SE468501B - APPLICATION OF DERIVATIVES OF PEROXIDIFOSOIC ACID FOR INHIBITION OF TUMOR DEVELOPMENT - Google Patents

Description

468 501 support the function of NK cells or where tumor formation does not occur to a sufficient degree to take over the function of NK cells and inhibit tumor development. Compounds that release active oxygen-containing groups generally do so very rapidly, whereas efficacy against tumor development in warm-blooded mammals such as humans appears to require at least a slower and more sustained release rate. Until the day of the present invention, this has not been possible. When the oxygen is released too quickly, both tumor and normal cells can be attacked.

U.S. Patent No. 4,041,149, issued August 9, 1977, discloses a composition in various forms comprising a dental tablet which inhibited the formation of a mouthpiece in which the active ingredient was a peroxide phosphate. Peroxide phosphate compounds differ from most other oxygen-providing compounds in that they do not provide an initial release of hydrogen peroxide. Instead, 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 available oxygen released by hydrogen peroxide. Furthermore, only about 50% of the active oxygen content is released for 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, etc.

An advantage of the present invention is that tumor development is inhibited on tumor cells in vitro and true malignant tumor development in vivo in warm-blooded animals, such as rodents and all the way up to humans.

A further advantage of the present invention is that tumor formation can be inhibited by the introduction of a slow oxygen releasing material into a living host.

According to certain aspects of the present invention, the invention relates to a composition comprising a dosage amount of about 0.1 - 10% of a nontoxic water-soluble pharmaceutically acceptable compound derived from peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical carrier.

Inhibition of tumor formation with malignant tumor cells occurs by administering a composition comprising a nontoxic dosage amount of about 0.1 - 6 g / kg body weight of a warm-blooded animal of a nontoxic water-soluble pharmaceutically acceptable compound derived from peroxydiphosphoric acid dissolved or dispersed. in a pharmaceutical carrier to a warm-blooded animal host by oral ingestion of a regimen which gives about 0.1 - 6 g / kg body weight of said warm-blooded animal per day.

One way to inhibit tumor formation is to systemically administer a composition comprising a nontoxic dosage amount of about 0.1 - 2 g / kg body weight of a warm-blooded animal having malignant tumor cells, of a nontoxic water-soluble pharmaceutically acceptable compound derived from peroxide phosphorus. acid dissolved or dispersed in a pharmaceutical carrier, to a warm-blooded animal value a dose of about 0.1 - 2 g / kg body weight of said warm-blooded animal.

The peroxydiphosphate compound (PDP) is in the form of a nontoxic pharmaceutically acceptable compound, which goes beyond the salt disclosed in the aforementioned U.S. Pat. No. 4,041,149.

The compounds include alkali metal salts (eg lithium, sodium and potassium), alkaline earth metal salts (eg magnesium, calcium and strontium), zinc and tin salts as well as organic peroxide diphosphate-C1-12 alkyl, adenylyl, guanylyl, cytosylyl and as well as quarters- CT * - CC! (fl CT! ... s near ammonium and similar salts. Alkali metal, especially potassium salt, is preferred over other inorganic cations.

The tetrachotassium peroxide diphosphate is a stable, odorless, finely divided, free-flowing, white non-hygroscopic crystalline solid with a molecular weight of 346.5 and an active oxygen content of 4.6%. Tetra potassium peroxide diphosphate is water soluble: in 47 - 51% at o - 61 ° C, but illegally in common solvents such as acetonitride, alcohols, ethers, cretones, dimethylformamide, dimethylsulfoxide and the like.

A 2% aqueous solution has a pH of about 9.6 and a saturated solution thereof has a pH of about 10.9. A 10% solution in water at 25 ° C showed no loss of active oxygen for 4 months; and at 50 ° C a 10% solution showed a loss of active oxygen of 3% for 6 months.

The organic salts may be particularly suitable for administration against malignant tumors. Of the organic esters, those which give hydrophobic properties, such as C1-12 alkyl radical, are preferred and they facilitate rapid uptake of the peroxide diphosphate moiety by the cells, such as adenylyl, guanyllyl, cytocylyl and thymylyl esters.

Pharmaceutical carriers suitable for oral administration are coated tablets composed of materials which degrade gastric acids at the pH present in the stomach (about 1-3), as the peroxide phosphate would be inactivated by such gastric acids. Instead, the carriers are dissolved in tableted granules of the solid peroxydiphosphoric acid salt material therein through intestinal fluids which have a higher pH (about 5 - 10) and which do not inactivate the peroxydiphosphate but leave it for enzymatic action of phosphatase present in man and others. 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 carnuba wax (typically about 15-25 and preferably about 20 parts by weight), fatty acid such as stearic acid (typically about 20-30, preferably parts by weight) and cellulose esters such as cellulose acetate phthalate (typically about 5-15 and 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 polyvinyl methyl ether. Such coatings differ from tablets which are broken down in the oral cavity 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 peroxydiphosphate salt are prepared by mixing about 30 to 50 parts by weight of the peroxyphosphate salt with about 45 to 65 parts by weight of a solid polyhydroxy sugar such as manitol and wetting with about 20 to 35 parts by weight of a solution of a polyhydroxy sugar compound such as sorbitol. sifting to size, mixing with about 20-35 parts by weight of a binder such as magnesium stearate and compacting the granules into tablets with a tablet press machine. The tableted granules are coated by spraying a foam of a solution of the coating material thereon and drying to remove solvent. Such tablets differ from dental tablets, which are typically pressed granules without any special protective coating.

An effective band with a prescribed regimen, when administered by oral ingestion, is about 0.1 - 6 g / kg body weight daily; when the administration is systemic, such as by intra-administration dose of peroxide diphosphate in co-muscular, intraperitoneal or intravenous injection, the dose is about 0.1 - 2 g / kg body weight daily. 468 501 Physiologically acceptable pyrogen-free solvents are suitable carriers for use in systemic administration.

Saline solution buffered with phosphate to a physiological pH of about 7 - 7.4 is the preferred carrier for systemic administration. Such solvents differ from water humectant vehicles typically used in toothbrushes.

Such a solution is prepared by sterilizing deionized distilled water, checking to ensure non-pyrogenicity using the limulus amoebocyte lysate (LAL) test described by Tsuji et al in "Pharmaceutical Manufacturing", October 1984, p. 35 - 41, with subsequent addition of phosphate buffer (pH eg about 8.5 - - 10) prepared from pyrogen-free sterile water and about 1-100 μm; peroxide diphosphate derivative compound and sodium chloride to a concentration of about 0.5 - 1.5% by weight. The solution can be packaged in bottles for use after being resterilized by passage through a microporous filter.

Alternatively, other solutions such as Ringer's solution may contain 0.86% by weight of sodium chloride 0i, 03% by weight of potassium chloride and 0.033% by weight of calcium chloride.

The peroxydiphosphate compound (PDP) slowly releases hydrogen peroxide in the presence of phosphatase enzymes according to the following equation: O 0 O ll ll ll 3 phosphates H 2 O H 2 O 2 + PO 4 I r O O O- Where X is a nontoxic pharmaceutically acceptable cation or terminates an organic ester group. Phosphatase that breaks down peroxide phosphate is present in saliva as well as in plasma, intestinal fluids and white blood cells. The slow oxygen release is particularly effective in enhancing the efficacy of I: 468 501 NK cells against malignant tumor cells, which respond to peroxide diphosphate therapy. When warm-blooded animals are treated with PDP according to the present invention, it is desirable to provide a cure, whereby the treatment continues at least until the tumors have recovered.

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

EXAMPLES Example 1: In vitro study of PDP tumor cytotoxicity.

This study examines the effects of PDP at different concentrations on the growth of murine myeloma (SP2 line) cells (Table 1). Human gingival fibroblasts were used as a control for normal cells (Table 2). The cells are grown in Dulbecco's modified Eagles medium supplemented with 10% fatty bovine serum, 1X MEM vitamins, 1XL-glutamine, 1X NEAA3, 1X gentamucine. They are incubated at 37 ° C in a humidified CO 2 atmosphere. Approximately 1 - 3 x 105 cells are placed in each wave by a 24-wave microtiter plate containing 2 ml of the medium. PDP (potassium salt) in varying concentrations is added.

After incubation, the viability of the cells is determined by removing samples from the waves over the time given in Table 1. The life force is determined by the "trypan blue exclusion test". New medium is added every wave, every day to maintain the necessary growth conditions. The inhibition was calculated by comparing the percentage of cells living in phosphate buffered saline (PBS) compared to PDP. Data are summarized in Table 1. 468 501 TABLE 1 Effects of PDP on murine myeloma (SP2-lü fi e) cells.

Treatment Control (PBS) PDP pH 7.0 100 mcg / ml 500 mcg / ml 1000 mcg / ml 2500 mcg / ml E nhnhnßuh Number of cells x 10 (after 72 hours) - 8.98 1.86 1.33 1.07 0.48 l + I + I + l + l + 0.14 0.14 0.03 0.17 0.15% viable cells 100% 47 33 29 12 These results show that compared to the buffer control potassium salt of PDP is highly cytotoxic and inhibitory against murine myeloma, cells (cancer).

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

TABLE 2 Effects of PDP on human gingival fibroblast.

Behanälin N Number of cells x 105% liVSdu¶li¶ä _________JE _ (after 72 hours) Eååiåå Control (Pass) 4 2.67 in 0.17 1oo PDP pH 7.0 100 mcg / ml 4 2.61 f 0.16 98 son mcg / ml 4 2.58 f 0.13 97 1ooo mcg / ml 4 2.12 3 0.15 79 2500 mcg / ml 4 1.97 i 0.11 74 V! 468 501 The data in Table 2 do not indicate a significant effect on cell growth at 10Û'500IM@hM.PDP but even on normal cells the viability is reduced at 100 and 2500 mcg / ml. It is remarkable that the effect on myeloma tumor cells (Table 1) even at high concentrations is more pronounced than the effect with normal cells (Table 2).

Similar results are obtained with lithium, sodium, magnesium, calcium, strontium, zinc and tin salts of PDP, organic peroxydiphosphate as well as C1-12 alkyl, adenylyl, guanyllyl, cytosylyl, fl nmyh fl esüßr and tetramethylammonP.

Example 2: Effects of PDP. potassium neurophosphate (KPP) and PBS (phosphate buffered saline) on tumor development in vivg- Seventy-five genetically identical Balb / C mice with an average weight of 20 g 1 3 g in groups of 25 animals each: (a) control treated with phosphate buffered saline (PBS) ; (b) treated with potassium peroxide diphosphate (PDP) and PBS, pH 7.0; and (c) potassium pyrophosphate (KPP) and PBS as a phosphate control. Each animal receives 0.2 ml of pristine intraperitoneally (I.P.) to prepare the animals for implantation of malignant SP2 cells (murine myeloma carcinoma tumor cells). After three weeks, the animals are put on a treatment comprising orally taking a regimen as follows: Group (a) receives via 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. Forty-eight hours after the third injection, each animal is inoculated (I.P.) with 2 to 3 x 10 6 cells SP2 (mouse tumor cells, murine myeloma). Thereafter, the animals are given their respective materials once daily for 5 days per week, i.e. (a) PBS, (b) PDP or (c) KPP. The animals are recorded with regard to tumor development and death every week. Data are analyzed with 468,501 using the Mantel-Haenszel procedure (Statistical Aspects of the Analyzes of Data from Retrospective Studies of Disease, J. National Cancer Institute, vol. 3, 719-748, 1959).

Data in Tables 3, 4 and 5 indicate that PDP is significantly effective in controlling tumor development in mice compared to PBS or KPP, thereby proving that the effects on inhibiting tumor development are due to the provision of active oxygen groups and non-phosphate.

TABLE 3 PBS * VS. CPP ** Ten-week study of tumor development VBCKA TREATMENT TUMOR AND PRAYER NO TUMOR IN RISKZONBN 1-4 PBs 11 14 25 KPP 10 15 25 PBs 4 10 14 KPP 4 11 15 6 PBs 5 5 10 KPP 2 9 11 7 PBS 2 3 5 KPP 4 5 9 8 PBs 0 3 3 KPP 1 4 5 9 PBS 2 1 3 KPP 3 1 4 PBS 1 0 1 KPP 0 1 1 Mantel-Haenszel chi-square = 0.36 with 1, df, P = 0.55 Odds ratio = 1.34 These results are not significant and do not prove a significant difference between PBS and CPP in reducing tumor development in the animals.

* PBS = phosphate buffered saline ** KPP = potassium pyrophosphate.

TABLE 4 Ten-week tumor study PBS * VS. PDP ** WEEK TREATMENT TUMOR AND DEATH NO TUMOR IN THE RISK ZONE 1-4 Bßs 11 14 25 PDP 2 23 25 Pßs 4 10 14 PDP 4. 19 23 6 Pas s S 10 PDP 5 14 19 7 Pßs 2 2 5 PDP 2 13 14 s Pas o _3 3 PDP 2 10 12 9 Pas 2 1 3 PDP 3 7 10 Pßs 1 0 PDP 1 6 Mantel-Haenszel chi-square = 10.40 med 1, df, P = 0.001.

Odds ratio = 3.66 These data indicate that the PBS control group develops tumors significantly faster than PDP-treated animals (P = 0.001).

* PBS = phosphate buffered saline ** PDP = potassium peroxide diphosphate 468 5531 12 TABLE 5 Ten week tumor study CPP * VS. PDP *** WEEK TREATMENT TUMOR AND DEATH NO TUMOR IN THE RISK ZONE 1-4 KPP 10 15 25 PDP 2 23 25 KPP 4 ll 15 PDP 4 19 23 KPP 2 9 11 PDP 5 14 19 7 KPP 4 5 9 PDP 2 14 14 3 KPP l 4 5 PDP 2 10 12 9 KPP 3 1 4 PDP 3 7 10 KPP 0 1 PDP 1 5 Mantel-Haenszel chi-square = 5.86 with 1, df, P = 0.02. oaas ratio = 2.60.

Data indicate that the KPP group develops tumors significantly faster than PDP-treated animals (P = 0.001).

* KPP = potassium pyrophosphate *** PDP = potassium peroxide diphosphate 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 giving a concentration of 1 mg / ml (0.1%) in a stable carrier of 45 parts of N-butyl stearate, 20 parts of carnauba wax, 25 parts of stearic acid and 10 parts of cellulose acetate phthalate. 468 501 13 Similar results are obtained with other inorganic salts of PDP, in particular lithium, sodium, magnesium, calcium, strontium, zinc and tin salts. Organic compounds of PDP, especially C1-12 alkyl, adenylyl, guanylyl, cytocylyl, thymylyl esters and tetramethylammonium salts are also effective in counteracting the growth of murine myeloma malignant tumor cells.

EXAMPLE 3,500 parts of potassium peroxide diphosphate and 641 parts of mannitol are mixed and wetted with 32.5 parts of a 10% solution of sorbitol to form a wet granulate, which is dried at 49 ° C and sieved through a 12 mesh US sieve (1, 68 mm sight openings). 35 parts of magnesium stearate are then added as binders and tableted granules are formed by pressing the composition on a tablet press machine.

The tablets are coated with an enteric coating solution having the following composition: Cellulose acetate phthalate 120 parts Carnauba wax 30 parts Stearic acid 10 parts 95% ethanol 450 parts Acetone Q.S. to 1000 parts The coating is carried out by a pouring procedure in a conventional coating vessel.

When the tablets thus prepared are swallowed, they pass through the stomach without decomposition and the coating is then dissolved by intestinal fluids. $> O \ CD CD ... x 14 EXAMPLE 4 Deionized distilled water is stabilized at atmospheric pressure for 20 minutes in an autoclave. After cooling, they are tested for non-pyrogenicity using Limulus-Amoebocyte-Lysate (LAL) as described by Tsuji et al in "Pharmaceutical Manufacturing", October 1984, p. 35 to 41. 50 parts of potassium peroxide diphosphate, sodium chloride in an amount corresponding to 0.9% of the solution and 0.1 M phosphate buffer containing KH 2 PO 4 and Na 2 HPO 4, pH 9.4 are added to the pyrogen-free sterile water. The solution is then sterilized by passage through a 0.5 micropore filter and then packaged in sterile packages. Although the invention has been described in connection with specific examples, it will be apparent to those skilled in the art that various modifications may be made which fall within the scope of the invention.

Claims (3)

468 501/5 P a t e n t k r a v Use of a pharmaceutically acceptable derivative compound of peroxydiphosphoric acid for the manufacture of an anti-cancer agent. Use according to claim 1 wherein the derivative compound of peroxydiphosphoric acid is a salt selected from the group consisting of alkali metal, alkaline earth metal, zinc and tin. Use according to claim 1 wherein the derivative compound of peroxydiphosphoric acid is selected from the group consisting of C 1-12 alkyl, adenylyl, guanylyl, cytosylyl, thymylyl ester and quaternary ammonium salt.