LU85976A1 - COMPOSITION FOR INHIBITING TUMOR DEVELOPMENT BASED ON A PEROXYDIPHOSPHORIC ACID DERIVATIVE - Google Patents

Description

*, * * 1 # The invention relates to the inhibition of the development of tumors, which concerns tumor cells in vitro and the real development of tumors in vivo in warm-blooded animals.

05 Cancer results from the development of malignant tumors. Considerable medical research has been done to reduce and defeat the scourge of cancer.

To date, a curative treatment for cancer has not been found. However, we have learned a lot about the irçëcanis-10 me by which warm-blooded animals avoid cancer. The invention contributes to this knowledge by providing a material which inhibits the development of tumors and a method of inhibiting this development.

Among the cells contained in the fluids of the mammalian organism are lymphocytes, monocytes, macrophages and polynuclear cells. These cells act as a natural surveillance system against the development of tumors from lower mammals, such as rodents, to humans. In recent years, it has been observed that a particular subpopulation of lymphocytes or lymphoid cells called natural killer cells ("Natural Killer" or "NK") destroys tumor cells and thus prevents the development of cancer.

The facts suggest that NK cells have cytolytic activity linked to the formation of an active oxygen species such as hydrogen peroxide (H202) or radicals containing oxygen-gene, for example the hydroxy anion (ΌΗ) and superoxide anion (02 *). KK cells and phenomena related to active oxygen are described by Herberman et al., Science, Vol. 214, October 2, 1981, pages 24-30; Roder et al., Nature, Vcl. 256, August 5, 1982, pages 35,569-572; Nathan et al., Journal of Iramunology, Vol.

129, No. 5, November 1582, pages 2 164-2171; and Ma vier et Coll., Journal cf Ir ~ unoiogy, Vol. 132, re 4 ", * f *> April 1984, pages 1 980- 1 986.

Of course, there are many compounds that release active oxygen species. However, this factor alone does not mean that such a compound can be introduced into the body to complete the function of NK cells or when the formation of a tumor is not sufficient to ensure the function of NK cells and inhibit tumor development. Compounds which release active oxygen species generally do so rapidly, while efficacy against tumor development in warm-blooded animals such as humans appears to require slower and more prolonged release. Until the invention this was not effectively achieved.

When the release of oxygen is too rapid, both tumor cells and normal cells can be attacked.

In US Patent 4,041,149 issued August 9, 1977, there is described a composition in various forms, including a dental tablet, which inhibits the onset of bad breath and whose active ingredient is peroxydiphosphate. Peroxydiphos-phate differs from most oxygen-providing compounds in that it does not produce an initial burst of hydrogen peroxide. On the other hand, it releases hydrogen peroxide slowly so that when comparisons are made at equivalent concentrations with hydrogen peroxide, the amount of oxygen released by peroxydiphosphate 30 is only one tenth that amount active oxygen released by hydrogen peroxide. In addition, only about 50% of 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 organisms of warm-blooded animals, including mice, rats, humans, etc.

A der r.unt = of the invention on the street the '"f 7% t * 3 tumor development is inhibited on tumor cells in vitro and in the real development of malignant tumors in vivo in warm-blooded animals ranging from rodents to humans.

Another advantage of the invention is that it provides methods for inhibiting the formation of tumors by introducing a material slowly releasing oxygen into a living host.

According to some of its aspects, the invention relates to a composition comprising a dose of approximately 0.1 to 10% of a non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydi-phosphoric acid dissolved or dispersed in a pharmaceutical vehicle.

According to some of its other aspects, the invention relates to a method for inhibiting the formation of tumors containing malignant tumor cells in which a composition comprising a non-toxic dose of about 0.1 to 6 g per kg of body weight. of a warm-blooded animal of a non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical vehicle is administered to a warm-blooded host animal, by oral ingestion, according to a po- 25 sology which provides about 0.1 to 6 g per kg of body weight of said warm-blooded animal per day.

According to some of its other aspects, the invention relates to a method for inhibiting the formation of tumors, wherein a composition comprising a non-toxic dose of about 0.1 to 2 g per kg of body weight of a warm-blooded animal having malignant tumor cells of a non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydiphosphoric acid dissolved or dispersed in a pharmaceutical vehicle, is administered systemically (general) to a host animal with blood hot according to t "-e rosolcmie which errrrre en-ri in 0.1 S 2 c because km of,> h * 4 1Γ body weight of said warm-blooded animal.

The compound of peroxydiphcsphate type (PDP) is in the form of a non-toxic pharmaceutically acceptable compound which goes further than a salt indicated 05 in the aforementioned patent US Pat. The compounds include the salts of alkali metals (e.g. lithium, sodium and potassium), alkali-no-earth metals (e.g. magnesium, calcium and strontium), zinc and tin, as well that the organic es-10 ters, of peroxydiphosphate of alkyl in â'adënylyl, <3e guanylyl, of cytosylyl and of thymylyl, as well as the quaternary ammonium salts and the like. Among the mineral cations, the alkali metals are preferred, in particular the potassium salts. Tetrapotassium peroxydiphosphate is a stable, odorless, finely divided, white, non-hygroscopic, crystalline solid with a molecular weight of 346.35 and an active oxygen content of 4.6%. Tetrapotassium peroxydiphosphate 20 is soluble at 47-51% in water between 0 ° and 61 ° C, but insoluble in ordinary solvents, such as acetonitrile, alcohols, ethers, ketones, dimethylformamide, dimethyl sulfoxide and similar. A 2% aqueous solution has a pH of about 9.6 and a saturated solution has a pH of about 10.9. A 10% aqueous solution in water at 25 ° C. shows no loss of active oxygen after four months; and at 50 ° C. a 10% solution has a loss of active oxygen of 3% in six months.

The organic salts may be particularly suitable for administration against malignant tumors. Among the organic esters, those having hydrophobic properties, such as those having an alkyl radical in ce5: which facilitate the fast fixing of the peroxydiphosphate fragment by the cells, such as the adenlylyic, euanylylic, cyto-spliclic and thymylylivesters. jt "5

The pharmaceutical vehicles or carriers suitable for oral ingestion are coated tablets "composed of a material which resists decomposition by gastric acids at the pH of the stomach (approximately 05 1-3), because peroxydiphosphate would be inactivated by these stomach acids. On the other hand, vehicles containing granules shaped into tablets of a solid peroxydiphosphoric acid salt, are dissolved by intestinal fluids which have a higher pH (approximately 10 5.5-10) and which do not inactivate peroxydiphosphate and let it undergo the enzymatic action of phosphatases present in humans and other warm-blooded animals. A suitable solution for coating the tablets is composed of 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 parts, preferably about 20 parts by weight), a fatty acid such as stearic acid 20 (typically about 20-30 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 25 400-900 parts). Other desirable coating materials include shellac and copolymers of maleic anhydride and ethylenic compounds such as polyvinyl methyl ether. These coated forms differ from tablets, which are fragmented in the oral cavity, the tablet material of which typically contains about 80 to 90 parts by weight of mannitol and about 30 to 40 parts by weight of magnesium stearate.

The granules formed into tablets of the peroxydiphosphate type salt are formed by mixing approximately 30 to 50 parts by weight of the peroxydiphosphate with approximately 45 to 65 parts by weight of a solid pclyhydro- * η 6 4 xylé sugar, such as mannitol, and moistening with about 20 to 35 parts by weight of a solution of a polyhydroxylated sugar, such as sorbitol, sieving to the desired size, mixing with about 20 to 35 parts by weight of a binding agent, such as magnesium stearate, and pressing the granules into tablets with a tablet shaping machine.

The granules formed into tablets are coated by spraying with a foam of a solution of the coating material and drying to remove the solvent. These tablets differ from dental tablets which are typically pressed granules which do not have a particular protective coating.

An effective dosage of peroxydiphosphate, when administered by oral ingestion, is about 0.1-6 g / kg of body weight per day; when the administration is carried out systemically, for example by intramuscular, intraperitoneal or intravenous injection, the dosage is approximately 0.1-2 g / kg of body weight per day.

Physiologically acceptable pyrogen-free solvents are suitable vehicles for use in conventional systemic administration. A phosphate buffered saline having a physiological pK of about 7 to 7.4 is the preferred vehicle for systemic administration. These solvents differ from vehicles consisting of water and a humectant typically used in toothpaste. Such a solution is typically prepared by sterilization of deionized distilled water, control to ensure the pyrogen-free character according to the test using a lysate of Limulus amibocytes (IAL) described by Tsuji and Coll, in "Pharmaceutical Manufao-35 turing ", October 1984, pages 35-41, then addition of phosphate buffer ÇpE for example approximately 8.5-105 consisting of sterile pyrogen-free water and approximately 1 i Ι0Γ 7 mg of a peroxydiphosphate derivative and sodium chloride at a concentration of about 0.5 to 1.5% by weight. The solution can be packaged in vials for use after further sterilization by passage through a microporous filter. Otherwise, other solutions can be used, such as Ringer's solution containing 0.86 I by weight of sodium chloride, 0.03% by weight of potassium chloride and 0.033% by weight of calcium chloride.

10 Peroxydiphosphate (PDP) slowly releases hydrogen peroxide in the presence of the phosphatase enzymes according to the following equation: ° jj * phosphatases jj * ^ 0 B ^ C ^ + PÛ ^ 15 X.-OPOOPO -> -oopo - ->

4 I I I

0 0 Ci- wherein X is a cation, or a fragment forming an organic ester, non-toxic and pharmaceutically acceptable. The phosphatase that breaks down peroxydiphosphate is present in saliva, as well as in plasma, intestinal fluids and white blood cells. The slow release of oxygen is particularly effective in supplementing the efficacy of NK cells against malignant tumor cells which are sensitive to treatment with peroxydiphosphate. When treating warm-blooded animals with a PDP according to the invention, it is desirable to establish a dosing regimen which continues at least until the tumors have regressed.

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

35 EXAMPLE 1

In vitro study of the cytotoxicity of PL? "is-g-vjs des *" 8

In this study, the effect of PDP was examined at various concentrations on the growth of murine myeloma cells (SPj line) (Table 1).

Human gingival fibroblasts were used as normal control cells (Table 2). The cells were cultivated in Eagle's medium modified according to Dulbecco, supplemented with 10% of fetal bovine serum, 1 X of vitamins MEM, 1 X of L-glutami "^ ne, 1 X of NEAA ^, and 1 X of gentamycin. They were incubated at 37 ° C in a humidified atmosphere. About 1 to 3 x 10 ^ cells were placed in each well of a 24 well microtiter plate containing 2 ml of medium. PDP (potassium salt) at various concentrations was added.

After incubation, the viability of the cells is examined by taking samples of the wells at the times indicated in Table 1. The viability is evaluated, according to the blue try-pan exclusion test. Fresh medium is added to each well every day to maintain the necessary culture conditions. The inhibition is calculated by comparing the percentage of living cells in phosphate buffered saline (PBS) versus PDP. The data are collated in Table 1. 25 Table 1

Effect of PDP on murine myeloma cells (sp2 line)

Treatment N Number of cells% of cells 30 5 x 103 (at 72 hours) viable Control (PBS) 4 8.98 ± 0.14 100% PDP pK 7.0 100 pg / rrtl 4 Ι, Εί IG, I (47 500 pg / ml 4 1.33 Z 0.02 33 1000 pg / ml 4 1.07 z ·.: "29 2500 nu '-: 4 r: i- 12 *» • € 9, The results show that, by compared to the buffer control, the potassium salt of PDP is very cytotoxic and inhibits murine myeloma cells (cancer).

05 Table 2 shows the effects on normal cells (human gingival fibroblasts)

Table 2

Effect of PDF on human gingival fibroblasts 10 Treatment N Number of cells% of viable cells (at 72 hours) viable Control (PBS) 4 2.67 * 0.17 100 PDP pH 7.0 15 100 jig / ml 4 2, 61 ± 0.16 98 500 pg / ml 4 2.58 ± 0.13 97 1000 jig / ml 4 2.12 i 0.15 79 2500 pg / ml 4 1.97 ± 0.11 74 20 Data in the table 2 show no significant effect of PDP on cell growth at 100-500 μg / ml but, even for normal cells, the viability is reduced to 1000 and to 2500 μg / ml. It should be noted that the effect on myeloma 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 tin salts of PDP, with organic peroxydiphosphates such as alkyl esters. adenylyl, guanylyl, cytosylyl. and thymylyl as well as the tetramethylammonium salt of PDP.

35 EXAMPLE 2

The effects of PDP, potassium pyrophosphate (PPE; d-fold) serum, physiological buffered.

it • 4 * 10 phosphate) on the in vivo development of tumors.

75 genetically identical Balb / C mice with an average weight of 20 4 3 g are used, divided into the following groups of 25 animals: 05 (a) a control group treated with phosphate buffered saline (PBS), (b) a group treated with potassium peroxydiphosphate (PDP) and PBS at pH 7.0, and (c) a control group for phosphate treated with potassium pyrophosphate (KPP) and PBS.

Each animal receives an intraperitoneal injection (i. P.) Of 0.2 ml of Pristane to sensitize it to the implantation of malignant SP2 cells (cells of a cancerous tumor of the murine myeloma type 15). After three weeks, the animals are subjected to the following oral ingestion treatments: 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. Forty-eight hours after the third injection, each animal (i. P.) Is inoculated with 2 to 3 x 10th SP2 cells (mouse tumor cells, murine myeloma). The animals are then subjected to the corresponding treatment once a day, five days a week, namely: (a) P3S, (b) PDP and (c) KPP. Each week, we note the development of the tumors of each animal and the deaths. The data are analyzed, according to the Mantel-Kaenszel method (Statistical Aspects of the 30 Analysis of Data from Retrospective Stucles of Disease, J. National Cancer Institute, Vol. 3, 719-748, 1959). The values in Tables 3, 4 and 5 indicate that PDP is significantly effective in controlling tumor development in mice compared to PBS or KPP, which establishes that the effects of inhibiting tumor development are due to the contribution of the species S acrif oxygen and | have·

Y

11 not phosphate.

Table 3 PBS * compared to KPP * *

Study of tumor development at 10 weeks 05

Week Treatment Tumors and No Risk of death tumors 1-4 PBS 11 14 25 10 KPP 10 15 25 5 PBS 4 10 14 KPP 4 11 15 15 6 PBS 5 5 10 KPP 2 9 11 7 PBS 2 3 5 KPP 4 59 20 8 PBS O 33 KPP 1 45 9 PBS 2 13 25 KPP 3 14 10 PBS 1 0 1 KPP 0 11 30 Chi-square test according to liantel-Haenszei = 0.36 with 1 degree of freedom, P = 0.55

Probability ratio = 1.34.

35 These results are not significant and do not establish a significant difference between PBS and KPP with regard to air.

ι 12 of tumor development in animals.

* PBS - phosphate buffered saline KPP = potassium pyrophosphate

Table 4 05 Study of tumors at 10 weeks PBS * compared to PDP ^ s

Week Tumor Treatment and No At Risk Death Tumors 10 --------------------------------------- ---------------- 1-4 PBS 11 14 25 PDP 2 23 25 5 PBS 4 10 14 15 PDP 4 19 23 6 PBS 5 5 10 PDP 5 14 19 20 7 PBS 2 2 5 PDP 2 13 14 8 PBS 0 3 3 PDP 2 10 12 25 9 PBS 2 13 PDP 3 7 10 10 PBS 1 0 1 30 PDP 16 7

Chi-square test according to Mantel-Haenszel = 10.40 with 1 degree of freedom,? = 0.001 35 Probability ratio = 3.66.

These values irire-e--: rua the group test.

13 PBS presents tumors significantly earlier than animals treated with PDP (P = 0.001) * PBS - saline phosphate buffered saline PDP = potassium peroxydiphosphate.

05 * Table 5

Study of tumors at 10 weeks KPP * compared to PDPt **

Week Treatment Tumors and No Risk 10 dead tumor 1-4 KPP 10 15 25 PDP 2 23 25 15 5 KPP 4 11 15 PDP 4 19 23 6 KPP 2 9 11 PDP 5 14 19 20 7 KPP 4 5 9 PDP 2 14 14 8 KPP 1 45 25 PDP 2 10 12 9 KPP 3 14 PDP 3 7 10 30 io KPP 0 11 PDP 1 67

Chi-square test according to Mantel-Haenszel = 5.86 with 1 degree of freedom, 35 p = 0.02

Probability ratio = 2.60.

% 14 *

The data indicate that the KPP group presents significant tumors sooner than animals treated with PDP (P = 0.001) ¥ KPP - potassium pyrophosphate 05 i ** PDP = potassium peroxydiphosphate.

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

Similar results are obtained with other PDP mineral salts, especially the lithium, sodium, magnesium, calcium, strontium, zinc and stannous tin salts. PDP organic compounds, especially C1-12 alkyl, aaënylyl, guanylyl, cytosylyl, thymylyl esters and tetramethylammonium salts are also effective in inhibiting the development of malignant murine myeloma tumor cells .

EXAMPLE 3 500 parts of potassium peroxydiphosphate and 641 parts of mannitol are mixed by moistening them with 32.5 parts of a 10% solution of sor-bitol to form wet granules which are dried at 49p C and qu '' sieved through a US sieve of 12 30 mesh (having mesh openings of 1.68 mm). 35 parts of magnesium stearate are then added as a binder and granules formed into tablets are formed by pressing the composition with a tablet shaping machine.

The tablets are coated with an enteric coating solution having the surviving composition t ^ I * 15

Cellulose acetate phthalate 120 parts

Carnauba wax * 30 parts

Stearic acid 10 parts

95% ethanol 450 parts 05 Acetone qs' 1000 parts.

The coating is carried out using a casting technique in a conventional coating device.

When the tablets thus formed are unmanaged, they pass through the stomach without degradation and the coating is then dissolved by the intestinal fluids.

EXAMPLE 4 15 Deionized distilled water is stabilized at atmospheric pressure for 20 minutes in an autoclave. After cooling, its pyrogen-free character is checked according to the Limulus amibocyte lysate (LAL) test described by Tsuji et Coll, in "Pharmaceutical Manufacturing" October 1984, pages 35-41. 50 parts of potassium peroxydiphosphate, sodium chloride, in an amount corresponding to 0.9% of the solution and of 0.1 M phosphate buffer containing 25 and ^ 2 ^ 0 are added to the sterile pyrogen-free water. ^, pH 9.4. The solution is then sterilized by passage through a filter having micropores of 0.5 μm, then it is packaged in sterile containers.

Claims (9)

1. Composition comprising a dose of approximately 0.1 to 10% of a non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydiphos phoric acid dissolved or dispersed in a pharmaceutical vehicle # the pharmaceutical vehicle being a phosphate buffered saline having a pH of about 7.0 to 7.4 or a coated tablet material which resists decomposition by stomach acids while being broken down by intestinal fluids at a pH of about 5, 5 to 10.. 2. Composition according to claim 1, characterized in that said pharmaceutical vehicle is said phosphate-buffered saline solution. 3. Composition according to claim 1, characterized in that said pharmaceutical vehicle is said material for coated tablets which resists decomposition by gastric acids while being decomposed by intestinal fluids at a pH of approximately 5.5 to 10. 4. Composition according to claim 3, characterized in that the coating of said tablet of the pharmaceutical vehicle comprises approximately 40 to 50 parts by weight of a fatty acid ester, approximately 15 to 25 parts by weight of a wax, approximately 20 to 30 parts by weight of a fatty acid and about 5 to 15 parts by weight of a cellulose ester. 5. Composition according to claim 4, characterized in that the fatty acid ester is n-butyl stearate, the wax is carnauba wax, the fatty acid is stearic acid and the ester of cellulose is cellulose acetate phthalate. 6. A composition according to claim 1, characterized in that the non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydiphosphoric acid is a salt which is chosen from among the alkali metal salts of retiv? *: - · - .. arreux, of "Jr *" S * 17 zinc and tin. I. Composition according to claim 1, characterized in that the non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydiphosphoric acid is chosen from the group consisting of alkyl esters of <3'adenylyl, of guanylyl, cytosylyl, thymylyl and a quaternary ammonium salt. B. Composition according to claim 6, 10 characterized in that said salt is the potassium peroxydiphos-phate. 9. Composition according to Claim 7, characterized in that the said derivative is an alkyl ester containing peroxidiphosphoric acid. 10. Composition according to claim 7, characterized in that said derivative is an adenosyl, guanylyl, cytosyl or thymylyl ester of peroxydiphosphoric acid-II. Application of a composition according to one of Claims 1 to 10 for obtaining a medicament intended to inhibit the development of malignant tumors in warm-blooded animals. 12_. Process for the preparation of granules shaped into tablets having a coating which is not broken down when passing through the stomach and which is dissolved by the intestinal fluids having a pH of 5.5 to 10, characterized in that it comprises mixing a non-toxic water-soluble and pharmaceutically acceptable derivative of peroxydiphosphoric acid with a solid polyhydroxylated sugar and humidifying the mixture with a solution of polyhydroxylated sugar, sieving to the desired size, mixing with a binding agent, pressing to form granules shaped into tablets and coating of said granules shaped into tablets by spraying a film of a coating solution which is not inactivated by acids by _____ -; .c cul est dir rouie rr 'es 11. qui des I »« «16 13- Process for the preparation of a solution of a non-toxic derivative soluble in water and pharmaceutically acceptable peroxydiphosphoric acid suitable for gastric administration, characterized 05 in that it comprises the sterilization of distilled water deionized to make it pyrogen-free and then adding thereto a phosphate buffer and said derivative of peroxydiphosphoric acid and sodium chloride "r