DK168191B1 - Use of peroxydiphosphates for the preparation of pharmaceutical tablets or pharmaceutical aqueous solutions - Google Patents

Description

DK 168191 B1

The present invention relates to the use of a non-toxic, water-soluble, pharmaceutically suitable derivative of peroxydiphosphoric acid as an active ingredient for the preparation of pharmaceutical tablets or pharmaceutical aqueous solutions.

5 The disease cancer is due to the development of malignant tumors. Much medical research has been aimed at reducing and overcoming the scourge of cancer. To date, no cure for cancer has been found. However, much has been learned about the mechanism by which warm-blooded animals avoid being haunted by 10 cancers. The present invention is based on this knowledge to provide a method for the manufacture of a drug which inhibits tumor development.

Among the cells contained in mammalian body fluids are lymphocytes, monocytes, macrophages, and polymorphonuclear cells.

These cells act as a natural surveillance system against tumor development in lower mammals such as rodents up to humans. In recent years, it has been observed that a particular subpopulation of lymphocytes or lymphoid cells called "natural killer" or "NK" cells destroys tumor cells and thereby prevents the development of cancer. The evidence suggests that "NK" cells have cytolytic activity that relates to the development of an active oxygen compound such as hydrogen peroxide (H 2 O 2) or oxygen-containing radicals, e.g. the hydroxyl anion (* OH) and the superoxide anion (02 '). The NK cells and active oxygen phenomena are described by Herberman et al., Science, vol. 214, October 2, 1981, pages 24 to 30, Roder et al., Nature, vol. 298, August 5, 1982, p. 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 30, 1984, page 1980-1986.

Of course, there are many compounds which release active oxygen compounds. However, that factor alone has not meant that such a compound could be introduced into a body to supplement the function of NK cells or where tumor formation does not appear sufficient to confer the functions of NK cells and inhibit tumor development. Compounds that release active oxygen types generally do so rapidly, while efficacy against tumor development in warm-blooded animals such as humans might appear to require at least a slower and more maintained rate of release. Until the present invention, this was not achieved effectively. When oxygen release is too fast, both tumorous and normal cells can be attacked.

In U.S. Patent No. 4,041,149, issued August 9, 1977, to the present inventors, an agent of various forms was disclosed, including as a dental tablet which inhibited mouth odor formation and wherein the active ingredient was a peroxydiphosphate. Peroxide diphosphate differs from most oxygen-producing compounds in that it does not produce an initial burst of hydrogen peroxide. Instead, it releases the hydrogen peroxide slowly, so that when equivalent concentrations are compared with hydrogen peroxide, the amount of oxygen released by the peroxide diphosphate is one tenth of the amount of 20 available oxygen released by hydrogen peroxide. Furthermore, only approx. 50% of the active oxygen in 20 hours at 25 ° C in the presence of alkaline phosphatase or acid phosphatase, each found in the bodies of warm-blooded animals, including mice, rats, humans, etc.

Furthermore, from GB-PS 2,116,035, it is known to prepare buffered aqueous compositions containing a peroxydiphosphoric acid derivative for topical treatment of skin lesions.

The object of the invention is to provide drugs that inhibit tumor development of tumor cells in vitro and the actual malignant tumor development in vivo in warm-blooded animals, from rodents and up to humans.

The object is achieved by using a derivative of peroxydiphosphoric acid, in particular an alkali metal, alkaline earth metal, zinc, tin or quaternary ammonium salt or an alkyl, adenylyl, guanylyl, cytosylyl or thymylyl ester, in a concentration of approx. 0.1 to approx. 10% by weight for the preparation of pharmaceutical compositions having an inhibitory effect on tuber formation, in particular tablets coated with a gastric acid decomposition material but dissolved by intestinal fluid at a pH of 5.5 to 10, or a non-buffered, non-pyrogenic aqueous solution for systemic use, preferably having a pH of 7.0 to 7.4.

10 The inhibition of tumor formation from malignant tumor cells is by orally administering a non-toxic dose amount of 0.1 to 6 g / ml. kg body weight of one warm-blooded animal per due to the non-toxic, water-soluble, pharmaceutically suitable derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical carrier in the form of a tablet, to a warm-blooded animal.

The inhibition of tumor formation is also achieved by systemically administering a non-toxic dosage amount of 0.1 to 2 g / ml. kg body weight of one warm-blooded animal per day of the non-toxic, water-soluble, pharmaceutically suitable derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical carrier in the form of an aqueous solution for a warm-blooded animal.

The peroxydiphosphate compound (PDP) is in the form of a non-toxic, pharmaceutically suitable compound which is not comprised of a salt as disclosed in the aforementioned U.S. Pat.

25 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, and organic peroxydiphosphate esters such as C1-12 alkyl, adenylyl, guanylyl, cytosylyl, and thymylyl esters and also quaternary ammonium salts Alkali metal, especially potassium salt, is preferred among the inorganic cations. , crystalline substance having a molecular weight of 346.35 and an active oxygen content of 4.6% .Tetracalcium peroxydiphosphate is 47% water-soluble at 0-6l ° C, but insoluble in ordinary solvents such as acetonitrile, alcohols, ethers, ketones, dimethyl formamide, dimethyl sulfide oxide, etc. A 2% aqueous solution has a pH of about 9.6 and a saturated solution thereof a pH of about 10.9. solution in water at 25 ° C showed no loss of active oxygen after 4 months and at 50 ° C a 10% solution showed an active oxygen loss of 3% in 6 months.

The organic salts may be particularly suitable for administration to malignant tumors. Among the organic esters, those which confer hydrophobic properties such as C1-12 alkyl radical and those which facilitate the rapid uptake of the peroxydiphosphate molecule into the cells such as the adenylyl, guanylyl, cytosylyl and thymylyl esters are preferred.

Pharmaceutical carriers suitable for oral ingestion are coated tablets composed of material that resists gastric acid degradation at gastric pH (about 1-3), as peroxydiphosphate would be inactivated by such gastric acids. Instead, the solid granulated carriers of the solid peroxy-diphosphoric acid salt are dissolved therein by the intestinal fluids, which have a higher pH (about 5-10) and do not inactivate the peroxydiphosphate but leave it to the enzymatic action of phosphatase found in humans. and warm-blooded animals. A desirable tablet coating solution is composed of a fatty acid ester such as N-butyl stearate (typically about 40-50, preferably about 45 parts by weight), wax such as carnauba wax (typically about 15-25, preferably about 20 parts by weight), fatty acid such as stearic acid (typically about 20-30 parts, preferably 25 parts by weight) and cellulose esters such as cellulose acetate phthalate (typically about 5-15, preferably about 10 parts by weight) and organic solvent (typically about 400-900 parts) . Other desirable coating materials include shellac and copolymers of maleic anhydride and ethylenic compounds such as polyvinylmethyl ether. Such coatings differ from tablets which decompose in the oral cavity, wherein the tablet material DK 168191 B1 typically contains approx. 80-90 parts by weight of mannitol and approx. 30-40 parts of magnesium stearate.

Tabulated grains of peroxydiphosphate salts are formed by mixing ca. 30-50 parts by weight of the peroxydiphosphate salt with approx. 45-65 5 parts by weight of a solid polyhydroxy sugar such as mannite and wetted with approx. 20-35 parts by weight of a polyhydroxy sugar solution, such as sorbit, sieved to the desired size, mixed with approx. 20-35 parts by weight of a binder, such as magnesium stearate, and compresses the granules into tablets with a tablet machine. The tabulated grains are coated by spraying a foam of a solution of the coating material thereon and drying to remove the solvent. Such tablets differ from dental tablets which are typically compressed grains without a special protective coating.

An effective dose of peroxydiphosphate with a prescribed therapy when administered by oral administration is approx. 0.1 to 6 g / kg body weight per day. When the administration is systemic, such as by intramuscular, intraperitoneal or intravenous injection, the dosage is approx. 0.1-2 g / kg 20 body weight per day.

Physiologically acceptable pyrogen-free solvents are suitable carriers for use in a well-known manner for systemic administration. Saline solutions buffered with phosphate to a physiological pH of approx. 7-7.4 is the preferred carrier for systemic administration. Such solvents differ from carriers of water and moisture binding agents typically used in dentifrices. Such a solution is typically prepared by sterilizing deionized distilled water, checking to ensure that there is no pyrogenicity using the Limulus amebocyte lysate (LAL) sample described by Tsuji et al. "Pharmaceutical Manufacturing", October 1984, pages 35-41 and then adding a phosphate buffer (pH e.g. 8.5-10) prepared in pyrogen-free sterile water and about, 1-100 mg of peroxydiphosphate compound derivative and sodium chloride to a concentration of about . 0.5-1.5 DK 168191 B1 6% by weight. The solution can be packaged in bottles for use after being re-sterilized by passage through a micropore filter.

Alternatively, other solutions such as Ringer's solution containing 0.86 wt% sodium chloride, 0.03 wt% potassium chloride and 0.33 wt% calcium chloride may be used.

The peroxydiphosphate compound (PDP) slowly releases hydrogen peroxide in the presence of phosphatase enzymes according to the following equation: 0 0 - 0 10 X4-0-P-0-0-P-0 phosphatase ^ -0-0-P-0-H2 ° 2 + P04 ~ 3 '0 0 0 "X is a non-toxic pharmaceutically acceptable cation or completes an organic ester molecule moiety. Phosphatase for degradation of the peroxydiphosphate is found in saliva and plasma, intestinal fluids and white blood cells. The slow oxygen release is particularly effective in supplementing the effectiveness of NK cells against malignant tumor cells responding to the peroxydiphosphate therapy When warm-blooded animals are treated with PDP in accordance with the present invention, it is desirable to provide a therapy in which treatment is continued, at least until tumors recede.

The following comparative examples illustrate the invention. All quantities are by weight unless otherwise stated.

Example 1: In vitro study of PDP tumor cytotoxicity In this study, the effects of PDP at various concentrations on the growth of murine myeloma (SP2li-nie) cells (Table 1) are examined. Human gingival fibroblasts are used as normal cell controls (Table 2). The cells are grown in Dul-becco's modified Eagles medium supplemented with 10% fetal calf serum IX MEM vitamins, IXL-glutamine, IX NEAA ^, IX gene tamycin. They are incubated at 37 ° C in a humidified CO 2 atmosphere. Ca.

DK 168191 B1 7 1 to 3 x 10 5 cells are placed in each well in a 24 well microtiter plate containing 2 ml of the medium. PDP (potassium salt) is added at varying concentrations.

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After incubation, cell viability is determined by taking aliquots from the wells according to the times listed in Table 1. Viability is assessed by the trypan blue nozzle closure test. Fresh medium is added to each well each day to maintain the necessary growth conditions. The inhibition was calculated by comparing% living cells in salt-10 water with phosphate buffer (PBS) with PDP. The data is summarized in Table 1.

Table 1

EFFECTS OF PDP ON MURINE MYELOMA (SP2 LINE) CELLS TREATMENT N NUMBER OF CELLS x 105% ViABLE CELLS

15 (AFTER 72 HOURS)

Control (PBS) 4 8.98 +/- 0.14 100% PDP pH 7.0 100 mcg / ml 4 1.33 +/- 0.14 47 500 mcg / ml 4 1.33 +/- 0.03 33 20 1000 mcg / ml 4 1.07 +/- 0.17 29 2500 mcg / 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 inhibitory to the murine myeloma (cancer) cells.

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

DK 168191 B1 8

Table 2

EFFECTS OF PDP ON HOMAN GINGIVAL FIBROBLAST

TREATMENT NUMBER OF LIVELY CELLS x 1Q5% LIVELY

(AFTER 72 HOURS) CELLS

Control (PBS) 4 2.67 +/- 0.17 100 PDP pH 7.0 100 mcg / ml 4 2.61 +/- 0.16 98 250 mcg / ml 4 2.58 +/- 0.13 97 1000 mcg / ml 4 2.12 +/- 0.15 79 10 2500 mcg / ml 4 1.97 +/- 0.11 74

The data in Table 2 show no significant effect on cell growth at 100-500 mcg / ml PDP, but that even on normal cells, viability is reduced at 1000 and 2500 mcg / ml. Remarkably, even at high concentrations, the effect on the myeloma tumor cells (Table 15 1) is more pronounced than the effect on normal cells (Table 2).

Similar results are obtained with lithium, sodium, magnesium, calcium, strontium, zinc and stannous salts of PDP, organic peroxydiphosphate and C1-12 alkyl, adenylyl, guanylyl, cytosylyl, thymylyl esters and tetramethylammonium salts .

Example 2: Effects of PDP. potassium pyrophosphate (KPP) and PBS (saline with phosoate buffer) on tumor development in vivo.

75 identical Balb / C mice with an average weight of 20 grams +/- 3 grams in groups of 25 animals each: (a) control treated with saline with phosphate buffer (PBS); (b) treated with potassium peroxydiphosphate (PDP) and PBS, pH 7.0, and (c) potassium pyrophosphate (KPP) and PBS as phosphate control. Each animal receives 0.2 ml of Pristan intraperitoneally (I.P) to prepare the animals for 30 malignant SP2 cell implantation (murine myeloma-car c in-tumor cells). After three weeks, the animals are subjected to the following oral treatment regimen: Group (a) receives via I. P. 0.2 ml of 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 hours after the third injection, every 5 animals (I.P.) are seeded with 2 to 3 x 10 6 cells SP2 (mouse tumor cells, murine myeloma). Then, the animals receive their respective materials once a day for 5 days / week. Ie (a) PBS, (b) PDP, or (c) KPP. The animals are assessed for tumor development and death each week. The data is analyzed using the Mantel-Haenszel approach (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 is significantly effective in controlling tumor development in mice compared to PBS or KPP, and thus shows that the 15 effects of inhibiting tumor development are due to the provision of active oxygen compounds and not phosphate.

Table 3 PBS * VS. KPP **

TUMOR DEVELOPMENT INVESTIGATION FOR 10 WEEKS 20 US TREATMENT TUMOR AND DEATH NO TUMOR MOUSE EXPOSED TO RISK

1-4 PBS 11 14 25 KPP 10 15 25 5 PBS 4 10 14 KPP 4 11 15 25 6 PBS 5 5 10 KPP 2 9 11 7 PBS 2 3 5 KPP 4 59 8 PBS 0 33 30 KPP 1 4 5 DK 168191 B1 10 9 PBS 2 1 3 KPP 3 14 10 PBS 1 0 1 0 11 5 Mantel-Haenszel chi-square = 0.36 with 1, df, P = 0.55 Odds ratio = 1.34

These results are not significant and show no significant difference between PBS and KPP in reducing tumor development in the animals.

10 * PBS = Saline with phosphate buffer.

** KPP = Potassium pyrophosphate.

Table 4 TUMOR STUDY FOR 10 WEEKS PBS * VS: PDP ** 15 WEEK TREATMENT TUMOR AND DEATH NO TUMOR RISK 1-4 PBS 11 14 25 PDP 2 23 25 5 PBS 4 10 14 PDP 4 19 23 20 6 PBS 5 5 10 PDP 5 14 19 7 PBS 22 5 PDP 2 13 14 8 PBS 03 3 25 PDP 2 10 12 9 PBS 21 1 PDP 3 7 10 10 PBS 10 1 DK 168191 B1 11 PDP 1 6 7

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

These data show that the PBS control group develops tumors significantly earlier than PDP-treated animals (P = 0.001).

* PBS = Saline with phosphate buffer ** PDP = Potassium peroxydiphosphate.

Table 5 THUMB SURVEY FOR 10 WEEKS 10 KPP * VS. PDP ***

WEEK TREATMENT TUMOR AND DEATH NO TUMOR EXPOSED TO RISK

1-4 KPP 10 15 25 PDP 2 23 25 5 KPP 4 11 15 15 PDP 4 19 23 6 KPP 2 9 11 PDP 5 14 19 7 KPP 459 PDP 2 14 14 20 8 KPP 14 5 PDP 2 10 12 9 KPP 314 PDP 3 7 10 10 KPP 0 11 25 PDP 16 7 DK 168191 B1 12

Mantel-Haenszel chi-square = 5.86 with 1, d.f., P = 0.02 Odds ratio = 2.60

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

5 * KPP = Potassium pyrophosphate *** PDP = Potassium peroxydiphosphate.

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

Similar results are obtained with other inorganic salts of PDP, especially the lithium, sodium, magnesium, calcium, strontium, zinc and stannous salts. Organic compounds of PDP, especially C1-12 alkyl, adenylyl, guanylyl, cytosylyl, thymylyl esters and tetramethylammonium salts are also effective in counteracting the growth of malignant murine myeloma tumor cells.

Example 3 20,500 parts of potassium peroxydiphosphate and 641 parts of mannite are mixed and wetted with 32.5 parts of a 10% solution of sorbit to form a wet granulate which is dried at 49 ° C and sieved through a 12 mesh US sieve (1.68 mm sieve openings). 35 parts of magnesium stearate are then added as a binder and tabulated 25 grains are formed by compressing the mixture on a tablet machine.

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

Cellulose acetate phthalate 120 parts 30 Carnauba wax 30 parts

Claims (7)

10 Deionized distilled water is stabilized at atmospheric pressure for 20 minutes in an autoclave. After cooling, it is tested for pyrogenicity using Limulus Amebocyte Lysate (LAL) as described by Tsuji et al. in "Pharmaceutical Manufacturing", October, 1984, pages 35-41. 50 parts of potassium peroxydiphosphate, 15 sodium chloride in an amount equal to 0.9% solution and 0.1 M phosphate buffer containing KH2PO4 and Na2HPO4, pH 9.4 is added to the pyrogen-free sterile water. The solution is then sterilized by passing it through a 0.5 micropore filter and then packing into sterile bottles. 20 Patent claims. Use of a non-toxic, water-soluble, pharmaceutically suitable derivative of peroxydiphosphoric acid as an active substance, in particular an alkali metal, alkaline earth metal, zinc, tin or quaternary ammonium salt or a C1-12 alkyl, adenylyl , guanylyl, cytosylyl or thymylyl ester, at a concentration of 0.1 to 10% by weight for the preparation of pharmaceutical compositions having inhibitory effect on tumor formation, more particularly, tablets coated with a material which resists gastric acid degradation but degrades of intestinal fluid at a pH value of 5.5 to 10, or a buffered, non-pyrogenic aqueous solution for systemic use, preferably with a pH of 7.0 to 7.4. Use according to claim 1, wherein for the preparation of tablets, the derivative of peroxydiphosphoric acid is mixed with a solid polyhydroxy sugar, moistened the mixture with a solution of a polyhydroxy sugar compound, aims at size, mixes a binder therewith, compresses the obtained mixture to tableted grains and coatings thereof by spraying a 10 film of a coating solution which is not inactivated by gastric acid and dissolved by domestic liquid having a pH of 5.5 to 10. Use according to claim 2, wherein the coating of the tablets is constructed from 40 to 50 parts by weight of a fatty acid ester, 15 to 25 parts by weight of a wax species, 20 to 30 parts by weight of a 15 fatty acid, and 5 to 15 parts by weight of a cellulose ester. Use according to claim 3, wherein n-butyl stearate is used as fatty acid ester, as wax is used carnauba wax, as fatty acid is using stearic acid and as cellulose ester is is using cellulose acetate phthalate. Use of potassium peroxydiphosphate according to any one of claims 2 to 4. Use according to claim 1, in which to prepare a solution for systemic use, the deionized distilled water is sterilized to be non-pyrogenic and then a phosphate buffer, the derivative of peroxydiphosphoric acid and sodium chloride is added. Use of potassium peroxydiphosphate according to claim 6. 30