FI84697B - A PROCEDURE FOR THE FRAMSTATION OF A PHARMACOLOGICAL COMPOSITION WITH DERIVATIVES OF PEROXIDE DOSPHOSPHORUS. - Google Patents

Description

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The present invention relates to a process for the preparation of a pharmacological composition.

Cancer is the result of the growth of malignant tumors. A huge amount of medical research has been sacrificed to alleviate and overcome cancer stigma. An anticancer drug has not yet been invented today. But much has been learned about the mechanism by which warm-blooded animals avoid cancer. The present invention is based on this information and provides a material for inhibiting tumor growth and a method for inhibiting such growth.

Cells in mammalian body fluids include lymphocytes, monocytes, macrophages, and polymorphonuclear cells. These cells act as a natural surveillance system against tumor growth from lower mammals such as rodents to humans. In recent years, it has been found that a particular subpolation of lymphocytes or lymphoid cells, called "Natural Killer" or "NK" cells, destroys tumor cells and thus prevents the development of cancer. Evidence suggests that NK cells have a cytolytic effect related to the action of active oxygen generators such as hydrogen peroxide (H2O2) or oxygen-containing radicals such as hydroxyl anion (.OH) and superoxide anion (02 *). NK cells and the phenomenon of active oxygen are described by Herberman et al., Science, Vol. 214, October 2, 1981, pages 24-30; Roder et al .; Nature, Vol. 298, August 5, 1982, pages 569-572; Nathan 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.

There are, of course, several compounds that release oxygen species. However, this fact alone does not suggest that such a compound could be introduced into the body to supplement NK cell function, or where tumor development is not yet strong enough, to induce NK cell function and inhibit tumor growth. Compounds that release active oxygen species usually do so rapidly, whereas efficacy against tumor development in warm-blooded animals would appear to require at least a slower and longer sustained release rate. Until the present invention, this has not been effectively accomplished. When the release of oxygen is too rapid, both tumor and normal cells may be affected.

U.S. Patent 4,041,149 discloses a mixture in various forms, e.g. as dental tablets which prevent the development of bad mouth and in which the active ingredient was peroxide-phosphate. The peroxide phosphate compound differs from most oxygen-generating compounds in that it does not cause an initial discharge of hydrogen peroxide. On the contrary, it releases hydrogen peroxide slowly, so that when equivalent concentrations are compared to hydrogen peroxide, the amount of oxygen released by the peroxide phosphate is one tenth of the amount of oxygen released by the hydrogen peroxide. In addition, only about 50% of the active oxygen is released within 20 hours at 25 ° C in the presence of alkaline phosphatase or acid phosphatase, both of which are present in the bodies of warm-blooded animals such as mice, rats, humans, and the like.

It is an advantage of this invention that tumor growth is inhibited in tumor cells in vitro and true malignant tumor development is inhibited in vivo in warm-blooded animals such as rodents to humans. The invention is thus mainly. .. characterized by what is said in the characterizing part of claim 1.

Another advantage of the invention is that it provides methods for inhibiting tumor formation by slowly introducing an oxygen scavenger into a living host animal.

In one aspect, the present invention relates to a method of inhibiting the formation of a tumor having 3,84697 malignant tumor cells by administering a non-toxic dosage amount of about 0.1 to 6 g per kilogram of body weight of a warm-blooded animal from non-toxic, water-soluble , a pharmaceutically innocuous compound which is a peroxydiphosphoric acid derivative dissolved or dispersed in a pharmaceutical carrier, is administered orally to a warm-blooded host animal as a course of administration in an amount of about 0.1 to 6 g per kilogram of body weight of said warm-blooded animals per day.

According to a further aspect of the present invention, it relates to a method of inhibiting tumor development by systemically administering a mixture of a non-toxic dosage amount of about 0.1 to 2 g per kilogram of body weight of a warm-blooded animal. per malignant tumor cells, a non-toxic water-soluble pharmaceutically acceptable compound which is a peroxydiphosphoric acid derivative dissolved or dispersed in a pharmaceutical carrier for a warm-blooded host animal as a regimen of about 0.1 to 2 g per body weight of said warm-blooded animal.

The Peroxide Phosphate Compound (PDP) is in the form of a non-toxic pharmaceutically safe compound which is superior to the salt mentioned in the aforementioned U.S. Patent 4,041,149. The compounds are alkali metal (e.g. lithium, sodium and potassium), alkaline earth metal (e.g. magnesium, calcium and strontium), zinc and tin salts *: as well as organic peroxide phosphate esters such as: alkyl, adenylyl, guanylyl , cytosyl and thymyl esters::: and also quaternary ammonium and other salts. Of the inorganic salts, alkali metal salts, especially potassium, are preferred. Tetrapotassium peroxide phosphate is a stable, odorless, finely divided, free-flowing, white, non-hygroscopic crystalline substance with a molecular weight of 346.35 and an active oxygen content of 4.6%. Tetrapotassium peroxide phosphate is 47-51% water soluble at 0 ° -61 ° C, but insoluble in common solvents such as acetonitrile, alcohols, ethers, ketones, dimethylformamide, dimethylsulfoxide, etc. The pH of a 2% aqueous solution is about 9.6 and its saturation is about 9.6. the pH of the solution is about 10.9. A 10% aqueous solution at 25 ° C showed no loss of active oxygen after four months; and at 50 ° C, the 10% solution lost 3% active oxygen over 6 months.

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Organic salts may be particularly suitable for administration against Maligne and tumors. Of the organic esters, those which confer hydrophobic properties such as the 2-allyl radical and those which facilitate the rapid uptake of the peroxide phosphate half of the cells, such as adenylyl, guanylyl, cytosyl and thymylyl esters, are preferred.

Pharmaceutical carriers suitable for oral administration include coated tablets made of a substance that can withstand gastric acids without breaking the stomach at pH (about 1-3), as these gastric acids would inactivate the peroxide phosphate. Carriers containing peroxide phosphoric acid salt solids in tableted granules are preferably allowed to dissolve in intestinal fluids of higher pH (about 5.5-10) and which do not inactivate the peroxide phosphate but leave it under the enzymatic action of phosphatase. , which is found in humans and other warm-blooded animals. A preferred tablet coating solution contains a fatty acid ester such as N-butyl stearate (typically about 40-50, preferably about 45 parts by weight), a wax such as carnauba wax (typically about 15-25, preferably about 20 parts by weight), a fatty acid such as stearic acid (typically about 20 parts by weight). 30 parts, preferably 25 parts by weight) and a cellulose ester such as cellulose acetate phthalate (typically about 5-15, preferably about 10 parts by weight) and an organic solvent (typically about 400-900 parts).

Other preferred coating materials include shellac and copolymers of maleic anhydride and ethylene compounds such as polyvinyl methyl ether. Such coatings are different from tablets that break in the oral cavity and in which the tablet material typically contains about 80-90 parts by weight of mannitol and about 30-40 parts by weight of magnesium stearate.

Tableted granules of the peroxide phosphate salt are formed by mixing about 30-50 parts by weight of the peroxide phosphate salt with about: 45-65 parts by weight of polyhydroxysugar crystals such as mannitol and moistening with about 20-35 parts by weight of a solution of the polyhydroxy sugar compound, e.g. , about 20 to 35 parts by weight of a binder such as magnesium stearate are mixed in and the granules are compressed into tablets by means of a compression machine. The tableted granules are spray sprayed by wetting the foam of the coating solution and drying to remove the solvent. Such tablets differ from dental tablets, which are typically compressed granules without a special protective coating.

When a regimen is prescribed for oral ingestion, an effective dosage of peroxide phosphate is about 0.1 to 6 g / kg body weight per day; when administered systemically, such as by intramuscular, intraperitoneal or intravenous injection, the dosage is about 0.1 to 2 g / kg of body weight per day.

Physiologically acceptable pyrogen-free solvents are suitable carriers for use in a manner known in the art for systemic drug administration. Saline buffered with phosphate to a physiological pH of about 7-7.4 is the preferred carrier for systemic drug delivery. Such solvents are different from the water wetting agents typically used in dentifrices. Such a solution is typically prepared by sterilizing deposited distilled water, monitored for non-pyrogenicity using the Limulus Amebocyte Lysate (LAL) test described by Tsuji et al., "Pharmaceutical Manufacturing", October 1984, pages 35.41, and then added. a phosphate buffer (pH, for example, about 8.5-10) in pyrogen-free sterile water and about 1-100 mg of the peroxide phosphate derivative and sodium chloride to a concentration of about 0.5-1.5% by weight. The solution can be packaged in vials for use after re-sterilization through a microporous filter. Alternatively, other solutions may be used, 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.

The peroxide phosphate compound (PDP) slowly releases hydrogen peroxide in the presence of phosphatase enzymes according to the following equation: 6 84697 «« phosphatases H H2 ° H2 ° 2 * P04 ~ 3 X.-O-P-O-O-P-O toostatases -0-0-P-0 ----->

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0- 0- wherein X is a non-toxic pharmaceutically harmless cation or completes an organic ester moiety. Phosphatase, which breaks down peroxide phosphate, is present in saliva as well as in plasma, intestinal fluids, and white blood cells. Slow oxygen release is particularly effective in supplementing the action of NK cells against malignant tumor cells that respond to peroxide phosphate therapy. When warm-blooded animals are treated with PDP in accordance with the present invention, it is preferred to follow a course of treatment in which treatment is continued at least until regression has occurred in the tumors.

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

Examples Example 1

In vitro study on the cytotoxic effect of PDP on tumor. This study investigates the effects of PDP at different concentrations on mouse myeloma cell growth (SP2 * line) (Table 1). Human gingival microblasts are used as controls for normal cells (Table 2). Cells are grown in Dulbecco's modified Eagles' medium supplemented with 10% fetal bovine serum, 1 X MEM vitamins, 1 XL glutamine, 1 X-NEAA, 1 X-gentamicin. They are incubated at 37 ° C in a humidified (X 2) atmosphere. About 1 to 3 x 10 cells are placed in each well of a 25 microtiter plate containing 2 ml of medium each. States.

'· · - After incubation, cell viability is determined by removing ·: ··· aliquots from the wells over the time shown in Table 1. Viability is determined using a trypan blue exclusion test. Fresh medium is added to each well ^ 84697 every day to maintain the conditions necessary for growth. Inhibition was calculated by comparing the percentage of viable cells in phosphate buffered saline (PBS) versus PDP. The data are summarized in Table 1.

Table 1 Effects of PDP on mouse myeloma cells (SP ^ line)

Treatment N Number of cells x 10 ^% viable _ _ _ (after 72 h) _cells

Control (PBS) 4 8.98 + 0.14 100% PDP pH 7.0 100 ug / ml 4 1.86 + 0.14 47,500 ug / ml 4 1.33 + 0.03 33 1000 ug / ml 4 1.07 + 0.17 29 2500 ug / ml 4 0.48 + 0.15 12 These results show that, compared to the buffer control, the potassium salt of PDP is highly cytotoxic and inhibits mouse myeloma (cancer) cells.

Table 2 shows the effects on normal cells (human gingival fibroblast cells).

Table 2 Effects of PDP on human gingival microblast

Treatment N% viable number of viable cells x 10 ”* cells _ _ (after 72 h) _ _

Control (PBS) 4 2.67 + 0.17 100 PDP pH 7.0 100 ug / ml 4 2.61 + 0.16 98,500 ug / ml 4 2.58 + 0.13 97 1000 ug / ml 4 2 , 12 _ + 0.15 79 2500 ug / ml 4 1.97 + 0.11 74

The data in Table 2 show no significant effect on cell growth at a concentration of 100-500 μg / ml PDP, but that even in normal cells viability is reduced at concentrations of 1000 and 2500 μg / ml. It should be noted that the effect on myeloma-8 84697 tumor cells (Table 1) even at high concentrations is more pronounced than the effect on normal cells (Table 2).

Similar results are obtained with the lithium, sodium, magnesium, calcium, strontium, zinc and stannous salts of PDP, with organic peroxide phosphate as well as with C 1-4 alkyl, adenylyl, guanylyl, cytosyl. , thymylyl esters and tetramethylammonium salt.

Example 2 Effects of PDP, potassium pyrophosphate (KPP) and PBS (phosphate buffered saline) on tumor growth in vivo

Seventy-five genetically identical Balb / C mice with an average weight of 20 g + 3 g in groups of 25 animals were treated with: a) phosphate buffered saline (PBS) for control; b) potassium peroxide phosphate (PDP) and PBS, pH 7.0; and c) potassium pyrophosphate (KPP) and PBS for phosphate control. Each animal receives 0.2 ml of Pristane intraperitoneally (i.p.) for implantation of malignant SP2 cells (mouse myelomacarcinoma tumor cells) into the animals. After three weeks, the animals are subjected to an oral regimen as follows: group a) receives i.p. 0.2 ml PBS; group b) receives 2.0 mg of PDP suspended in 0.2 ml of PBS, and group c) receives 2.0 mg of KPP in 0.2 ml of PBS, for three consecutive days. 48 h after the third injection, each animal is inoculated (i.p.) with 2-3 x 10 2 S 2 cells (mouse tumor cells, mouse myeloma).

The animals are then administered the same substances, once a day for five days / week, i.e. a) PBS, b) PDP or c) KPP. Animals are examined for Tumor Development and Deaths weekly. Data were analyzed using the Mantel-Haenszel method (Statistical: Aspects of the Analysis of Data from Retrospective Studies of

Disease, J. National Cancer Institute, Part 3, 719-748, 1959).

The data in Tables 3, 4, and 5 indicate that PDP is clearly effective in controlling tumor growth in mice when compared to PBS or KPP, proving that the tumor growth inhibitory effect is due to the development of an active oxygen species and not to phosphate.

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Table 3 PBS1 versus KPP2

A ten-week study of tumor development

Week Treatment Tumor and Non-tumor Risk group _ _ death _ _ 1-4 PBS 11 14 25 KPP 10 15 25 5 PBS 4 10 14 KPP 4 11 15 6 PBS 5 5 10 KPP 2 9 11 7 PBS 2 35 KPP 4 59 8 PBS O 33 KPP 1 45 9 PBS 2 13 KPP 3 14 10 PBS 1 0 1 KPP O 11

Mantel-Haenszel chi-square = 0.36 and 1, d.f., P = 0.55 Separation ratio = 1.34 These results are not relevant and do not prove a significant difference between PBS and KPP in reducing tumor development in animals.

PBS = Phosphate buffered saline 2 KPP = Potassium pyrophosphate 10 84697

Table 4

Ten-week tumor study PBS1 versus PDP2

Week Treatment Tumor and No tumor At risk _ _ death _ _ 1-4 PBS 11 14 25 PDP 2 23 25 5 PBS 4 10 14 PDP 4 19 2 3 6 PBS 5 5 10 PDP 5 14 19 7 PBS 2 25 PDP 2 13 14 8 PBS 033 PDP 2 10 12 9 PBS 213 PDP 3 7 10 10 PBS 1 O 1 PDP 167

Mantel-Haenszel chi-square = 10.40 and 1, d.f., P = 0.001.

Separation ratio = 3.66.

These data indicate that in the PBS control group, tumors develop ---- significantly faster than in PDP-treated animals (P = 0.001).

PBS = phosphate buffered saline 2 PDP = potassium peroxide phosphate.

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Table 5

Ten-week tumor study KPP * versus PDP ***

Week Treatment Tumor and No tumor At risk _ _ death _ _ 1-4 KPP 10 15 25 PDP 2 23 25 5 KPP 4 11 15 PDP 4 19 23 6 KPP 2 9 11 PDP 5 14 19 7 KPP 45 9 PDP 2 14 14 8 RDP 14 5 PDP 2 10 12 9 RDP 31 4 PDP 3 7 10 10 RDP 0 1 1 PDP 16 7

Mantel-Haenszel chi-square = 5.86 and 1, d.f., P = 0.02.

Separation ratio = 2.60.

The data showed that tumors in the KPP group developed significantly ... faster than in PDP-treated animals (P = 0.001).

* KPP = potassium pyrophosphate *** PDP = potassium peroxide phosphate

Similar results can be observed when PBS, KPP and PDP are each administered intramuscularly and intravenously at the same concentrations in PBS or orally at a concentration of 1 mg / ml (0.1%) in a stable vehicle containing 45 parts of N-butyl stearate. , 20 parts of carnauba wax, 25 parts of stearic acid and 10 parts of cellulose acetate phthalate.

Similar results are obtained with other inorganic salts of PDP, especially lithium, sodium, magnesium, calcium, strontium, zinc and stannous salts. Organic salts of PDP, especially C 1-4 alkyl, adenylyl, guanylyl, C 124697 cytosyl, thymylyl esters and tetramethylammonium salts are also effective in inhibiting the growth of mouse myeloma malignant tumor cells.

Example 3,500 parts of potassium peroxide phosphate and 641 parts of mannitol are mixed and moistened with 32.5 parts of a 10% solution of sorbitol to form a moist granulate which is dried at 49 ° C and sieved through a 12 mesh U.S. sieve (1.68 mm sieve holes). .

35 parts of magnesium stearate are then added as a binder and the tableted granules are formed by compressing the mixture with a tablet press.

The tablets are coated with an enteric coating solution having the following composition:

120 parts of cellulose acetate phthalate

30 parts of carnauba wax

Stearic acid 10 parts of 95% ethanol 450 parts

Acetone q.s. Coating in 1000 parts is carried out by the casting method in a conventional coating pan.

When the tablets thus prepared are taken orally, they pass through the stomach without breaking and the coating then dissolves in the intestinal fluids.

Example 4

Deionized distilled water is stabilized at atmospheric pressure for 20 min in an autoclave. After cooling, its non-pyrogenicity is tested using Limulus Amebocyte Lysate (LAL) as described by Tsuji et al., "Pharmaceutical Manufacturing", October, 1984, pages 35-41. 50 parts of potassium peroxide phosphate, sodium chloride in an amount corresponding to a 0.9% solution and 0.1 M phosphate buffer containing K 2 PO 4 and β 2 O 2, pH 9.4, are added to 134697 pyrogen-free sterile water. . The solution is sterilized by passing it through a 0.5 micropore filter and then packaged in sterile bottles.

Although the invention has been described based on specific examples, it will be apparent to those skilled in the art that various modifications may be made within its scope.

Claims (2)

A process for the preparation of a pharmaceutical composition, characterized in that a dosage amount of about 0.1-10% by weight of a non-toxic water-soluble pharmacologically acceptable substance derivative of peroxydiphosphoric acid is mixed with a pharmacological carrier which is a coated tablet material which resists digestion by gastric acids but decomposed by intestinal fluid at a pH of about 5.5-10, wherein said substance derivative of peroxydiporphoric acid is mixed with a solid polyhydroxy sugar compound and the mixture is wetted with a solution of a polyhydroxy sugar compound, filtered to a desired size, and a binder mixed, pressing into tablet-shaped grains is carried out and the tablet grains are coated by spraying with a film of coating solution, which film does not decompose or dissolve by the gastric acids, but dissolves in intestinal fluids, the pH of which is about 5.5-10, the coating of said pharmacological tablet carrier material. comprises about 40-50 parts by weight of fatty acid ester, preferably desvis N-butyl stearate, about 15-25 weight parts of wax, preferably carnauba wax, about 20-30 parts by weight of fatty acid, preferably stearic acid, and about 5-15 parts by weight of cellulose ester, preferably cellulose acetate phthalate. 2. A process according to claim 1, characterized in that the non-toxic water-soluble substance derivative of the peroxydiphosphoric acid is its 0-O 2 O 3 -β1β1 5ΐ®Γ / adenylyl ester, guanilyl ester, cytosylyl ester, thymylyl ester or quaternary ammonium or quaternary ammonium to a metal which is an alkali metal, alkaline earth metal, zinc or tin, preferably potassium peroxide phosphate.