RU2695326C1 - Antitumour agent for treating prostate cancer in the form of sachet - Google Patents

Abstract

FIELD: medicine; pharmacology.SUBSTANCE: invention refers to medical chemistry and pharmacology, and concerns an antitumour agent for treating prostate cancer in form of 2-sachet'-{[(E)-3β-hydroxy-androst-5-en-17-ylidene]methyl}-4',5'-dihydro-1',3'-oxazole, modified with oleic acid to obtain a pharmaceutical substance – (3S,10R,13S,E)-17-((4,5-dihydrooxazol-2-yl)methylene)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl oleate, wherein the antitumour agent additionally contains phospholipid and maltose in the following ratio of components: pharmaceutical substance: phospholipid: maltose 1:10:39.EFFECT: use of the invention enables higher clinical effectiveness with reduced dose and reduced toxic effect on healthy tissues.1 cl, 4 dwg, 5 tbl, 6 ex

Description

This invention relates to the field of medical chemistry and pharmacology and relates to an antitumor agent for the treatment of prostate cancer in the form of a sachet, including the oxazoline derivative pregn-17 (20) -ene embedded in phospholipid nanoparticles.

Prostate cancer (PCa) is the most common cancer in men. The incidence of prostate cancer is steadily increasing worldwide. According to statistics of the Ministry of Health of the Russian Federation, the number of cases increases by 8% -9% every year (which is 34 thousand new cases). Currently, the Russian indicators of the increase in the incidence of prostate cancer are 4 times higher than in the United States and 2.5 times higher than in the European population [http://impotencija.net/rak-predstatelnoj-zhelezy]. Androgens play a key role in the development and progression of prostate cancer, and a decrease in the level of androgens in the tumor is the basis of the current treatment strategy for this disease. The key enzyme for androgen synthesis is cytochrome P450 17A1 (17α-hydroxylase-17,20-lyase, CYP17A1). Currently, CYP17A1 is considered as the main target for the development of drugs for the treatment of prostate cancer. Steroid and non-steroid compounds are known that are capable of inhibiting the activity of CYP17A1, inhibiting the growth of prostate carcinoma cells and exhibiting antitumor activity in vivo. As steroid compounds, which are CYP17A1 inhibitors, 3-pyridyl-, 2-pyridyl, 4-pyridyl-, 1-pyrazolyl-, 1-imidazolyl-, 1,2,3-triazol-1-yl-, 1,2 are used 3-triazol-2-yl-, 1,2,4-triazol-1-yl-, 1H-benzimidazol-1-yl-containing derivatives of androst-16-ene [1-6]. A known CYP17A1 inhibitor is androsta-5,16-dien-17- (3-pyridyl) -3β-ol (abiraterone), which is a drug against prostate cancer. Abiraterone acetate (trade name Zytiga) was recommended for widespread use as a medicine in 2011, and its analogue, galeterone (androsta-5.16-diene-hydroxy-17- (1H-benzimidazol-1-yl) -3β -ol, TOK-001, VN / 124-1) is currently undergoing phase III clinical trials. Inhibition of CYP17A1 activity by abiraterone and galetonone inhibits the androgen biosynthesis, slows the growth of prostate tumor cells and tumor development in mice, and also prolongs the life of patients suffering from complicated forms of prostate cancer in the final stages of the disease [2, 3].

The synthesis of abiraterone and galeronone is complicated and expensive, since: 1) it is carried out in five stages; 2) includes the use of the hormonal drug dehydroepiandrosterone as a starting material; 3) includes the use of a palladium-based catalyst. Oxazoline-containing derivatives of pregna-5.17 (20) -diene have been described [4, 5], but information on their biological activity has not been reported previously.

Known nanosystem based on plant phospholipids for the inclusion of biologically active compounds and the method for its preparation (options) (RF patent 2391966), including plant phosphatidylcholine (78-95%), maltose and a drug in the form of phospholipid nanoparticles with a size of 10-30 nm in the following the ratio of components: phosphatidylcholine - 20-43 wt. %, maltose - 57-80 wt. %, the drug is 2-8 wt. %

Also known is a composition for incorporating drug substances into a lipid matrix, a drug composition with a phospholipid fatty acid system and methods for their preparation (RF patent 2463056), including plant-derived phosphatidylcholine (73-94%), a fatty acid salt and maltose in the following ratio of components: phosphatidylcholine - 19-25 wt. %, fatty acid salt - 1.5-10 wt. %, maltose - 67-78 wt. %, drug substance - 1-10 wt. %, in the form of freeze-dried nanoparticles with a size of 10-20 nm. The disadvantage of the composition is the introduction of an additional component - salts of fatty acids.

The closest in technical essence (prototype) to the proposed antitumor agent for the treatment of prostate cancer are derivatives of pregn-17 (20) -ene, exhibiting antitumor activity [1], which are derivatives of pregn-17 (20) -ene of the general formula (I ), where R is 4 ', 5'-dihydro-1', 3'-oxazol-2'-yl- (Ia), or benzo [d] -oxazol-2'-yl- (Ib)

The inventive antitumor agent differs from the object disclosed in the prototype in that, in order to increase the effectiveness of the dose while reducing the toxic effects on healthy tissues, it is further modified with oleic acid to produce a pharmaceutical substance and contains phospholipids and maltose in the following ratio of components: pharmaceutical substance : phospholipids: maltose 1:10:39, made in the form of a sachet.

The present invention is the development of an antitumor agent for the treatment of prostate cancer, including the oxazoline derivative pregn-17 (20) -ene embedded in phospholipid nanoparticles.

At the Scientific Research Institute of Biomedical Chemistry named after V.N. Orekhovich (IBMH) developed an original three-stage scheme for the synthesis of new nitrogen-containing derivatives of 17 (20) E] -pregn-5.17 (20) -diene from available 3β-acetoxy-17α-bromo-21-iodpregn-5-en-20-one , characterized by simplicity and economy in comparison with the known schemes for the synthesis of nitrogen-containing derivatives of androst-5,16-diene. The structure of the synthesized compounds meets all the requirements of the well-known pharmacophore for CYP17A1 inhibitors (a set of elements in the structure of a compound necessary for its binding to a molecular target).

1 - 2 '- {[((E) -3β-hydroxyandrost-5-en-17-ylidene] methyl} -4', 5'-dihydro-1 ', 3'-oxazole; 2 - 2 '- {[((E) -3β-hydroxyandrost-5-en-17-ylidene] methyl} -benzo [d] -oxazole

Based on a detailed comparative study of the physicochemical properties of compounds 1 and 2, development of methods for their synthesis, the results of a preliminary study of specific antitumor activity obtained for the further development of a drug for the treatment of PCa, compound 1 - 2 '- {[(E) -3β- hydroxyandrost-5-en-17-ylidene] methyl} -4 ', 5'-dihydro-1', 3'-oxazole, because it had greater inhibitory and specific activity against LNCaP prostate carcinoma cells and 17a-hydroxylase-17,20-lyase (CYP17A1) [1].

Studies have shown that the selected compound 1 was poorly soluble in water. In order to increase the solubility, compound 1 was modified with oleic acid to obtain the pharmaceutical substance (PS) - (3S, 10R, 13S, E) -17 - ((4,5-dihydrooxazol-2-yl) methylene) -10,13-dimethyl- 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-3-yl oleate with its subsequent inclusion in phospholipid nanoparticles as a transport system. The preparation of a derivative modified with oleic acid also facilitated the incorporation of PS into phospholipid nanoparticles.

According to the invention, the finished dosage form is a powder for preparing a solution in a sachet bag of the following composition:

Active substance

Oxazoline derivative pregn-17 (20) -ene, modified with fatty acid (FS) 0.05 g

Excipients

Phospholipids 0.50 g Maltose Monohydrate 1.95 g

Conditionally called Alseviron, which has an antitumor effect.

The drug Alseviron does not apply to generics or to generic drugs. The active component of this drug is a new compound.

The main component of the used plant soybean phospholipid is phosphatidylcholine, the content of which is not less than 78-95% of the mass. The phospholipid also contains small amounts of other phospholipid components (lysophosphatidylcholine up to 4 wt%, other phospholipids - trace amounts).

As an excipient, the composition also contains maltose. Maltose is a cryoprotectant and is used to obtain freeze-dried powder, capable of completely recovering its structure (in particular, particle size) after dissolution in physiological saline or water.

The invention is characterized by the following examples.

Materials and methods

The following materials were used in the work:

1. Soya phospholipid brand Lipoid C 100

Lipoid GmbH, Germany

2. Maltose monohydrate (TU 6-09-2108-77)

3. Water for injection (according to FS No. 42-4587-95)

Example 1. The method of producing PS - (3S, 10R, 13S, E) -17 - ((4,5-dihydrooxazol-2-yl) methylene) -10,13-dimethyl-2,3,4,7,8, 9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-3-yl oleate.

Oleic acid (0.123 ml, 110 mg, 0.39 mmol) was dissolved in toluene, dicyclohexylcarbodiimide (144 mg, 0.7 mmol) was added, then 2 '- {[(E) -3β-hydroxyandrost-5-en-17- ylidene] methyl} -4 ', 5'-dihydro-1', 3'-oxazole ^* (132 mg, 0.37 mmol) and dimethylaminopyridine (226 mg, 1.85 mmol). The solution was stirred at room temperature for 3.5 hours, then the reaction mixture was evaporated and the residue was flash chromatographed on silica gel in a petroleum ether-acetone-acetic acid system (75: 24: 1). Fractions containing PS were evaporated. FS yield 105 mg (0.17 mmol, 46%). HRMS calculated for [C ₄₁ H ₆₆ NO ₃ ] ⁺ : 620.5043, found: 620.5032; ¹ H NMR: 0.83 (3H, s, H-18); 0.87 (3H, t, J = 6.8 Hz, CH ₃ , oleate); 1.03 (3H, s, H-19); 2.25 (2H, t, J = 7.4 Hz, αCH ₂ , oleate); 2.74 (2H, m, H-16); 3.88 and 4.22 (each 2H, m, CH ₂ , oxazoline); 4.60 (1H, m, H-3); 5.33 (2H, m, CH, oleate); 5.37 (1H, m, H-6); 5.64 (1H, t, J = 2.4 Hz, H-20).

Example 2. A method of obtaining a finished dosage form (GLF).

975 mg (2.84 mmol) of soy phospholipid and 25.0 mg (0.04 mmol) of FS were added to the 975 mg maltose feed solution. The mixture was homogenized by rotor-stator homogenization for 3 minutes. at a temperature not exceeding 45 ° C until a homogeneous primary suspension is obtained. The resulting suspension was transferred to a receiving container of a high pressure homogenizer. Homogenized cyclically at a pressure of 1000 bar ± 10%. The solution between subsequent cycles was cooled by passing through a water-cooled refrigerator, preventing it from heating above 55 ° C.

The homogenization process was continued until the solution reached a transparency of 50-60%. The transmission control was carried out at 660 nm in a cuvette with an optical path length of 1 cm.

Then, a standardizing filtration was performed on a Millipore Corporation installation (USA), sequentially passing the solution through 1 μm fiberglass filters and a 0.22 μm membrane membrane.

The resulting ultra-thin emulsion was poured into stainless steel trays with a thickness of about 15 mm and lyophilized. The resulting freeze-dried mass (yellowish powder) was a drug composition, from which, after grinding in a ball mill, an oral drug was prepared in the form of a powder for the treatment of prostate cancer in the form of a powder for the preparation of a solution in 2.5 g sachets .

After rehydration of the contents of the sachet (drug Alseviron), its physicochemical characteristics were determined.

The main characteristics of SLEF Alseviron are presented in table 1.

A series of experiments was carried out with different ratios of components: PS, phospholipid and maltose. The data obtained are presented in table 2.

It has been established that the ratio of the components of PS: phospholipid: maltose 1:10:39 is the most optimal for producing small-sized phospholipid particles with the substance included. Therefore, for further research and experiments on animals, we selected the composition under number 2 in table 2.

In addition to standard quality control methods regulated by GF, the following methods for characterizing GLF samples were selected for characterizing a pharmaceutical substance (FS): light transmission method, HPLC with optical and mass spectrometric detection on Agilent 1100 and Agilent 1200 Series chromatographs (Agilent Technologies, USA), respectively.

To measure light transmission, the contents of the sachet bag were diluted to 10 ml with distilled water, the resulting sample was poured into a quartz cuvette and light transmission at a wavelength of 660 nm was determined. In FIG. Figure 1 shows a typical light transmission spectrum of one of the GLF samples. In FIG. 2 shows the chromatograms obtained on a chromatograph with optical and mass spectrometric detection.

Example 3. Data on the specific activity of Alseviron.

Preclinical studies of the specific activity of the drug Alseviron were performed in parallel with the substance Abiraterone acetate (Abiraterone), which is the active substance of the antitumor drug Zitiga, on signal cell lines of prostate cancer (in vitro) in the concentration range of 0.001-1000 μg / ml on the line of cancer cells human prostate gland RS-3. Cytotoxicity was judged by the generally accepted indicator IC50 (half maximal inhibitory concentration) in a standard MTT test.

Assessment of cytotoxic activity. The test compounds (samples of Alseviron and Abiraterone) were dissolved in dimethyl sulfoxide (DMSO). The concentration of compounds in stock solutions is 1 mg / ml. To obtain working solutions, a series of ten-fold serial dilutions were made in sterile 0.9% NaCl solution in triplets.

As a test cell culture, a transplantable line of human prostate adenocarcinoma cells RS-3 (from the bank of the FSBI NNC Oncology named after N.N. Blokhin of the Ministry of Health of Russia) was used in the phase of logarithmic growth. Cells at a concentration of 4.2 × 105 cells / ml resuspended in RPMI-1640 medium supplemented with 1% serum from cow embryos, 2 mM L-glutamine, 100 U / ml penicillin and 100 μl / ml streptomycin (all - PanEco, RF ), scattered through the wells of a 96-well plate (Nung) in 180 μl, to which 20 μl of working solutions of the studied substances in tetraplets were added. 20 μl of 0.9% NaCl solution was added to control wells. A cell culture with test substances was incubated at 37 ° C and 5% CO2 for 4 days.

The effect of the studied substances on the survival of tumor cells was determined using the MTT test. The optical density of the colored solution was evaluated at a wavelength of 540 nm versus 690 nm on a Multiscan MS flat panel photometer (ThermoLabsystems, Finland). The final test result was calculated by the formulas:

Cytotoxicity (Cytotoxicity),% = 100-Survival

According to the results of calculating the cell culture survival in tetraplets in the Combenefit software program, the EC50 indicator was calculated - the effective concentration of the test compound, which caused the death of 50% of the cells.

Comparative evaluation of the cytotoxic activity of Alseviron and Abiraterone. In FIG. 3 shows the results of the calculation of the EC50. The results indicate that Alseviron has the highest activity in comparison with both Abiraterone samples, effectively (by more than 50%), inhibiting the viability of the culture of tumor cells of the PC3 line at a concentration of more than 7.1 μg / ml compared to 19.2 and 20 , 6 μg / ml Abiraterone, respectively.

In FIG. 4 shows the results of the calculation of the cytotoxicity of solutions with different concentrations of active substances. The given discrete values show that of the tested compounds, Alseviron at concentrations of 100 and 1000 μg / ml had the broadest effective range of concentrations mediating a significant cytotoxic effect leading to the death of more than 50% of cells in culture.

According to the results of the studies, it was proved that Alseviron has a higher cytotoxic activity in comparison with Abiraterone. The death of more than 50% of tumor cells was observed as a result of their co-incubation with Alseviron at a concentration of 50 μg / ml and higher, while Abiraterone even at a concentration of 100 μg / ml induced the death of only 41 ± 3.4% of cells. It should be noted that the cytotoxic activity of Alseviron at a concentration of 25 μg / ml was also rather high - 47 ± 2.2%, while the activity of Abiraterone at this concentration corresponded to 34 ± 2.2%. Calculation of the approximation curves of the average effective concentration of the drug, which causes the death of 50% of the cells during coincubation, showed that the EC50 of Alseviron on average corresponds to 7.7 μg / ml, and Abiraterone 19-20 μg / ml, i.e. Alseviron causes a cytotoxic effect of cells at a concentration 2.5 times lower than Abiraterone.

Tables 3-5 show data for assessing the cytotoxicity of Alseviron and Abiraterone in a wide range of concentrations.

Example 4. The study of acute toxicity of the drug

For experiments, animals were randomly assigned to groups using body weight as a criterion, so that in the group the individual animal weight did not differ by more than 10% from the average weight of animals of the same sex. The studied drug Alseviron was administered to mice of both sexes (n = 72) and rats of both sexes (n = 72) intragastrically. The animals were injected with the maximum possible volume during intragastric administration: mice 0.5 ml / 20 g, rats 4.0 ml / 200 g. In order to achieve the volumes necessary for administration, the minimum dose was diluted with water for injection to the appropriate animal body weight. Doses for mice 5.0 - 10.0 - 15.0 - 20.0 - 25.0 ml / kg in dosage form; doses for rats: 5.0 - 10.0 - 15.0 - 20.0 - 25.0 ml / kg in dosage form. The determination of toxicity parameters in a single administration was carried out using the two-stage method. At the first stage, the approximate LD50 was established by the method of Kerber and Miller, or Deichmann and Leblanc. At the second stage, the exact indices of LD16, LD50 ± m, and LD84 were determined by probit analysis according to Litchfield and Wilcoxon. Manifestations of intoxication: no mortality cases were noted in any of the groups. After the introduction of intragastric maximum doses of the drug Alseviron, there were no visible differences in the motor and nutritional behavior of animals, in the state of the external integuments and visible mucous membranes, in reactions to external stimuli in comparison with the control groups. The total duration of observation of animals after drug administration was 14 days. On the first day, the observation was continuous, because this day is the highest point of intoxication. The daily registration of the clinical picture of intoxication was carried out visually in the cage and in the hands. As a result of studies on white outbred mice and rats, it was found that with a single intragastric administration of the maximum doses of the drug Alseviron, the death of animals was not observed. LD50 for mice and rats was not achieved with the maximum possible dose of 25.0 ml / kg in the dosage form or 62.50 mg / kg in the active substance FS (oxazoline derivative 17 (20) E-pregna-5.17 (20) diene in the form of a conjugate with oleic acid), which is 88 times more than the therapeutic dose for humans 0.71 mg / kg. There was no effect on the mass coefficients of organs with a single intragastric administration to white outbred mice. There was no difference in weight gain between the groups with the administration of the study drug and the control groups with a single intragastric administration to white outbred rats in acute doses. In the experiments, no gender and interspecific differences were found.

Example 5. The study of chronic toxicity of the drug

To conduct an experiment to study chronic toxicity, Wistar rats (160 animals) and Chinchilla rabbits (96 animals) were used.

Studies were conducted with intragastric administration of the drug for 180 days daily in three doses.

Single dose for a person: 50 mg / 70 kg = 0.71 mg / kg (1TV).

Since the drug does not have high toxicity (LD50 was not achieved), ETD was taken to calculate the doses to rats and rabbits.

1 ETD for rats weighing 200 g = 0.71 × 39 / 6.5 = 4.26 mg / kg (39 is the dose conversion factor for humans weighing 70 kg, 6.5 is the dose conversion factor for rats weighing 200-250 g );

5 ETD (intermediate) = 4.26 × 5 = 21.3 mg / kg;

10 ETD (maximum) = 4.26 × 10 = 42.6 mg / kg;

1 ETD for rabbits weighing 2 kg = 0.71 × 39 / 12.8 = 2.16 mg / kg (39 is the dose conversion factor for humans weighing 70 kg, 12.8 is the dose conversion factor for rabbits weighing 2000 g);

5 ETD (intermediate) = 2.16 × 5 = 10.8 mg / kg;

10 ETD (maximum) = 2.16 × 10 = 21.6 mg / kg.

The following analysis methods were used:

- General condition (dynamics of body weight, rectal temperature). Appearance is assessed by daily inspection of animals. Weighing, measuring rectal temperature, water and feed intake is performed once a week;

- The behavior of rats (motor and research activity);

- Hematological parameters (the number of red blood cells, white blood cells, platelets, hemoglobin level, white blood cell count);

- myelogram;

- Blood coagulability;

- Biochemical parameters and activity of serum enzymes (total protein, creatinine, urea, glucose, cholesterol, total lipids, total and associated bilirubin, activity of alkaline phosphatase, lactate dehydrogenase, aspartate and alanine aminotransferases, calcium, potassium, sodium).

- The functional state of the cardiovascular system of rats (ECG study in II standard lead, systolic pressure);

- The functional state of the central nervous system of rats (behavior, sum-subthreshold indicator);

- Functional activity of rat kidneys (by urine analysis);

- Pathomorphological examination, including: necropsy, macroscopic examination, weighing and histological examination of internal organs.

Local irritant effect with pathomorphological (macroscopic and histological) description of the injection site.

Results. Within 180 days, there was no death of animals with intragastric administration of Alseviron in equitherapeutic and doses exceeding the equitherapeutic by 5 and 10 times. No local irritating effect of the studied drug Alseviron with its multiple intragastric administration was detected, there were no violations of the functional state of the main organs and body systems.

The results of a study of subchronic toxicity in rats and chinchilla rabbits showed that repeated intragastric administration of the studied drug Alseviron in subtoxic doses did not cause significant disturbances in the functional state of the main organs and systems of the body.

Example 6. The study of the specific toxicity of the drug

The study of the specific toxicity of the drug Alseviron in equitherapeutic doses and doses exceeding the equitherapeutic by 10 times showed:

- the absence of allergic properties (study for the presence of anaphylactic activity during skin testing and in the conjunctival test);

- lack of immunotoxic activity (the drug does not cause activation of the humoral and cellular immunity in both males and females);

- the absence of mutagenic activity when testing mutations of base pair substitution in a DNA molecule in the Ames test on strains of S. typhimurium TA98, TA 100, TA1535, TA1537 and E. coli strains pKM101 and uvrA, in variants with and without metabolic activation of S9, does not have cytogenetic activity in the test for accounting for chromosomal aberrations in mammalian bone marrow cells, did not exert a genotoxic effect on lymphocytes and liver cells of BALB / c mice 6 and 24 hours after intragastric administration.

The innovative antitumor drug for the treatment of prostate cancer based on the phospholipid composition of the oxazoline derivative pregn-17 (20) -ene, developed by IBMC, was obtained using the original technology. The drug is planned to be obtained in the form of freeze-dried powder with a long shelf life. However, the technology is not complex and well reproducible. Preliminary preclinical studies have shown that the developed drug has undeniable competitive advantages compared with foreign counterparts both from the economic and medical side.

LITERATURE

1. RF patent 2617698

2. US 5604213 A.

3. Stulov SV, Tkachev YV, Novikov RA, Zavialova MG, Timofeev VP, Misharin AY. Synthesis of 21-nitrogen substituted pregna-5.17 (20) -dienes from pregnenolone, Steroids 2012; 77: 77-84.

4. Stulov SV, Mankevich OV, Novikov RA, Tkachev YV, Timofeev VP, Dugin NO, Pozdnev VF, Fedyushkina IV, Scherbinin DS, Veselovsky AV, Misharin AY. Synthesis and molecular modeling of (4``R) - and (4''S) - 4 '' - substituted 2 '' - {[(E) -androst-5-en-17-ylidene] -methyl} -oxazolines . Steroids 2013; 78: 521-527.

5. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth. 1983, 65, 55-63.

Tables

Claims (1)

An antitumor agent for treating prostate cancer in the form of a sachet containing 2 '- {[(E) -3β-hydroxyandrost-5-en-17-ylidene] methyl} -4', 5'-dihydro-1 ', 3'- oxazole, characterized in that 2 '- {[((E) -3β-hydroxyandrost-5-en-17-ylidene] methyl} -4', 5'-dihydro-1 ', 3'-oxazole is modified with oleic acid to give pharmaceutical substance - (3S, 10R, 13S, E) -17 - ((4,5-dihydrooxazol-2-yl) methylene) -10,13-dimethyl-2,3,4,7,8,9,10, 11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-3-yl oleate, the antitumor agent additionally containing phospholipid and maltose in the following the general ratio of components: pharmaceutical substance: phospholipid: maltose 1:10:39.

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