WO2006110119A1 - Antineoplastic preparation - Google Patents

Abstract

The inventive antineoplastic preparation is based on a gluconic acid and exhibits an apoptotic and anti-proliferative effect preserving healthy cells. In the first variant, said preparation is embodied in the form of at least one type of gluconate, wherein a salt-forming agent is selected from a group comprising magnesium, sodium, potassium, η-aminobutyric acid, ethylenediamine or the mixture thereof. In the second variant, the perpetration is embodied in the form of a co-ordination compound which is based on a magnesium gluconate in which ligands are selected from a group consisting of a η-aminobutyric acid, 3-hydroxy-η-aminobutyric acid, ethylene diamine tetraacetic acid and an ethylene glycol tetraacetic acid. In the third variant, the composition is embodied in the form of a binary salt selected from a group consisting of a magnesium butyrate-gluconate, magnesium oxybutyrate-gluconate, magnesium glucarate-gluconate, calcium butyrate-gluconate, butyrate-gluconate oxybutyrate-gluconate, calcium glucarate-gluconate and the combination thereof.

Description

 ANTINEOPLASTIC DRUG (options)

Technical field

The invention relates to the composition of such antineoplastic preparations based on rluconic acid, which exhibit a proapoptotic and antiproliferative effect.

State of the art

Oncological diseases as a cause of disability and mortality in people take second place after cardiovascular diseases (.Putay M. L, Dear

A.P. Illness and mortality in the form of twigs and blood circulation in Ukraine // Nova Medicina. - 2002, NaZ; in Russian: Lutay M. I., Dorogiy A.P. Morbidity and mortality from diseases of the circulatory system in Ukraine // New Medicine. - 2002, NsЗ).

It is already universally recognized that the prerequisite for malignancy of cells is the disruption of the functioning of the genome under the influence of various chemical, physical and biological mutagenic factors. Every day in the body of an adult, up to 10 ⁶ mutant cells arise (Perederni V. G., Bychkova H. G. Popular immunology. - Kiev: “Haykova Dymka”, 1990).

In a healthy body, such cells die almost completely (usually due to apoptosis, the auto-initiation of which is now fairly well understood).

The main role in it is played by the immune system. After recognition of foreign (in particular, mutated) cells, its cells produce chemical agents called cytokines, which “shut down” the multi-stage apoptosis process.

However, often the body does not recognize the danger due to immunodeficiency, which occurs, in particular, under the influence of factors produced by cancer cells. Therefore, in clinical practice, such antineoplastic preparations are increasingly used that themselves can cause apoptosis.

These include (see CD Mosbu's, 2003) antibiotics of the anthracycline group: doxorubicin, otherwise referred to as adriamycin (A. SWadapowski & J. Kopora. Adriatus apditus pogroptochelpocillosophosompochem. 46, 375)), and similar to it bleomycin ^ dactinomycin, daunorubicin, idarubicin, etc.

According to many researchers, doxorubicin and its analogues induce apoptosis by inhibiting topoisomerase (the enzyme responsible for malignant cells). Unfortunately, therapeutic agents of this kind can also induce apoptosis in healthy cells. Thus, the cytotoxic effect of doxorubicin for the human body as a whole occurs with a cumulative dose of more than 20 mg / kg. On the background of cytotoxicosis, concomitant organic lesions develop (often with a fatal outcome). The main ones are thrombocytopenia, neutropenia, anemia and cardiopathy, leading to the death of 5-20% of patients treated with doxorubicin (CD Mosbu's, 2003) and other well-known drugs of the same class (Ez-assess kur opsologogu. Corea UPHAR PHARM, 2004, p. 107).

Naturally, the search and introduction into clinical practice of less dangerous inducers of apoptosis remains an urgent task. It is already known that apoptosis is caused by: betulinic acid (Betulipis acid ipodus aortosis ip humapulobell cellulitis: M.L.Schmidt, et al .; Eur. J. Sapser 33, 2007, 1997), tiuramdisulfide (daupi of arthotisis b thiurаmdisulfιdes, thе rеаtivе metаbоlitеs оf dithiosarbаmаtеs, thоough соrdіпаtіпо moduliop оf NFkarra, with fоs / с-уппо иппо ипо ипо ипо ипо ипо ипо ипо ипо ипо ипо ипо ипо ипо ипп и ипп ипп ипп ипп ипп ипп и ипп ипп ипп ипп ипп иппо ипп ипп ипп ипп ипп ипп ипп ипп ипп и ипо, of arthotisis bu gallic acid as ip lups sapper sells: Y. Ohpo, et al .; Aptisapser Drugs 10, 845, 1999), epigallic acid and its derivatives (1. A tumo romotropi pattwau ap its Fuhpiklis,: .; Sapser Test. Rev. 189, 1 (1994); 2. IPhibitiop Of the tumor-rotor-assisted activator-1 aptivati-ap-bell-trpsurmapiul, (-) -epigallocatechip-gal, Dax-Dal, 1997, Dl. ipodеce mоее heratis arthоsis: rssiblе ipvolvеmept with tumo songstasy, but with caspase-3 support: Y. Itakazu, et al .; Biol. Pharm. BuII. 22, p. 1127, 1999).

All of them are toxic to the body as a whole and are not introduced into clinical practice.

Less dangerous are cytosphatic antineoplastic drugs, which slow down, and often completely suppress, the proliferation (growth) of cancerous tumors (or Еz Еz-assess kur opsologogu, p. Z). The most famous of them are: platinum preparations (cis-platinum, oxyplatinum, carboplatin); alkylating agents (cyclophosphamide, busulfan, dacarbazine, thiotepa, etc.); antimetabolites (cytarabine, fluorouracil, methotrexate, mercaptopurine, etc.); herbal preparations (docetaxel, paclitaxel, vincristine, vinblastine, etc.); hormonal agents (chlormadinone, diethylstilbestrol, flutamide, etc.).

However, almost all cytostatics inhibit the maturation of blood cells, causing anemia, thrombocytopenia and neutropenia, which worsens the condition of patients. Therefore, there is an urgent need for such antineoplastic drugs that would be practically non-toxic to healthy cells.

So, drugs based on interferon and a specific protein TNF (tumor necrotic factor) are known (Vozianov A.F., Butenko AK, Zak K.P. Cytokines. Biological and antitumor properties. - Kiev: Naukova Dumka, 1998). However, TNF acts through specific receptors, which in cancer cells often work differently than in normal cells. Therefore, the practical use of TNF is limited to cases when tumors are obviously sensitive to its action (opcit). Moreover, the technology for producing TNF is very laborious. And finally, some cytokines exhibit noticeable side effects, which limits their effects. change (op sit).

Thus, the task of developing commonly available non-toxic antineoplastic drugs with a wide spectrum of action that can induce apoptosis and inhibit the proliferation of cancer cells remains relevant. In particular, a preparation based on gluconic acid, namely calcium gluconate, which is closest to the invention in technical essence, is known. It has been established that it is useful in chemotherapy of malignant neoplasms (Bogush T.A., Smirnova GB, Vikhpyantseva H.O., Syrkin AB, Effect of calcium gluconate on the toxicity and antitumor activity of doxorubicin in mice. Antibiotics and chemotherapy, 2002).

However, calcium gluconate, taken alone, weakly inhibits the growth of cancerous tumors.

SUMMARY OF THE INVENTION

The basis of the invention is the task of changing the composition to create a drug based on gluconic acid, which, when introduced into the human body, would exhibit a pronounced antineoplastic effect against the background of the preservation of healthy cells.

The task in the first embodiment is solved in that the antineoplastic preparation based on gluconic acid according to the invention is at least one such salt in which the salt-forming agent is selected from the group consisting of magnesium, sodium, potassium, γ-aminobutyric acid (hereinafter GABA ), ethylenediamine or mixtures thereof.

Indeed, as will be shown in the examples below, the practical application of any of the possible combinations of glucdnate anions with the indicated substances, which possess the properties of bases, simultaneously provides an apoptotic and antiproliferative effect.

The first and second additional differences, respectively, are that the antineoplastic preparation is magnesium gluconate, or potassium gluconate. This drug is preferably used for oncological diseases of arbitrary etiology against a background of deficiency of potassium or magnesium ions in the body.

The task in the second embodiment is solved in that the antineoplastic preparation based on gluconic acid according to the invention is a coordination compound based on magnesium gluconate, in which the ligands are selected from the group consisting of GABA, 3-hydroxy-GAMK, ethylene diamine tetraacetic acid (hereinafter EDTA) ) and ethylene glycol tetraacetic acid (hereinafter referred to as EGTA). These drugs are preferred for neoplastic changes in epithelial tissue, especially against a background of deficiency of magnesium ions in the body.

An additional difference is that the antineoplastic preparation is the coordination compound diHAMK-Mg-gluconate. This prep It is preferred to use rats for oncological diseases of the respiratory organs, brain and intestines.

The task in the third embodiment is solved in that the antineoplastic preparation is a double salt selected from the group consisting of magnesium butyratogluconate, magnesium hydroxybutyrate gluconate, magnesium glucarate gluconate, calcium butyratogluconate, calcium hydroxybutyrate gluconate, calcium glucarate gluconate and their combination. These drugs are preferably used for neoplasty.

processes in the presence of deficiency of magnesium or calcium ions in the body.

An additional difference is that the antineoplastic drug is magnesium glucaratogluconate, which is preferable for cancer of arbitrary etiology against the background of a deficiency of magnesium ions in the body.

BEST MODES FOR CARRYING OUT THE INVENTION Hereinafter, the essence of the invention is explained: a description of methods for producing preparations based on gluconic acid according to the invention and their experimental dosage forms. a description of experiments on IP vitro models and the results obtained in comparison with the results of the action of conventional antineoplastic drugs. preliminary guidelines for the use of drugs according to the invention for the treatment of cancer.

1) Methods for preparing the preparations according to the invention Gluconic acid (empirical formula C ₆ H ₁₂ O ₇ ) as such, magnesium gluconate (empirical formula dihydrate C ₁₂ H ₂₂ MgO ₁₁ ^ H ₂ O), GABA (empirical formula C ₄ H ₉ NO ₂ ), 3-hydroxy-GAMK (empirical formula C ₄ H ₉ NO ₃ ), EDTA (empirical formula C ₁₀ H ₂ o0 ₁₀ N ₂ ), EGTA (empirical formula Ci ₄ H ₂₄ N ₂ Oi ₀ ) and glu- carboxylic acid (empirical formula C ₆ H ₈ O ₈ ) are available on the market as chemical reagents, which for the implementation of the invention must have a quality of at least “xh” (chemically pure).

If desired, magnesium gluconate can be obtained by neutralizing gluconic acid (usually taken as an aqueous solution of the easily hydrolyzable d-gluconolactone) with an almost equimolar amount of magnesium oxide (or hydroxide).

Normal gluconic acid salts are prepared by a neutralization reaction, in particular by adding a calculated amount of a base (or amphoteric compound) to a (usually aqueous) acid solution. The target products are isolated, as a rule, by evaporation of the solvent and drying the residue to constant weight (and, optionally, additional recrystallization).

Magnesium gluconate coordination compounds are prepared from magnesium gluconate and a source of selected ligand as follows. First in distilled When stirring and heating in a water bath, a certain amount (for example, 0.1 mole) of magnesium gluconate is slowly dissolved. The solution is cooled to. temperature 35-4O ⁰ C and gradually, with stirring, the calculated amount of the ligand is introduced in a molar ratio to magnesium r-jonate 2: 1. The finished product is isolated from the solution by spray drying.

In the synthesis of coordination compounds of magnesium gluconate with EDTA or EGTA, solutions of acids in a molar ratio of 1: 2 or 1: 3 with respect to gluconic acid are used with the calculated amount of magnesium salt added to the reaction mixture. Compounds are isolated by recrystallization from alcohol. Thus, compounds of the following composition were obtained:

Mg ₂ (C ₁ oH ₂ o0 ₁₀ N ₂ ) (C ₆ H ₁₂ 0 ₇ ) ₂ (Mg ₃ (C ₁₀ H _2O O _1O N ₂ ) (C ₆ H ₁₂ O ₇ ) _S OH), Mg ₂ (C ₁₄ H ₂₄ N ₂ O ₁₀ ) (C _b H ₁₂ O ₇ ) ₂ and (Mg ₃ (C ₁₄ H ₂₄ N ₂ O ₁₀ ) (C ₆ H ₁₂ O ₇ ) ₃ OH). These compounds may contain sodium or potassium ions as a counterion.

DiHAMK-Mg-gluconate (empirical formula of dihydrate C ₂₀ H ₃₈ MgN ₂ O ₁₈ ^ H ₂ O) is obtained by dissolving magnesium gluconate in water when heated in a water bath. After cooling the solution to 35-4O ⁰ C, the calculated amount of GABA in molar ratio to magnesium gluconate 2: 1 is added with stirring. The desired product is isolated by spray drying.

Coordination compounds of calcium gluconate are obtained from calcium gluconate and the corresponding ligand according to a scheme that is similar to the above.

For the synthesis of coordination compounds of calcium gluconate with EDTA or EGTA, solutions of these acids are used in a molar ratio of 1: 2 or 1: 3 calculated on the gluconate anion and the calculated amount of calcium gluconate is added to such a solution. The products are isolated by recrystallization from ethanol. Thus, the compounds of the following composition were obtained:

Ca ₂ (C ₁ oH ₂ o0 ₁₀ N ₂ ) (C ₆ H ₁₂ 0 ₇ ) ₂ (Ca ₃ (C ₁₀ H ₂ o0 ₁ oN ₂ ) (C ₆ H ₁₂ 0 ₇ ) ₃ OH)

Ca ₂ (C ₁₄ H ₂₄ N ₂ O ₁₀ χC ₆ H ₁₂ O ₇ ) ₂ and (Ca ₃ (C ₁₄ H ₂₄ N ₂ O ₁₀ ) (C ₆ H ₁₂ O ₇ ) ₃ OH).

These compounds as a counterion may contain sodium or potassium ions. Double magnesium salts are obtained in this way. Initially, an almost 50% aqueous solution of an equimolar (calculated as a cation) mixture of gluconic and one of the above (butyric, or hydroxybutyric, or glucaric) acids is prepared. Then, an equimolar amount of magnesium oxide (or hydroxide) is gradually introduced into such a solution with vigorous stirring. After assimilation of the magnesium source, the reaction mixture is cooled in an ice bath, the remaining water is separated on a Shot filter, and the precipitate dried to constant weight.

Double calcium gluconate salts are prepared in a similar manner. Experimental dosage forms are prepared by dissolving said compounds in an isotonic aqueous solution of sodium chloride or glucose. 2) Experiments on laboratory models of cancer cultures.

The effectiveness of the antineoplastic preparations according to the invention was studied on three types of tumor cell cultures (laboratory lines): A-549 (cells obtained by biopsy of a large cell lung cancer of a person), A-549-R (cells of the same type, resistant to interferon at a concentration of 10,000 IU / ml) and U-937 (cells sensitive to known apoptosis inducers). The control was healthy cells of the same tissues, not exposed to antineoplastic drugs.

Cells of each indicated line and corresponding healthy cells were cultured at 37 ^° C in complete nutrient medium RPMM 640 (SIGMA, USA) with the addition of 2 mM L-glutamine, 10% fetal serum, and 40 μg / ml gentamicin in a humidified atmosphere with volumetric concentration of CO ₂ 5%. The indicated medium was replaced every 2-3 days. After the formation of a dense monolayer of cells on a substrate (4-5 days of growth), cell suspensions were prepared and 24-well plates were seeded at a concentration of 3 * 10 ⁴ cells per well. The control drug doxorubicin and preparations according to the invention, indicated in the table attached at the end of the text, were used as additives to liquid nutrient media at the same concentration of 10 ^"3 mM / L. During the experiments, the nutrient medium was replaced daily.

To visually distinguish between living and dead cells, they were stained with trypan blue. Counting of surviving cells was carried out on a remocytometer after 24 hours, 48 hours and 72 hours, based on the actual number of living cells at the beginning of the corresponding day.

It has been established that all tested compounds based on gluconic acid are in principle (albeit of varying degrees) suitable for the treatment and prophylaxis of oncological diseases. Indeed, it follows from the table that these compounds simultaneously exhibit antiproliferative activity and proapoptotic activity against cancer cell cultures, but do not affect healthy cells.

Naturally, part of the preparations according to the invention exhibits either predominantly antiproliferative activity or predominantly proapoptotic activity and that the cumulative antineoplastic effect depends both on the type of tumor and on the composition and structure of the preparation and on its concentration and / or duration of action on tumor cells.

However, diGAMK-Mg-gluconate, di-3-hydroxyGAMK-Mg-gluconate, diGAMK-Ca- gluconate, di-3-hydroxyGAMK-Ca-gluconate, magnesium butyratogluconate and calcium butyrato-gluconate showed a fairly balanced and pronounced antiproliferative and proapoptotic activity. The same compounds are low toxic. So, in the course of standard experiments on rats, it was found that their LD _5O is at least 700 mg / kg (and for diGAMA-Mg-gluconate, approximately 3000 mg / kg).

Therefore, they can be used in very high single, daily and course doses to success without a noticeable risk of complications.

Along with the serial experiments described above and taken into account in the table, the dependence of the antineoplastic efficacy of diHAMK-Mg-gluconate on its concentration in cell suspension was studied on two lines of cancer cells A-549 and U-937. Typical examples of such studies are briefly described below.

Example 1. Cell suspensions based on the A-549 line were obtained and seeded in four 24-well plates at a concentration of 3 × 10 ⁴ cells per well, as described above. DiHAMK-Mg-gluconate was added to the culture media in dilutions of 10 ^{~ 1} , 10 ^"2 , 10 ^, and 10 ^ mmol / L.

This preparation at a dilution of 10 ^{~ 1} mM / L after 24 hours caused the death of 85% of cancer cells, and at a minimum dilution of 10 ^"4 mM / L within 72 hours, death of 50% of the cancer cells from their initial number was achieved.

Example 2. Cell suspensions based on the line U-937 were obtained and seeded in four 24-well plates at a concentration of 3-10 ⁴ cells per well, as described above. DiHAMK-Mg-gluconate was added to the culture media in dilutions from 10 ^{~ 1} to 10 ^"4 mM / L.

This preparation at a dilution of 10 ^{~ 1} mM / L after 24 hours caused the death of 95% of the cancer cells, and at a minimum dilution of 10 ^"4 mm / L, within 72 hours, the death of 60% of the cancer cells from their initial number was achieved.

In the control on suspensions of healthy cells, it was found that their death under the action of diHAMK-Mg-gluconate in a dilution of 10 ^{~ 1} mM / L for 72 hours did not exceed 7%, that is, at the level of the acceptable estimation error.

3) Preliminary guidelines for the use of drugs according to the invention for the treatment of cancer. The proposed drugs based on gluconic acid can be used for the prevention and treatment of neoplastic processes. The prophylactic use of drugs is especially justified against the background of immunodeficiency of arbitrary etiology. It can be assumed that these drugs will be most effective for cancer of the lungs, brain, kidneys and gastrointestinal tract (especially with parenteral or oral use).

In the case of skin cancers, their local (local) use is possible (in particular, in the form of applications of solutions, ointments or liniment, or trans dermally).

The use of the preparations according to the invention in clinical practice can be combined with conventional chemo- or interferon therapy. This combination is especially useful against the background of the development of resistant ™ tumor tissue to interferons. Industrial applicability

The antineoplastic preparations according to the invention can be easily manufactured industrially and used for the prevention and outpatient or clinical treatment of cancer. They are easily absorbed and practically harmless to the human body, even in such single doses, which significantly exceed the therapeutically necessary doses.

Table

ANTIPROLIFERATIVE AND PROPOPTOSIS ACTIVITY

PREPARATIONS ON CULTURES OF CANCER CELLS ΪP vito

(indicated number of viable cells in%)

Exposure, hours Type of cancer cell culture

A-549 A-549-R U-937 Test drugs 24 48 72 24 48 72 24 48 72

Control 100 100 100 100 100 100 100 100 100

Doxorubicin 60 50 40 60 50 50 40 50 50

Gluconic acid 80 80 70 80 80 70 80 80 70

Magnesium Gluconate 50 50 60 50 50 60 50 50 60

Potassium gluconate 60 60 70 60 60 70 60 60 70

Ethylene diamine gluconate 40 50 50 40 50 50 40 50 50

GABA-gluconate 50 50 60 50 50 60 50 50 60

DiGAMK-Mg-gluconate 40 45 55 40 40 55 10 20 35

Di-3-hydroxy GAMK-Mg-gluconate 50 45 50 50 45 50 50 45 50

Coordination connection

EDTA-Mg-digluconate 50 40 40 50 40 40 50 40 40

Coordination connection

EGTA-Mgg-di-gluconate 35 40 40 35 40 40 70 80 70

Coordination connection

EDTA-Mg-triglyconate 40 40 50 40 40 50 60 50 65

Coordination connection

EGTA-Mg-triglyconate 30 40 40 30 40 40 60 60 60

Magnesium butyratogluconate 40 50 40 40 50 40 60 60 60

Magnesium oxybutyrate gluconate 50 50 45 50 50 45 50 50 45

Magnesium glucarate-gluconate 60 50 50 50 40 40 60 60 50

DiGAMK-Ca-rluconate 60 50 50 60 50 50 60 50 50

Di-3-hydroxy GAMK-Ca-gluconate 50 50 55 55 50 55 55 50 55

Coordination connection

EDTA-Sag-Digluconate 40 50 50 40 70 90 40 70 90

Coordination connection

EGTA-Sag-digluconate 30 40 50 30 70 70 60 80 75

Coordination connection

ZDTA-Saz-triluconate 40 40 50 40 40 50 60 40 50

Coordination connection

EGTA-Saz-triglyconate 40 50 70 40 50 40 60 65 70

Calcium butorate gluconate 40 50 50 40 50 50 40 30 40

Calcium Oxybutyrate Gluconate 50 50 60 50 50 60 50 50 60

Calcium Glucaratogluconate 60 70 70 60 70 70 60 70 70

Claims

CLAIM 1. Antineoplastic preparation based on gluconic acid, characterized in that it is at least one such salt thereof, in which the salt-forming agent is selected from the group consisting of magnesium, sodium, potassium, γ-aminobutyric acid (hereinafter GABA), ethylenediamine or mixtures thereof. 2. The antineoplastic preparation according to claim 1, characterized in that it is magnesium gluconate. 3. The antineoplastic preparation according to claim 1, characterized in that it is potassium gluconate. 4. Antineoplastic preparation based on gluconic acid, characterized in that it is a coordination compound based on magnesium gluconate, in which the ligands are selected from the group consisting of GABA, 3-hydroxy-GAMK, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (ETH) ) 5. The antineoplastic preparation according to claim 4, characterized in that it is a coordination compound diHAMK-Mg-gluconate. 6. An gluconic acid-based antineoplastic preparation, characterized in that it is a double salt selected from the group consisting of magnesium butyratogluconate, magnesium hydroxybutyratogluconate, magnesium glucaratogluconate, calcium butyratogluconate, calcium hydroxybutyrate gluconate, calcium glucoratogluconate and ichiogluconate. 7. An antineoplastic preparation according to claim 6, characterized in that it is magnesium glucaratogluconate.