with about 20 to 35 parts by weight, sieved to a uniform size, blended with about 20 to 35 parts by weight of a binder (e.g., magnesium stearate), and compressed into tablets in a granular tablet machine. Manufactured by

The tabletted granules are coated by spraying them with a foam of a solution of coating material and drying to remove the solvent. This type of tablet differs from compressed dental tablets, which generally do not have special protective coatings.

When administration is by oral ingestion, the effective dosage of pequoxodiphosphate in the manner indicated is approximately 0.05 mg.
1-69/Kf (body weight) for 7 days; if administration is systemic, such as by intramuscular, intraperitoneal or intravenous injection, the dose is about 0.1-29/Kf (body weight) It is the 7th.

Physiologically acceptable pyrogen-free solvents are suitable carriers for use in an 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 is different from the humectant vehicles commonly used in dentifrices. This type of liquid preparation is generally made by sterilizing deionized distilled water, and Tsuji et al.
(October 1984, pp. 35-41) A test to confirm pyrogen-free conditions was performed using the Limulus amoeba-like cell lysate (LAL) test described by K. Phosphate buffer (
ItipH about 8.5-10) and beroxodiphosphate derivatives about 1-100, and about 0.5-100
It is prepared by adding sodium chloride to a concentration of 1.5% by weight. This solution is passed through a perforated filter and re-sterilized by K. and then filled into vials for use. Alternatively, other solutions such as Ringer's solution containing 0.86% by weight of sodium chloride, 0.03% by weight of potassium chloride and 0.033% by weight of calcium chloride may be used.

Hydrogen peroxide is gradually released according to the following formula in the presence of beroxodiphosphate compound (FDP)ti phosphatase enzymes.

In the above formula, X is a non-toxic, pharmaceutically acceptable cation or completes an organic ester moiety. Phosphatases that break down verokundiphosphate are present in saliva, as well as plasma, intestinal fluid, and white blood cells. Slow oxygen release complements NK cell efficacy against malignant tumor cells associated with beloxodiphosphate therapy
Particularly effective. When treating warm-blooded animals with FDP according to the present invention, it is desirable to employ a modality in which treatment is continued at least until the point at which the tumor regresses.

The invention is illustrated by the following examples. All amounts are by weight unless otherwise specified.

Example I In vitro study of tumor cytotoxicity of FDP.
The effect of DP was investigated at various concentrations (Table 1). Human gingival fibroblasts were used as control normal cells (Table 2
). Cells contain 10% bovine serving serum, MEM vitamin is,
L-Glutamine IZ% NEAA3 1 z1 Gentamai'/71Z was grown in Dulbecko's modified Eagle's medium supplemented. This was incubated at 37°C in a humidified CO2 atmosphere. Approximately 1 to 3 x 10 cells in each well of a 24-well microtiter plate containing medium 2-
cells were placed in the wells, and various concentrations of FDP (potassium salt) were added.

After incubation, cell viability was determined by removing aliquots from the wells at the times indicated in Table 1.

Viability was assessed by trypan blue exclusion test. Fresh medium was added daily to each well to maintain the necessary growth conditions. The inhibition rate was calculated by comparing the ratio of cells surviving in phosphate buffered saline (PBS) to that in FDP. The data are summarized in Table 1.

Table 1 Control (PBS) 4 8.98±0.14 100%
PDP pH7.0 100mtJ/vt 41.86±〇, 1447500
/ 41.33±0.03331000 //
4 1.07±ζ17292NJ00 〃40.48
±0.1512 These results demonstrate that the potassium salt of FDP is highly cytotoxic and inhibitory to murine myeloma (cancer) cells compared to the control buffer.

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

Table 2 Control (PBS) 4 2.67±0.17 100cm PDP pH7.0 100mc'i/ml 4 2.61±0.16
98500 7 4 2.58±0.13 97
1000 // 4 2.12±0.15
792500 // 4 1.97±0.11
74 The data in Table 2 is for PDP 100-500t
Although nc'j/vrl has no significant effect on cell proliferation, 1000 and 2500 mcq7- show a decrease in survival rate even for normal cells. It is noteworthy that the effect on myeloma cells (Table 1) is more pronounced than on normal cells (Table 2) even at high concentrations.

FDP lithium salt, sodium salt, magnesium salt,
Similar results were obtained for the calcium, strontium, zinc and stannous salts, as well as the Cl-12 alkyl, adenylyl, guanylyl, cytosilyl, thymyryl ester and tetramethylammonium salts of organic beroxodihonuphate or PDP. It was done.

Example 2 Effect of FDP, Potassium Pyrophosphate (KPP) and PBS (phosphate buffered saline) on tumor progression in vivo Mean body weight 20g ± 39, genetically equivalent valves CBatb)/in groups of 25 animals each Seventy-five C mice were treated with (α) control phosphate buffered saline (PBS) and (b) potassium peroxodiphosphate (FDP) and PBS (pH
7,0) and (C) potassium pyrophosphate (
KPP) and PBS (as a phosphate control). Pristang for each animal
) 0.2− was administered intraperitoneally to the animals to induce malignant SP2 cells (
The cells were prepared for inoculation (murine myeloma carcinoma cells). After 3 weeks, animals were treated by oral feeding in the following manner in group 0 (c
L) Intraperitoneal administration of PBS O,2-; Group (b)
Administer FDP2.0rnf suspended in PBSO,2-; and group (c) KPP2 in PBSO12-
．． Administration of 0fFIjl (for 3 consecutive days) 48 hours after the third injection, each animal received KSP2 cells (mouse tumor cells,
(Murine myeloma) 2-3 x 106 cells were inoculated (intraperitoneally)
. The animals were then administered their respective substances once a day for 5 days/week.

i.e. (α) PBS, (6) FDP or (C) KP
It is P. Animals were scored weekly for tumor progression and death. Data were collected using the Mantel-Hefuer method (Statistical aspects of the analysis of data obtained from retrospective surveys of diseases, Journal of National Cancer Institute, 3).
Vol., pp. 719-748, 1959). The data in Tables 3, 4 and 5 show that PDP is significantly more effective in controlling tumor progression in mice compared to PBS or KPP, and thus the effect of inhibiting tumor progression is This is proven to be due to the supply of oxygen species and not due to phosphate.

Table 3 Mantel-Hentzel X square = 0.36.1, d, f,
, P = 0.55, Otuzu = 1.34 These results were not significant, indicating that PB
No significant difference was shown between S and KPP.

*PBS = Phosphate Buffered Saline **KPP = Potassium Pyrophosphate Table 4 Mantel-Haenszel X Square = 10.40,1, d, f
, P = 0.0010 Otsuzu-3,660 These data indicate that the control PBS group had significantly more rapid tumor progression than FDP-treated animals (P = 0.0010).
1).

*PBS = Phosphate Buffered Saline FDP = Potassium Bekuoxoniphosphate Table 5 Mantel-Haenszel X Square = 5.86.1, d, f,
, P = 0.020 = 2.60° These data indicate that the tumors progressed significantly more rapidly in the KPP group than in the PDP-treated animals.

*KPP=potassium pyrophosphate***PDP=potassium peroxodiphosphate BS,K
PP and PDP were administered intramuscularly and intravenously at the same concentration in PBS, respectively, or orally with 45 parts of N-butyl stearate, 20 parts of carnauba wax, 25 parts of stearic acid, and 10 parts of cellulose hyacetate phthalate. One of the most stable carriers! Similar results were obtained when administered at a concentration of /m 2 (0.1 h).

Other inorganic salts of PDP, especially lithium salts, sodium salts,
Similar results were obtained using magnesium salts, calcium salts, stomintyram salts, zinc salts and stannous salts of oPDP organic compounds, especially CT-12 alkyl, adenylyl, guanylyl, cytosilyl, thymyryl esters,
and tetramethylammonium salts are also effective in combating the proliferation of murine myeloma malignant tumor cells.

Example 3 500 parts of potassium peroxodiphosphate and 641 parts of mannitol were blended and 32 parts of a 10 thiorbitol solution was prepared.
．． 5 parts to form wet granules, which were dried at 49°C and 12 mesh US m < sieve opening 1.68 mg
) sieved with 0 then magnesium stearate 35
The tablets were coated with the enteric coating solution described below.

Cellulose acetate phthalate 120 parts Carnauba wax 30 parts Stearic acid 10 parts 95 L'' 450 parts Coating was carried out by pouring in a typical coating tank.

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

Example 4 Deionized distilled water is heated to atmospheric pressure in an autoclave for 2 hours.
After sterilizing and cooling for 0 minutes, Tsuji et al.
Limulus ameboid cytolysin (LAL) was tested for pyrogen incompatibility using the Limulus ameboid cytolysin (LAL) described in 1984, October 1984, pp. 35-41).

50% potassium peroxodiphosphate, an amount of sodium chloride corresponding to 0.9 t of solution, and KKH2PO4
and 0.1 M phosphate buffer (pH 9.4) containing Na2HPO4 in sterile pyrogen-free water @, voL.
The solution was then sterilized by passing through a 0.5 microporous filter and then filled into sterile files.

Claims (26)

[Claims] (1) Dissolved or dispersed in a pharmaceutical carrier;
The pharmaceutical carrier is a phosphate buffered saline solution having a pH of about 7.0 to 7.4, or by intestinal fluids that resist degradation by gastric acid but has a pH of about 5.5 to 10. A non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate, which is a disintegrating coating tablet material, about 0
．． Compositions consisting of doses of 1-10%. (2) The composition of claim 1, wherein the formulation carrier is a phosphate buffered saline solution. 3. The composition of claim 1, wherein the pharmaceutical carrier is a coated tablet material that resists degradation by gastric acid but is degraded by intestinal fluids at a pH of about 5.5-10. (4) The coating of the tablet with the pharmaceutical carrier comprises about 40-50 parts by weight of fatty acid ester, about 15-25 parts by weight of wax, about 20-30 parts by weight of fatty acid, and about 5-15 parts by weight of cellulose ester. The composition according to item 3. (5) The composition 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. . (6) The non-toxic, water-soluble, pharmaceutically acceptable derivatives of peroxodiphosphate include alkali metals, alkaline earth metals,
A composition according to claim 1, which is a salt selected from the group consisting of zinc and tin. (7) A non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphoric acid is selected from the group consisting of C_1_-_1_2 alkyl, adenylyl, guanylyl, cytosylyl, thymylyl esters and quaternary ammonium salts. The composition according to item 1. (8) The composition according to claim 6, wherein the salt is potassium peroxodiphosphate. (9) C_1_-_1_ whose derivative is peroxodiphosphoric acid
8. The composition of claim 7, which is a 2-alkyl ester. (10) The composition according to claim 7, wherein the derivative is an adenylyl, guanylyl, cytosylyl, or thymylyl ester of peroxodiphosphoric acid. (11) Approximately 0.1 to 6 g/kg body weight of a warm-blooded animal of a nontoxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate that resists degradation by gastric acid but p
About 0.1 to 6 g per kg body weight of a warm-blooded animal per day of a composition consisting of a coated tablet material dissolved or dispersed in a pharmaceutical carrier that is disintegrated by intestinal fluids of about 5.5 to 10 H A method for inhibiting the formation of malignant tumor cells in a warm-blooded animal, the method comprising administering to a warm-blooded animal host by oral ingestion in a manner such that the host is provided with the following: (12) The tablet coating consists of about 40 to 50 parts by weight of N-butyl stearate, about 15 to 25 parts by weight of carnauba wax, about 20 to 30 parts by weight of stearic acid, and about 5 to 15 parts by weight of cellulose acetate phthalate. , the method according to claim 10. (13) Claim 11, wherein the non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphoric acid is a salt of a metal selected from the group consisting of alkali metals, alkaline earth metals, zinc and tin. The method described in. (14) The non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate is C_1_-_1_2 alkyl;
selected from the group consisting of adenylyl, guanylyl, cytosilyl, thymylyl esters and quaternary ammonium salts,
A method according to claim 11. (15) The method according to claim 13, wherein the salt is potassium peroxodiphosphate. (16) C_1_-_1 whose derivative is peroxodiphosphoric acid
15. The method of claim 14, wherein the _2 alkyl ester. (17) the derivative is an adenylyl, guanylyl, cytosylyl or thymylyl ester of peroxodiphosphoric acid;
A method according to claim 14. (18) A nontoxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate at a nontoxic dose of about 7.0 to 7.4 g per kg of body weight of a warm-blooded animal. Dissolved or dispersed in a pharmaceutical carrier at physiological pH, approximately 0.1 kg body weight per day for warm-blooded animals.
A method for inhibiting the formation of malignant tumor cells in a warm-blooded animal, comprising administering systemically to a warm-blooded animal host in such a manner that ~2 g is delivered. (19) Claim 18, wherein the non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphoric acid is a salt selected from the group consisting of alkali metals, alkaline earth metals, zinc and tin. The method described in. (20) the non-toxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate is C_1_-_1_2 alkyl;
selected from the group consisting of adenylyl, guanylyl, cytosilyl, thymylyl esters and quaternary ammonium salts,
A method according to claim 18. (21) The method according to claim 19, wherein the salt is potassium peroxodiphosphate. (22) C_1_-_1 whose derivative is peroxodiphosphoric acid
21. The method of claim 20, wherein the _2 alkyl ester. (23) the derivative is an adenylyl, guanylyl, cytosylyl or thymyryl ester of peroxodiphosphoric acid;
A method according to claim 20. (24) The method of claim 18, wherein the physiologically acceptable pyrogen-free solvent is phosphate buffered saline. (25) A nontoxic, water-soluble, pharmaceutically acceptable derivative of peroxodiphosphate is blended with a solid polyhydroxy sugar, and the blend is wetted with a solution of the polyhydroxy sugar compound and sieved to a uniform size. and blended with a binder and compressed into compressed granules, which are not defeated by stomach acid and have a pH of about 5.
coating by spraying a film of a coating solution that is dissolved by intestinal fluids at a pH of about 5.5 to 10 and does not decompose when passing through the stomach.
A method for producing compressed granules having a coating that is dissolved by intestinal fluids. (26) non-toxic peroxodiphosphate, comprising sterilizing deionized distilled water to make it pyrogen-free and then adding thereto a phosphate buffer and a peroxodiphosphate derivative and sodium chloride; A method for preparing a solution suitable for intragastric administration of a water-soluble pharmaceutically acceptable derivative.