WO2013126031A1

WO2013126031A1 - Antineoplastic agent

Info

Publication number: WO2013126031A1
Application number: PCT/UA2012/000043
Authority: WO
Inventors: Евгенний Иванович СУСЛОВ; Татьяна Петровна ПОДГАЕВСКАЯ
Original assignee: Suslov Yevgeniy Ivanovich; Pidgaievska Tetiana Petrivna
Priority date: 2012-02-20
Filing date: 2012-04-20
Publication date: 2013-08-29
Also published as: UA103694C2

Abstract

The invention relates to medicine, and specifically, to the field of oncology, and can be used for treating benign and malignant tumours. The proposed antineoplastic agent is a mixed-ligand complex of calcium with ethylenediaminetetraacetate and gluconate. The novelty resides in the fact that the components are used in the following ratio: calcium salt of gluconic acid 1 g\mol and disodium salt of ethylenediaminetetraacetic acid 1 g\mol. The agent does not contain excesses of starting substances. The claimed agent is non-toxic (LD50 on intraperitoneal administration to non-pedigree white rats is 8000.0 mg/0.1 m). The main characteristic of said agent is the capability of retarding the growth of benign and malignant tumours and metastases thereof and of activating apoptosis of cancer cells. All this makes it possible to group said agent with modern new-generation antineoplastic agents which may be widespread in the future in pharmacology.

Description

Antitumor agent

The invention relates to medicine, namely to the branch of oncology and can be used to treat benign, malignant tumors and their metastases to internal organs.

Known agent for the treatment of malignant tumors containing a mixture of components: calcium gluconate - 120.0-150, (g / l); ethylenediaminetetraacetic acid disodium salt (EDTA) 13.3-16.7 (g / L); sodium hydroxide 7.1-8.9 (g / l) and distilled water added to 1 liter. (see Patent of Ukraine N ° 76671, MPK7, A 61K31 / 185, A 61 ZZ / 06, A61P35 / 00, Publ. 15.08.2006).

The disadvantages of this antitumor agent are:

- the ratio of the two main ingredients - disodium salt of ethylenediaminetetraacetic acid - 13.3-16.7 (g / l) and calcium gluconate - 120.0-150, (g / l), which proportionally corresponds to 1: (8.8-9 , 2) (weight%), is not chemically balanced: it is not “attached” to their equimolecular ratio (1: 1 g / mol), at which their complete chemical interaction in solution occurs;

- due to the extremely poor solubility of the mixture with an excess of calcium gluconate in it, it is impossible to create infusion forms of the drug;

- the tablet form of the drug has low efficiency;

- excess calcium gluconate overloads the blood, which does not exclude possible negative side effects of somatic symptoms in the form of hypercalcemia, increased heart rate, increased blood pressure, increased blood clotting, and the development of uric acid diathesis.

The closest analogue of the claimed antitumor agent is the "Suslov-Podgayevsky means for the treatment of malignant tumors", which contains calcium gluconate and disodium salt of ethylenediaminetetraacetic acid in a ratio of 1: 1 (or 50: 50 (weight%,) see: Patent of Ukraine Ш 42244, IPC 7 A 61K31 / 191, A 61K31 / 185, A61R35 / 00, Publ. 15.10.2001)

The disadvantages of this analogue include:

- unbalanced ratio of the main components. This is explained by the following: if you make a mixture in the specified proportion, then in 1 liter of solution may contain, for example:

Option 1 - 100.0 g of one + 100.0 g of another component, or

Option 2 - 400.0 g of one + 400.0 g of another component (a total of 800.0 g). Considering that the molecular weight of disodium salt of ethylenediaminetetraacetic acid = 372, calcium gluconate = 223, then only in the aqueous solution of the 2nd option (under certain conditions) can one chelate compound form - a mixed ligand complex of calcium with ethylenediaminetetraacetate and gluconate (in the ratio of 1: 1 (g / mol), or 372.0 g + 223.0 g). Thus, in 1 liter of such a solution, the ballast of unreacted both disodium ethylenediaminetetraacetic acid and calcium gluconate (28.0 g and 177.0 g, respectively) remains, which will be in a dissociated state. The solubility of the compound, weighing 800.0 g of the substance in 1 liter of water, is very small, that is, weak, which will require the addition of other chemical compounds to the solution, the use of high-temperature treatment, risky autoclaving under high pressure, or something else;

- all excess unreacted mixture of substances among themselves in the solution, getting into the blood during the intravenous treatment of the patient, overload it and become toxic due to their high concentration. They can form derivative complexes, combining with biomolecules of decaying tissues of malignant tumors, becoming cytotoxic toxins and creating a risk of thrombosis;

- the possibility of developing negative side somatic symptoms in the form of hypercalcemia, increased heart rate, increased blood pressure, increased blood coagulation, development of uric acid diathesis, sclerosis of the walls of blood vessels, and liver intoxication is not ruled out.

The basis of the invention is the task - to create such a means in which, by changing the technological conditions, the indicated disadvantages are eliminated - excesses in the solution of unreacted masses of the main components, thereby avoiding intoxication of the body and achieving a high antitumor effect in the treatment of patients.

In the course of solving this task during the development of the invention, it was scientifically substantiated the preparation of a new effective, non-toxic chemically correct complex (chelate) individual compound, changing the technological conditions and having worked out the only correct ratio the specified ingredients of the mixture in their proportion - 1: 1 (g / mol) (372.0: 223.0 (g), respectively). Thus, a new tool was found mixed calcium ligand complex with ethylene diamine tetraacetate and gluconate. The invented agent has a high antitumor effect.

After conducting preclinical experimental studies on three models of cancer in animals and developing an AED to create an infusion form of the claimed drug, a mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate with the absence of an excess of the main components in the infusion solution, this can eliminate the possibility of side (toxic) effects on the patient’s body from their exposure, its effectiveness has been proven to activate cancer cell apoptosis ..

It is known that apoptosis of foreign bioagents, parasitic and cancer cells is a natural, genetically determined process that occurs at physiological body temperature. The main action of the claimed antitumor agent is the activation of apoptosis of cancer cells at the genetic level with a parallel increase in the natural killer activity of lymphocytes of the T-cell immune system (killer cells).

Mebifon and Cisplatin can be considered prototypes for the antitumor activity of the claimed drug. But due to the lack of calcium in them, they cannot deliver its ionized form (Ca ²⁺ ) to the nuclei of cancer cells, where it takes part in the activation of the apoptosis enzyme - Ca ²⁺ -ATPase (calcium-dependent adenosine triphosphatase). Mebifon and Cisplatin belong to cytotoxic chemotherapeutic agents of antitumor action (so, LD of so Mebifon is 160.0 mg / m).

In preclinical trials of the claimed antitumor agent on several cancer models, its non-toxicity was established (the LD50 value of the mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate when administered intraperitoneally to white non-linear mice was 80,000.0 mg / m). The main oncopharmacological action of a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate is inhibition of tumor growth, a decrease in the number of metastases and the percentage of metastases of malignant tumors> 70% after treatment.

All this allows us to attribute it to modern non-toxic antitumor drugs of a new generation, belonging to the class of antimetabolites, which have a great future in the field of oncopharmacology.

In experiments on white nonlinear mice and black mice C57 line

B16 on lung cancer models (see Table 1, 2), the drug was administered in a pre-determined average therapeutic dose of 400.0 mg / kg body weight. Histologically examined organs and tissues of animals. So, in the lungs, it was found that under the influence of a mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate, the antitumor and antimetastatic effect occurs through the active apoptosis of cancer cells, since ionized Ca ²⁺ enters there, which is necessary for the activation of Ca-ATPase, which triggers apoptosis .

The study of the Z - potential (mV) of tumor cells in an experiment by electrophoresis of a tumor cell culture in a polyacrylamide gel before and after treatment of animals with the indicated agent showed the presence of a negative Z - potential (mV) of tumor cell culture in untreated animals and a significant increase in the positive Z - potential in culture tumor cells in the group of animals treated with a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate: 16.74 ± 1.06 (mV) versus 27.42 ± 1.41 (mV), respectively (p 0.001).

Clinical trials of the antitumor effect of the claimed drug were carried out over 10 years in 54 volunteers, mainly with the Sh-th and IV-th stage of cancer. These were patients with cancer of the lungs, breast and rectum with metastases to the lymph nodes, mediastinal organs, skeleton bones. After an analytical assessment of the data of clinical examinations, the general condition of patients, laboratory, tumor markers and instrumental examinations, the volunteers received a solution of a mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate intravenously in a therapeutic dose of 300.0 mg / kg body weight x 48 hours x 10 injections. Statistical processing of the obtained results of clinical trials established significant inhibition of the growth of the main malignant tumor and a decrease in the number of tumor metastases in the internal organs and lymph nodes in all patients (p <0.05). There are observations of survival of patients from 3 until the 10 year period. It was found that the inventive mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate softens the lymph nodes, removes foci of liver metastases and heals tumor metastases in the bones of the skeleton.

Example 1. Volunteer Gurinets (G-ts) M.A., 1929/73 History of the University Ν ° not specified, from

May 3 to June 29, 2002, Chernihiv Regional Oncology Center. Diagnosis: Cancer of the left lung T ₄ N ₂ M ₃ , II class. column with metastases to the right, lung, liver, cervical, thoracic and lumbar spine, bones of the right lower leg, with fractures of the cervical vertebrae, to the lymph nodes of the bronchi and mediastinum. Somatic condition of the patient was very difficult: cough with purulent sputum, he could not get out of bed on his own, a corset was put on his neck so that his head would not fall to the side, he moved to the toilet in a wheelchair with outside help, or with a stick. During the 1st course of treatment with the claimed antitumor agent in the form of a 15% aqueous solution of a mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate, which was obtained at the rate of 1: 1 (g / mol) of the main ingredients, the patient received the drug by intravenous drip after 48 hours in the average therapeutic dose of 300.0 mg / kg body weight. After the third injection of the drug, the patient became much stronger, started to get out of bed on her own and move around the room without a wheelchair. After the end of the 1st course of treatment (10 droppers), the patient was discharged from the Oncology Center home, to the village of Palchiki, in good condition: pain in the liver and in the bones of the skeleton disappeared. A wet cough with purulent sputum stopped. It can be seen from the discharge epicrisis that metastases were scarred in the vertebrae of all parts of the spine. Metastases in the lungs decreased significantly, their growth was inhibited. At home, the patient began to lead an active way of life - to do housework. Through the weather (in October 2002), she decided to take the second course of treatment on the same conditions and in the same Oncology Center. The treatment was carried out under the active supervision of the inventors. Discharged in good condition, found scarring of metastases in the vertebrae, lymph nodes, liver and lower leg bones. Found the disappearance of foci of the tumor process in the lungs, in the liver. To this day (on! 1.02. 2012, the patient is alive, her age is ^“ 83 years old), has not sought help, has not taken repeated courses of antitumor therapy, leads an active lifestyle. It has been observed by the authors of the claimed antitumor agent for 9.5 years. Assessment of the general condition of the former patient good, according to the ECOG-WHO system before and after treatment - 4 - 2.

Example 2. Volunteer Malyuga (Ma). V.A., born in 1937/70, applied for help 01.03.2005. b-no. J b 9901, archival jN 24 dated 03.04.2007, Kiev, the Main Military Clinical Hospital. Diagnosis: Prostate adenocarcinoma T ₂ N in M], II class. gr., pain in the lumbar region. Anemia. Oncomarker Diagnosis — The PSA test is critically positive. He took the course of treatment with the claimed agent in the form of a 15% aqueous solution of a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate at a rate of 1: 1 (g / mol) of the main ingredients: calcium gluconate - 1.0 g / mol, disodium EDTA - 1.0 g / mol, in an average therapeutic dose of 300.0 mg / kg of body weight intravenously drip after 48 hours. After the 1st, the patient's condition improved significantly: anemia, pain were gone, the PSA test returned to normal. No complaints, oncotest negative.

After a year, the patient turned for re-treatment due to a weakly positive oncotest and a slight anemia. No complaints. The fifth (repeated) course of treatment was taken for prophylaxis. The treatment was carried out by the claimed agent in the form of a 17% aqueous solution at the rate of 2.5: 1 (g / mol) of the components (calcium gluconate - 2.5 g / mol, disodium EDTA - 1.0 g / mol, respectively), the average therapeutic dose of 400.0 mg / kg of body weight intravenously drip after 48 hours. After the second course of treatment, the patient's condition improved: PSA test within the physiological norm. Oncotest is negative. Complaints were about increased heart rate and infrequent aching pain in the kidney area. On examination, Pasternatsky’s symptom on the right is positive. Ultrasound revealed sand and small calculi in the parenchyma of the right kidney. Diuretic and anti-inflammatory uroseptics are prescribed. After a course of anti-inflammatory therapy, the patient's condition returned to normal, the pain went away, ultrasound did not determine sand and calculi in the kidneys, the oncotest was negative. Thus, it can be assumed that an excess of calcium gluconate (almost 2 times greater than 1 g / mol) could cause (or exacerbate) the inflammatory process in the kidneys and uric acid diathesis. After recovering from complications, in the future the patient did not contact the authors of the application for an invention, since he did not present any complaints. It has been observed for 5 years. Assessment of the general condition of the former patient is good, according to the ECOG-WHO system before and after treatment - 4 - 2. Example 3. Volunteer Gumenyuk (GK) A.I., 1966/39. Medical report of the LCC from 08.25.2005: Diagnosis: Cancer of the left breast of the PA Art. with metastases to regional lymph nodes, T ₂ N i Mo, III class. gr., condition after left-sided mamectomy from 07/27. 2005 and polychemotherapy. Somatic condition of the patient was very poor: alopecia, severe cachexia, anemia, pallor of the skin and mucous membranes after undergoing polychemotherapy. Complained of pain in the lumbar region, in the chest cavity, in the internal organs. The patient herself refused further courses of traditional polychemotherapy. In 2006, she took the 1st and after a year Fifth treatment with the claimed antitumor agent - a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate, which was made from the calculation: disodium salt of EDTA - 2.5 g / mol, calcium gluconate - 1, 0 g / mol (in a ratio of 2.5: 1 (g / mol).

After the end of the 1st course of treatment, the patient's condition improved anemia and the pain syndrome disappeared. Oncological markers returned to normal. I started a working schedule of a normal life, working in the field of jurisprudence. In the first three months, she complained of aching pain in the liver. A biochemical study of liver samples revealed minor deviations from the reference parameters. After she was prescribed anti-inflammatory hepatoprotective therapy, the pain in the liver disappeared, and her liver test results returned to normal. Thus, it can be assumed that an excess of disodium EDTA in the body, more than 1 g / mol per kg of body weight (2.5 g / mol) could cause an inflammatory process in the liver and biliary tract. After 6 months, the patient again had a positive oncotest. The volunteer decided to repeat the course of treatment with the claimed agent, which was made up in an equimolecular ratio of the main ingredients (1: 1 g / mol, respectively). After conducting the I-th course of treatment with this agent - a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate, negative symptoms (consequences) or inflammatory processes from the internal parenchymal organs, including the liver, were not noted, liver tests were normal. The patient's survival today is 6.5 years, no complaints. To prevent a possible relapse of the disease, she recently decided to take the third course of treatment with the claimed drug - mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate. Assessment of the general condition of the former patient is good, according to the ECOG-WHO system before and after treatment was 4 - 2.

FINDINGS

• The antitumor agent that is claimed is an individual substance - a non-toxic mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate (see Structural formula in Table 4), which is obtained by the reaction of sequential interaction in the equimolecular ratio of calcium gluconate and disodium salt of ethylenediaminamine acid 1 : 1 (g / mol), respectively, in an aqueous medium, controlling the reaction process, up to the receipt of a chelating substance (mixed ligand complex) with stable hydrochloric structure without excess in the infusion solution the main active reagents and their derivatives which adversely affect the process of treatment (Table 3; examples:. 2,3).

• Treatment of patients with the infusion form of the claimed drug showed a pronounced antitumor effect in both non-adjuvant and adjuvant therapy during preclinical (Table 2) and clinical trials (Examples: 1, 2, 3). This indicates the possibility and relevance of its use as a non-toxic antitumor drug in the treatment of cancer patients at all stages of the disease.

• An excess of non-toxic to the body in the treatment of patients with a mixed ligand complex of calcium with ethylene diamine tetraacetate and gluconate, which is declared as an individual chemical substance in an equimolecular ratio of 1: 1 (g / mol) of its components in infusion form, there may be a small amount of excess of only one of the two ingredients - calcium gluconate, which is not more than 0.5 (g / mol) (Table 3, column 1.). Large doses of excesses of both ingredients are not permissible, as they exhibit symptoms of intoxication and slagging of the patient’s body (Examples: 2,3). Such patients will need additional repeated long-term drug treatment for complications - inflammatory and destructive-dystrophic disorders of the body. Table 1

The antitumor activity spectrum of the claimed agent is a mixed ligand calcium complex with ethylenediaminetetraacetate and gluconate in comparison with the antitumor activity analogue - Mebifon

table 2

The antitumor activity of the claimed drug is a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate with non-adjuvant and adjuvant therapy on three models of experimental tumors in the spectrum of data from the group of untreated (control) and in the groups of treated animals

%%

Medium Medium

Koli- Dose brake Volume brake

Weight Mass Index Quality Average

sob of a tumor of the tumor in the effect of the animal P of the growth of the pelvis in growth

enter in the group of experimentally benign tumors in mg / kg (V ³ )

Contraction of the group of animals (V ³ )

basins

Lewis Epidermoid Carcinoma, Adjuvant Therapy

Cont 4.17 ±

role 11, 21

0.53

fifteen

20 400.0 r / h 0.99 ± 0.53 <0.001 76.3 4.2 0.08 99.28 0

Lewis Epidermoid Carcinoma, Adjuvant Therapy

Kont-4, 17 ±

role 11, 21

0.53

fifteen

20 400.0 r / h 1, 05 ± 0, 13 <0.001 74.9 3.97 0.07 99.37 0

Melanoma B ₁₆

Cont- 2.87 ± 6.57 ±

role

0.53 0.46

8

1, 03 ±

200.0 r / h 0.83 ± 0.46 <0.001 71 D 3.45 84.4 0

0.46

Ehrlich carcinoma

Count 9.31 ±

role 2.97

0, 18

6

0.92 ±

9 400.0 r / h 0.75 <0.05 74.8 3.96 90, 12 0

0.27 Table 3

The results of preclinical trials of the antitumor effect of the claimed drug is a mixed ligand calcium complex with ethylenediaminetetraacetate and gluconate in comparison with the analogue agent (Pat. U A 42244) on a model of transplantable Lewis carcinoma with lung metastases in black mice with non-adjuvant therapy

Table 4

The structural formula of the proposed drug is a mixed ligand calcium complex with ethylene diamine tetraacetate and gluconate

COONa

Claims

FORMULA

An antitumor agent, which is a mixed ligand calcium complex with ethylenediaminetetraacetate and gluconate, as an individual chemical substance, which is characterized in that it contains the calcium salt of gluconic acid and the disodium salt of ethylenediaminetetraacetic acid in an equimolecular ratio of 1: 1 (g / mol, respectively) mixtures of the starting ingredients.