KR880002266B1 - Composition for inhibiting tumor development - Google Patents

Abstract

A method for preparing an anti-tumor agent is consists of a compn. contg. 20 parts cellulose acetate phthalate, 30 parts carnauba wax, 10 parts stearic acid, 450 parts 96%-EtOH, and 390 parts acetone which has an inhibitory effect against bone marrow.

Description

[Name of invention]

Tumor growth inhibiting composition

Detailed description of the invention

The present invention is directed to inhibiting the development of existing tumors in vivo in tumor cells and in warm-blooded animals.

Cancer is a disease caused by the development of malignant tumors. There have been many medical efforts to overcome and reduce cancer. To date, no cure for cancer has been found. However, much is known about the mechanism of avoiding the pain of cancer in warm-blooded animals. The present invention provides a substance for inhibiting tumor development and a method for inhibiting such development.

Among the cells contained in mammalian fluids are lymphocytes, monocytes, macrophages and polymorphic nuclear cells. These cells serve as natural surveillance systems for tumor development from lower mammals such as rodents to humans. Recently, it has been observed that certain lymphocytes or lymphocyte populations, called natural killer or NK cells, destroy tumor cells and thus inhibit cancer development. It has been suggested that NK cells have cellular solution activity associated with the generation of active oxygen-containing substances such as hydrogen peroxide (H ₂ O ₂ ) or oxygen-containing radicals such as hydroxyl anions (OH) and peroxide anions (O ₂ ). .

NK cells and reactive oxygen phenomena are mentioned in the literature (by Herbrman et al, Science.Vol. 214, 2 October 1981, pages 24-30, Roder et al, Nature, Vol. 298.5 August 1982. pages 569-572; Nature et al; Journal of Immunology, Vol. 129, No. 5, November, 1982, pages 2164-2171; and Mavier et al, Journal of Immunology, Vol. 132, No. 4, April, 1984, pages 1980-1986 .)

Of course there are many compounds that release free radical species. However, one such factor does not mean that these compounds are introduced in vivo to aid the work of NK cells or to make NK cells work and inhibit tumor development so that tumors do not develop. Compounds that release reactive oxygen species generally work rapidly but have been shown to require at least a slower and more sustained release rate to be effective on tumor development in warm-blooded animals such as humans. This was not effectively accomplished until the present invention. If oxygen release is too rapid, both tumor cells and normal cells can be attacked.

U.S. Pat. No. 4,041,149 (August 9, 1977, the inventors and co-inventors) mentions compositions of various forms, including oral tablets that support the formation of bad breath, wherein the active ingredient is peroxydiphosphate. Peroxydiphosphate compounds differ from most oxygen supply compounds in that they do not initially provide hydrogen peroxide release. Rather, it slowly releases hydrogen peroxide and the oxygen released by peroxydiphosphate is 1/10 the effective oxygen released by hydrogen peroxide when compared to the hydrogen peroxide equivalent concentration. In addition, only about 50% of the active oxygen is released for 20 hours at 25 ° C. in the presence of alkaline phosphatase or acid phosphatase present in warm-blooded animals including mice, rats, humans and the like.

The advantages of the present invention are in the development of existing malignant tumors in vivo in warm-blooded animals, from rodents to humans, and in preventing tumor growth in tumor cells in vitro.

Another advantage of the present invention is to provide a method of inhibiting tumor formation by introducing a substance that slowly releases oxygen into the body.

According to one aspect of the invention, the invention relates to a composition containing about 01-10% amount of a non-toxic water soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid dissolved or dispersed in an agent carrier.

According to yet another aspect of the present invention, the present invention provides a composition comprising a nontoxic dose of about 0.1-6 g / kg body weight of a warm-blooded animal, a non-toxic water-soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid dispersed in a pharmaceutical carrier. The present invention relates to a method for inhibiting tumor formation in warm-blooded animals having malignant tumor cells consisting of oral administration to warm-blooded animals in a dosage of about 0.1-6 g / kg of warm-blooded animal per day.

According to another aspect of the present invention, the present invention corresponds to about 0.1-2 g / kg of warm-blooded animal having a malignant tumor cell with a non-toxic water-soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid dispersed or dissolved in a pharmaceutical carrier. It relates to a method for inhibiting tumor formation consisting of systemic administration of a composition composed of a non-toxic dose to a warm-blooded animal in a dosage of about 0.1-2 g per kg of the warm-blooded animal.

Peroxydiphosphate compound (PDP) is a non-toxic pharmaceutical acceptable compound form that is superior to the salts shown in the aforementioned US Pat. No. 4,041,149. Compounds, organic peroxydicarbonates phosphate C ₁ - ₁₂ alkyl, adenylyl vanillyl, nine not reel, Saito silyl and tea pushed esters as well as alkali metal (such as lithium, sodium and potassium), alkaline earth metal salts (e.g. magnesium, calcium and the host tontyum ), Zinc and tin salts and quaternary ammonium and other salts of the same kind. Among the inorganic cations, alkali metals, in particular potassium salts, are preferred.

Tetrapotassium peroxydiphosphate is a finely divided free flowing white non-absorbent crystalline solid that is stable, odorless, has a molecular weight of 346, 35 and an active oxygen content of 4.6%.

Tetrapotassium peroxydiphosphate is 47-51% water soluble at 0-61 ° C. but insoluble in solvents such as acetonitrile, alcohols, ethers, ketones, dimethyl formamide and dimethyl sulfoxide. The 2% aqueous solution has a pH of about 9.6 and its saturated solution has a pH of about 10.9. The 10% aqueous solution at 25 ° C. did not show any free radical loss after 4 months and the 50 ° C. 10% solution showed 3% free radical loss after 6 months.

Organic salts may be particularly suitable for administration to malignant tumors. Among organic esters C ₁ - is an ester that can facilitate the rapid absorption of the peroxydicarbonate phosphate component by the cells, such as those adenylate vanillyl, not old reel, Citrus silyl and tea pushed capable of providing a hydrophobic as ₁₂ alkyl radical, preferably Do.

Pharmaceutical carriers suitable for oral administration are suitable for refining tablets composed of substances that withstand disintegration by gastric acid at gastric pH (about 1-3) because peroxydiphosphate is inactivated by such gastric acid. Tablet carriers obtained by tableting granules of peroxydiphosphorate solids therein have a higher pH (about 5.5-10), are soluble in intestinal fluids that do not inactivate peroxydiphosphate and are human or other temperate animals Enzyme action by phosphatase present in Preferred tablet coatings include fatty acid after ethers such as N-butylstearate (usually about 40-50, preferably about 45 parts by weight) waxes such as carnauba wax (usually about 15-25, preferably 25 parts by weight) such as stearic acid. Fatty acids (usually about 20-30 parts, preferably 25 parts by weight and cellulose esters such as cellulose acetate phthalate (usually about 5-15, preferably about 10 parts by weight and organic solvents (usually about 400-900 parts). Other preferred materials include copolymers of ethylene compounds such as shellac and polyvinyl methyl ether and maleic anhydride, these coatings are distinct from tablets that disintegrate in the oral cavity and tablets that disintegrate in the globular phase are usually about 80-90 weight. Part of mannitol and about 30-40 parts by weight of magnesium stearate.

The granule tablet of peroxydiphosphate salt is mixed with about 30-50 parts by weight of peroxydiphosphate salt with about 45-65 parts by weight of polyhydroxy saccharide solid such as mannitol and then wetted with about 20-35 parts by weight of polyhydroxy saccharide compound solution such as sorbitol. It is prepared by crushing to a certain size, mixed with about 20-35 parts by weight of a binder such as magnesium stearate and compressed into tablets in a tablet press. Granular tablets are coated by spraying a foam of coating material solution in place and drying the solvent. These tablets are different from oral tablets made by simply compressing granules without a special protective coating.

For oral administration, the effective dose of peroxydiphosphate is about 0.1-6 g / kg body weight per day and the dose at systemic administration such as intramuscular, intraperitoneal or intravenous injection is about 0.1-2 g / kg body weight / day.

Solvents that do not contain physiologically acceptable pyrogenic substances when used in systemic administration are suitable carriers. Saline with pH7-7.4, a physiological pH buffered with phosphate, is a preferred carrier for systemic administration. These solvents are different from the excipients that are wetted by the water normally used in toothpaste. These solutions are sterilized with deionized distilled water and then checked for the absence of pyrogenic substances using the Limulus amebocyte lysate test (Tsuji et al in "Pharmaceutical Manufacturing", October, 1984, p35-41). It is prepared by adding a phosphate round solution (pH such as about 8.5-10) made from sterile water free of acid, about 1-100 mg of the peroxydiphosphate compound derivative, and sodium chloride at a concentration of about 0.5-1.5% by weight. The solution can be sterilized by passing through a single filter and then filled into vials. Other solutions may also be used, such as Ringer's solution containing 0.86% by weight sodium chloride, 0.03% potassium chloride and 0.033% calcium chloride.

The peroxydiphosphate compound (PDP) slowly releases hydrogen peroxide in the presence of the phosphatase enzyme according to the following scheme.

Where X is a non-toxic pharmaceutically acceptable cation or organic ester component. Phosphatases that disintegrate peroxydiphosphate are present in plasma, serous and white blood cells as well as saliva. This slow release of oxygen is particularly effective in assisting the effects of NK cells on malignant tumor cells in response to peroxydiphosphate therapy. If warm-blooded animals are treated with PDP according to the present invention, it is preferred to use a therapy in which the treatment can continue at least until the tumor is arrested.

The following comparative examples illustrate the invention. All amounts are by weight unless otherwise indicated.

[Examples]

In vitro test for PDP tumor cytotoxicity

In this study, PDPs were held at different concentrations and examined the effect on the growth of myeloma (SP ₂ line) cells in animals (Table 1). Human gingival fibroblasts were used as normal cell controls (Table 2). Cells were grown in Dulbe Cox mododide eagles medium supplemented with 10% fetal calf serum, IXMEM vitamins, IXL-glutamine, IX NEAA ₃ , IX gentamicin. (Dulbecco's modified Eagles's medium) They were incubated at 37 ° C. under controlled humidity CO ₂ atmosphere. About 1-3x10 ⁼ cells were placed in each well of a 24-well micro titer plate containing 2 ml of medium. PDP (potassium salt) was added at various different concentrations. Cell culture was determined by taking a partial standard from the wells at the time indicated in Table 1 after incubation. Viability was assessed by trypanche exclusive test. Clean medium was added to each well daily to maintain the necessary growth conditions. The percent inhibition was calculated by comparing the percentage of viable cells in phosphate buffered saline (PBS) with that in PDP. The results are summarized in Table 1 below.

TABLE 1

Effect of PDP on Murine Myeloma (SP ₂ Line) Cells

These results show that the potassium salt of PDP shows greater cytotoxicity and inhibitory effects on murine myeloma (cancer) cells compared to the buffer control.

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

TABLE 2

Effect of PDP on Human Hemorrhoidal Fibroblasts

The data in Table 2 did not show a significant effect on cell growth at PDP 100-500mcg / ml concentrations, but growth was reduced even at normal cells at 1000 and 2500mcg / ml. It is worth noting that at high concentrations the effect on myeloma tumor cells (Table 1) is superior to the effect on normal cells (Table 2).

PDP of C ₁ - ₁₂ alkyl, adenylyl vanillyl, not old reel, Citrus silyl, T pushed ester and tetramethylammonium salts, as well as organic peroxydicarbonate phosphate and PDP lithium, sodium, magnesium, calcium, strontium, zinc and tin similar in The result was obtained.

Example 2

: Effects of PDP, potassium pyrophosphate (KPP) and PBS (phosphate) buffered saline on tumor development in vivo. Each group consisted of 25 75% genetically identical Balb / C mice with an average body weight of 20g ± 3g. have.

(a) Control group treated with phosphate buffered saline (PBS) (b) PBS and potassium pyrophosphate (KPP) as a control group (c) phosphate control treated with potassium peroxydiphosphate (PDP, pH 7.0).

Each animal was administered 0.2 ml of Pristane intraperitoneally (IP) for culture of transplantation of malignant SP ₂ cells (murine myeloma carcinoma tumor cells). Three weeks later, the animals were orally administered to the animals as follows: (a) group was intraperitoneally administered 0.2 ml of PBS, (b) group was administered 2.0 mg of PDP suspended in 0.2 ml of PBS, and (c) 2.0 mg of KPP dissolved in 0.2 ml of PBS was administered for 3 days. 48 hours after the third injection, each animal was inoculated with 2-3 × 10 ⁶ SP ₂ cells (mouse tumor cells, murine myeloma) (IP). The animals were then administered (a) PBS, (b) PDP (c) and KPP once daily for five days a week. Each week, the tumor growth and mortality rate of the animals was checked. Data were classified using the Mantel-Haenszei method (Statistical Aspects of the Analysis of Data from Retrospective Studies of Disease, J. National Cancer Institute.Vol. 3, 719-748, 1959).

The data in Tables 3, 4 and 5 show that PDP has a significant effect on controlling tumor growth in mice when compared to PBS or KPP, so the effect of inhibiting tumor growth is not by phosphate but by active oxygen species. Proved to be due.

TABLE 3

Mantel-Haenszel chi-square = 0.36 with 1, d.f., P = 0.55 Odds ratio = 1.34

These results are not significant and indicate that there is no significant difference between PBS and KPP in reducing tumor development in animals.

* PBS = phosphate buffered saline

** KPP = Pyrocalcium Phosphate

TABLE 4

10 week tumor study

PBS ^* : KPP ^**

Mantel-Haenszel chi square = 10.40 with 1, d.f.,

P = 0.001.

Rain heat ratio = 3.66

These data show that PBS control spreads tumors faster than animals treated with PDP (P = 0.001)

^* PBS = phosphate buffered saline

^** PDP = Calcium Peroxydiphosphate

TABLE 5

10 week tumor study

KPP ^* : PDP ^***

Mantel-Haenszel chi square = 5.86 with 1, d.f.,

P = 0.02.

Dominance ratio 2 = 2.60.

This data indicates that the KPP group developed tumors significantly faster than PDP treated animals.

^* KPP = pyro potassium phosphate

^*** PDP = potassium peroxydiphosphate.

1 mg / of a stable carrier consisting of 45 parts N-butyl stearate, 20 parts canubawax, 25 parts stearic acid and 10 parts cellulose acedate phthalate, when intramuscular and seminal administration of PBS, KPP and PDP are each administered at the same concentration Similar results were observed when administered orally at a concentration of 0.1 ml).

Similar results have been obtained with other inorganic salts of PDP, in particular the lithium, sodium, magnesium, calcium, strontium, zinc and tin salts. An organic compound of a PDP, particularly C ₁ - ₁₂ alkyl, adenylyl vanillyl, not obtain reel, silyl cytokines, T pushed ester and tetramethyl ammonium salt was also effective in inhibiting the growth of murine myeloma tumor cell.

Example 3

500 parts of potassium peroxydiphosphate and 641 parts of mannitol were mixed and wetted with 32,5 parts of a 10% sorbitol solution to produce wet granules, dried at 49 ° C., and separated through a 12 mesh sieve (screen hole 1.68 mm). After adding 35 parts of magnesium stearate as a binder, the granules were compressed into tablets in a tablet press to obtain granule tablets.

Tablets were coated with a coating solution having the following composition.

Cellulose acetate phthalate 120parts

Carnauba wax part 30

10 parts stearic acid

450 parts of 95% ethanol

Appropriately until 1000 parts of acetone

Coating was carried out by injection in conventional coating pans.

Claims (26)

Non-toxic pharmaceutically acceptable compound derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical carrier, about 0.1-10% dose, the pharmaceutical carrier is phosphate buffered saline with a pH of about 7.0-7.4 or disintegrated by gastric acid And is a peeling material that disintegrates in semen at a pH of about 5.5-10. The composition of claim 1, wherein said pharmaceutical carrier is a different phosphate buffered saline. The composition of claim 1, wherein the pharmaceutical carrier is a coating material that disintegrates in the intestinal fluid having a pH of about 5.5-10 while resisting disintegration by gastric acid. 4. The pharmaceutical carrier of claim 3, wherein the coating material of the pharmaceutical carrier consists of 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. Composition. The composition of claim 4 wherein said fatty acid ester is N-butyl stearate, said wax is carnauba wax, said fatty acid is stearic acid and said cellulose ester is cellulose acetate phthalate. The composition of claim 1 wherein said non-toxic water soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid is a salt selected from the group consisting of alkali metals, alkaline earth metals, zinc and tin. 2. The method of claim 1, wherein the peroxy depot cephalosporin acid of the non-toxic water-soluble pharmaceutically acceptable compound derivative is a C ₁ - ₁₂ alkyl, adenylyl vanillyl reel cytokines silyl not obtain tea from the pushed, esters and quaternary group consisting of ammonium salt Composition selected. The composition of claim 6 wherein said salt is potassium peroxydiphosphate. The method of claim 7, wherein said compound derivative is a peroxy acid depot of cephalosporin C ₁ - ₁₂ ester composition. 8. The composition of claim 7, wherein said compound derivative is adenosyl, guanylyl, cytosyl or thymilyl ester of peroxydiphosphoric acid. Non-toxic, water-soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical carrier in a repellent tablet that withstands disintegration to gastric acid and is disintegrated by intestinal fluids at a pH of about 5.5-10 per kg body weight of warm-blooded animals A method of inhibiting the production of malignant tumor cells in a warm blooded animal consisting of administering a composition consisting of about 0.1-6g to a warm blooded animal in an oral regimen provided about 0.1-6 g / kg of said warm blooded animal per day. The tablet component of claim 11 wherein the coating component of the tablet is about 40-50 parts by weight of N-butyl stearate, about 15-25 parts by weight carnauba wax, about 20-30 parts by weight stearic acid and about 5-15 parts by weight cellulose acetate The method consists of phthalates. The method of claim 11 wherein said non-toxic water soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid is a salt selected from the group consisting of alkali metals, alkaline earth metals, zinc and tin. 12. The method of claim 11, wherein the peroxy depot cephalosporin acid of the non-toxic water-soluble pharmaceutically acceptable compound derivative is a C ₁ - from ₁₂ alkyl, adenylyl vanillyl, not old reel, Citrus silyl, T pushed esters and quaternary group consisting of ammonium salt The method chosen. The method of claim 13, wherein said salt is potassium peroxydiphosphate. 15. The method of claim 14, wherein the compound derivative of the cephalosporin C ₁ Depot peroxy acid - ₁₂ method as alkyl esters. 15. The method of claim 14, wherein said compound derivative is adenylyl, guanylyl, cytosylyl or thymilyl ester of peroxydiphosphoric acid. A non-toxic, water-soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical carrier having a physiological pH of about 7.0-7.4. A composition comprising a nontoxic dose of about 0.1-2 g / kg body weight of a warm-blooded animal. A method of inhibiting the production of malignant tumor cells in a warm blooded animal consisting of systemic administration to the warm blooded animal in a regimen wherein about 0.1-2 g / kg of warm blooded animal per day is provided. 19. The method of claim 18, wherein said non-toxic water soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid is a salt selected from the group consisting of alkali metal, alkaline earth metal zinc and tin. 19. The method of claim 18, wherein the peroxy depot cephalosporin acid of the non-toxic water-soluble pharmaceutically acceptable compound derivative is a C ₁ - ₁₂ alkyl, adenylyl vanillyl, not old reel, Citrus silyl, T pushed esters and quaternary group consisting of ammonium salt Selected from. The method of claim 19, wherein said salt is potassium peroxydiphosphate. The method of claim 20, wherein said compound derivative is a peroxy Depot C ₁ of the cephalosporin acid-way ₁₂ alkyl ester. The method of claim 20 wherein said compound derivative is adenyl, guanylyl, cytosylyl or thymilyl ester of peroxydiphosphoric acid. 19. The method of claim 18, wherein said solvent that does not contain a physiologically acceptable pyrogenic substance is a phosphate buffered saline solution. The non-toxic water-soluble pharmaceutically acceptable compound derivative of peroxydiphosphoric acid is mixed with a polyhydroxy saccharide solid, and then the mixture is wetted with a polyhydroxy saccharide compound solution, separated to size, mixed with a binder and compressed to granulate After the tablets were formed, the coating consisting of coating the granule tablets by spraying a coating solution film dissolved in the intestinal fluid at pH about 5.5-10 without being inactivated by gastric acid was dissolved in the intestinal fluid of 5.5-10. A method of making granules with a coating that does not decompose while passing through the stomach. Non-toxic water-soluble pharmaceutical solution of peroxydiphosphoric acid suitable for intragastric administration consisting of sterilizing deionized distilled water to remove pyrogenic substances and then adding sodium chloride and phosphate buffer and the aforementioned compound derivatives of peroxydiphosphoric acid A process for preparing a phase acceptable compound derivative solution.