RU2849628C1 - Method for obtaining osteogenic agent - Google Patents

Abstract

FIELD: pharmacy; biomedicine.

SUBSTANCE: invention relates to a method for producing an osteogenic agent intended for use in traumatology, orthopaedics, maxillofacial surgery, and reconstructive and restorative surgery for the prevention and treatment of bone tissue damage and diseases. The proposed method for producing an osteogenic agent in the form of calcium chelidonate involves dissolving chelidonic acid, synthesised from diethyl oxalate and acetone, by heating in distilled water at a ratio of 1 g of acid to 100 ml of water, °adding a 10% solution of sodium hydroxide to the resulting solution, dissolving calcium chloride anhydrous in distilled water, gradually adding the helidonate acid solution to the aqueous calcium chloride solution, maintaining the resulting mixture at a temperature of 20-25 °C for 12 hours, then at a temperature of 3-4 °C for 12 hours, filtering the resulting crystals, washing them with distilled water, and drying them at 120 °C for 1 hour. then for 12 hours at a temperature of 3-4 °C, filtering the resulting crystals, washing them with water and drying. In this case, chelidonic acid that has not undergone recrystallisation after its synthesis is used, a sodium hydroxide solution is added until a pH of 5-6 is reached, calcium chloride is dissolved in water at a ratio of 1 g of calcium chloride to 75 ml of water, and the weight ratio of chelidonic acid to calcium chloride is 1:3.

EFFECT: ability to synthesise calcium chelidonate with improved technological (uniform crystallinity with maximum yield of the target product) and biomedical (high bone cell survival) parameters compared to the prototype.

2 cl, 11 dwg, 4 tbl, 10 ex

Description

Field of technology

The invention relates to pharmacy and biomedicine, more specifically to a method for producing an osteogenic agent intended for use in traumatology, orthopedics, maxillofacial surgery, reconstructive surgery for the prevention and treatment of bone tissue damage and diseases.

Prior art

Enhancing regenerative processes and improving bone tissue condition are important in cases of injuries (fractures, wounds), bone diseases (osteoporosis, osteomalacia, osteomyelitis, etc.), osteosynthesis, and bone arthroplasty. Stimulating bone matrix regeneration and mesenchymal stem cell activity are crucial in the treatment and prevention of bone pathologies, as calcium is deposited only in the new collagen matrix synthesized by osteoblasts [1].

The currently existing osteogenic drugs (bisphosphonates, denosumab, calcitonin, bone morphogenetic proteins), with all their advantages, do not meet the needs of both therapists and surgeons in the fight against damage and diseases of bone tissue, solve narrow problems, have a number of serious side effects (osteonecrosis of the jaw, musculoskeletal pain, atypical fractures of the femur), often leveling out the benefits of specific osteogenic activity [2], [3].

Recent studies have shown that small molecules with osteogenic properties, including chelidonic acid and its derivatives, hold promise. The ability of chelidonic acid to chelate vital metal ions through coordination bonds has allowed the synthesis of various derivatives, including calcium chelidonate [4], [5]. The osteogenic activity of calcium chelidonate has been experimentally demonstrated in mesenchymal stem cells in vitro [5] and in vivo following oral administration [5], [6]. A method for obtaining calcium chelidonate from natural plant sources has been described [6]. However, natural sources of calcium chelidonate are extremely limited, and the extraction technology is labor-intensive and expensive.

A method for producing calcium chelidonate from synthesized chelidonic acid is also described [7], which is a prototype of the proposed invention. The known method is as follows. A 10% sodium hydroxide solution is added to an aqueous solution of chelidonic acid recrystallized from hot water to a pH of 7-8. The resulting chelidonic acid solution is added to an aqueous solution of anhydrous calcium chloride and left for 2 hours at 20-25°C, then kept for 12 hours at 3-5°C. The resulting calcium chelidonate is filtered, washed with water and dried. As indicated by the authors, the yield of the target product is approximately 100% of the initial mass of chelidonic acid. However, the technological parameters of the obtained sample are not specified and the cytotoxicity, which is possible due to the production of calcium chelidonate from synthetic raw materials and the recrystallization process, has not been studied.

The essence of the invention

The objective of the present invention is to obtain calcium chelidonate with its maximum yield, improved technological parameters (uniform crystallinity) and low cytotoxicity.

The said problem is solved in that in the method for producing an osteogenic agent in the form of calcium chelidonate, which includes dissolving chelidonic acid synthesized from diethyl oxalate and acetone by heating in distilled water at a ratio of 1 g of acid per 100 ml of water, adding a 10% sodium hydroxide solution to the resulting solution, dissolving anhydrous calcium chloride in distilled water, gradually adding the chelidonic acid solution to an aqueous solution of calcium chloride, maintaining the resulting mixture at a temperature of 20-25 ° C until a precipitate forms, then for 12 hours at a temperature of 3-4 ° C, filtering off the resulting crystals, washing them with water and drying, according to the present invention, chelidonic acid is used that has not been recrystallized after its synthesis, a sodium hydroxide solution is added until a pH of 5-6 is reached, calcium chloride is dissolved in water at a ratio of 1 g calcium chloride per 75 ml of water, and the weight ratio of chelidonic acid and calcium chloride is 1:3.

In addition, the resulting mixture is kept at a temperature of 20-25°C for mainly 30 minutes.

Brief description of the drawings

Fig. 1 - appearance of a sample of calcium chelidonate obtained according to Example 1.

Fig. 2 - appearance of a sample of calcium chelidonate obtained according to Example 2.

Fig. 3 - appearance of a sample of calcium chelidonate obtained according to Example 3.

Fig. 4 - appearance of a sample of calcium chelidonate obtained according to Example 4.

Fig. 5 - appearance of a sample of calcium chelidonate obtained according to Example 5.

Fig. 6 - appearance of a sample of calcium chelidonate obtained according to Example 6.

Fig. 7 - appearance of a sample of calcium chelidonate obtained according to Example 9.

Fig. 8 - appearance of a sample of calcium chelidonate obtained according to Example 10.

Fig. 9 - appearance of a sample of calcium chelidonate obtained from chelidonic acid that has not been recrystallized after its synthesis according to the present invention.

Fig. 10 - appearance of a sample of calcium chelidonate obtained from chelidonic acid recrystallized from water after its synthesis.

Fig. 11 - appearance of a sample of calcium chelidonate obtained from chelidonic acid recrystallized from ethanol after its synthesis/

Implementation of the invention

To validate the proposed method for producing crystalline calcium chelidonate, several parameters were varied: the ratio of chelidonic acid to calcium chloride by weight, the volume of water used to prepare the calcium chloride solution, the pH of the chelidonic acid solution, and the crystallization time. Synthetic chelidonic acid (CAS: 6003-94-7, Acros Organics) with a 96% content and anhydrous calcium chloride of analytical grade were used in the experiment.

Since the chelidonic acid content in the synthetic sample typically does not exceed 96%, it was further purified by preliminary recrystallization from hot water with cooling and from an aqueous solution with ethanol. Calcium chelidonate samples obtained from crude chelidonic acid (KH-1), recrystallized from water (KH-2), and recrystallized from ethanol (KH-3) were studied for cytotoxicity.

The optimality criteria were: improved technological parameters (uniform crystallinity of the sample with maximum yield) and biological safety (absence of cytotoxicity) in comparison with the prototype [7].

Examples 1-5. The ratio of chelidonic acid to calcium chloride was varied by weight (Table 1, Figs. 1-5). A weighed sample of chelidonic acid was dissolved in distilled water with heating in a ratio of 1:100 (g:ml), a 10% sodium hydroxide solution was added to pH 5-6. A weighed sample of anhydrous calcium chloride was dissolved in distilled water in a ratio of 1:50 (g:ml). The resulting chelidonic acid solution was gradually added to a calcium chloride solution, kept for 30 min at room temperature (20-25°C), then 12 h at 3-4°C. The resulting crystals were filtered and washed with distilled water.

The maximum yield of calcium chelidonate was obtained at a ratio of 1:3 (chelidonic acid: calcium chloride) by weight, but the appearance of the obtained sample is not uniform; inclusions of amorphous substance are observed in the microcrystalline powder (Table 1, Fig. 3).

Table 1

The yield of calcium chelidonate depending on the ratio of chelidonic acid (CA) : calcium chloride (CaCl ₂ ) (the results are presented as the standard deviation (M±SD) and the mean value.

Example number Ratio
HC (g): CaCl ₂ (g) Output, g Yield, % of the initial mass of helid acid Crystallinity /
sample amorphism 1 0.05:0.05 0.027±0.001 54.0 amorphous. 2 0.05:0.10 0.038±0.002 76.4 amorphous. 3 0.05:0.15 0.051±0.003 101.4 amorphous+cryst. 4 0.10:0.05 0.066±0.003 66.0 amorphous+cryst. 5 0.15:0.05 0.069±0.004 46.4 amorphous.

Examples 3, 6-8. The pH of the chelidonic acid solution was varied (Table 2, Figs. 3, 6). 0.05 g of chelidonic acid was dissolved in 5 ml of distilled water with heating, and a 10% sodium hydroxide solution was added to a specific pH. 0.15 g of anhydrous calcium chloride was dissolved in 7.5 ml of distilled water. The resulting chelidonic acid solution was gradually added to the calcium chloride solution and kept for 30 min at room temperature (20-25°C), then for 12 h at 3-4°C. The resulting crystals were filtered and washed with distilled water.

Shifting the pH toward the alkaline side reduces the yield of calcium chelidonate and impairs its process parameters. Shifting the pH toward the acidic side is impossible, as free chelidonic acid crystallizes and complex formation does not occur. The optimal pH of a chelidonic acid solution is 5-6.

Table 2

Calcium chelidonate yield depending on the pH of the chelidonic acid solution (results are presented as standard deviation (M±SD) and mean value).

Example number pH Output, g Yield, % of the initial mass of helid acid Crystallinity /
sample amorphism 3 5-6 0.051±0.003 101.4 amorphous+cryst. 6 7-8 0.044±0.002 88.8 amorphous. 7 9-10 0.029±0.001 58.0 amorphous. 8 11-12 0.016±0.001 31.4 amorphous.

Examples 3, 9-10. The volume of water used to obtain a calcium chloride solution was varied to determine the optimal volume for crystallization (Table 3, Fig. 1). 0.05 g of chelidonic acid was dissolved in 5 ml of distilled water with heating, and a 10% sodium hydroxide solution was added to pH 5-6. 0.15 g of anhydrous calcium chloride was dissolved in distilled water in varying ratios. The resulting chelidonic acid solution was gradually added to the calcium chloride solution and kept for 30 min at room temperature (20-25 °C), then for 12 h at 3-4 °C. The resulting crystals were filtered and washed with distilled water.

Increasing the volume of the crystallization mixture improves the technological parameters of calcium chelidonate. The optimal calcium chloride to water ratio is 1:75 (g:ml).

Table 3

The yield of calcium chelidonate depending on the volume of crystallization solution (the results are presented as both the standard deviation (M±SD) and the mean value).

Example number Ratio
CaCl ₂ (g): H ₂ O (ml) Output, g Yield, % of the initial mass of helid acid Crystallinity /
sample amorphism 3 1:50 0.051±0.003 101.4 amorphous+cryst. 9 1:75 0.053±0.003 105.4 crystal 10 1:100 0.042±0.002 84.4 crystal

To evaluate the effect of crystallization time, crystallization was carried out according to Example 9 in two modes: 30 min at 20-25 °C, then 12 h at 3-4 °C; 60 min at 20-25 °C, then 24 h at 3-4 °C. In both modes, the yield of calcium chelidonate was approximately 105% of the initial mass of chelidonic acid, with uniform crystallinity. In addition, the effect of changing the sequence of operations according to Example 9 was studied: adding a chelidonic acid solution to a calcium chloride solution or vice versa, a calcium chloride solution to a chelidonic acid solution, which did not affect the yield of calcium chelidonate or its process parameters.

Then, following Example 9, calcium chelidonate samples were obtained from additionally crude chelidonic acid (KH-1), recrystallized from water (KH-2), and recrystallized from ethanol (KH-3). This parameter did not affect the yield of the target product, but it did influence the process parameters. Optimum process parameters were observed in samples KH-1 (uniformly crystalline cream-colored powder) and KH-3 (finely crystalline beige powder) (Figs. 9, 11).

Next, in vitro cytotoxicity testing of the synthesized substance samples was conducted using the provisions of ISO 10993-5 (Biological evaluation of medical devices. Part 5: Tests for in vitro cytotoxicity). Dry calcium chelidonate substances obtained from additionally crude chelidonic acid (KH-1), from chelidonic acid recrystallized from water (KH-2), and recrystallized from ethanol (KH-3) were compared with each other and with natural calcium chelidonate (KH-0) and chelidonic acid (X-0) substances. A suspension of human bone cells of the MG-63 line, obtained from the cell bank of the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg, Russia), was used in in vitro cytotoxicity experiments.

The cell suspension was incubated at a concentration of 50× ¹⁰³ cells in 1 ml of a medium containing 90% alpha-MEM, 10% fetal bovine serum, 40 mg/ml gentamicin, and 2 mM L-glutamine. The test substances were added to the cell suspension at a final concentration of 100 mg/l, after which the cell cultures were incubated for 24 h at 35°C, 100% humidity, and 5% _CO2 .

After incubation for 24 h, the proportion of viable cells was estimated using 0.4% trypan blue solution in phosphate-buffered saline on a Countness II FL counter (Thermo Fisher Scientific, USA) according to the instrument manual and ISO 10993-5 recommendations. Nine wells of 24-well culture plates were used for each observation group. The in vitro testing results presented in Table 4 showed that additionally crude synthetic chelidonic acid (X-1), synthetic chelidonic acid recrystallized from water (X-2), and, to a greater extent, synthetic chelidonic acid recrystallized from ethanol (X-3) showed some cytotoxicity, increasing the number of trypan blue-stained (dead) bone cells by 8-17% compared to natural chelidonic acid (X-0).

In contrast, synthetic calcium chelidonate (KH-1) obtained by the proposed method reduced the content of stained (dead) cells by more than 2 times (from 36% to 17%) compared to natural calcium chelidonate (KH-0; p < 0.001) (Table 4). This indicates an increase in bone cell survival in the presence of KH-1 from 64% (KH-0) to 83%. At the same time, synthetic forms of calcium chelidonate obtained from chelidonic acid recrystallized from water (KH-2) or ethanol (KH-3) did not exhibit a similar cytoprotective effect. The percentage of dead cells exceeded the 30% threshold for in vitro cytotoxicity recommended by the international standard ISO 10993-5.

Table 4

The percentage of dye-stained (dead) bone cells after 24-hour in vitro incubation with natural or synthesized chelidonic acid or calcium chelidonate obtained by different methods. Results are presented as mean and standard deviation (M±SD).

Substance X-0 X-1 X-2 X-3 KH-0 KH-1 KH-2 KH-3 Group number 1 2 3 4 5 6 7 8 Percentage (%) of dead cells 17 ± 7 26.5±4.5 28 ± 4 34 ± 3 36 ± 2 17 ± 3 39 ± 3 32 ± 5 Level of statistical differences between groups* p ₁₂ = 0.031 p ₁₃ = 0.015 p ₁₄ < 0.001
p ₂₄ = 0.009
p ₃₄ = 0.045 p ₅₆ < 0.001 p ₆₇ < 0.001 p ₆₈ < 0.001 * according to Student's t-test

Thus, the samples of synthetic calcium chelidonate obtained according to the present invention from additionally unrefined chelidonic acid, i.e. not subjected to recrystallization (KH-1) after its synthesis, showed the best biomedical result (low cytotoxicity) in comparison with natural and other synthesized calcium salts of chelidonic acid.

Since the osteogenic properties of calcium chelidonate are known from the existing state of the art [5], [6], [7], the synthetic calcium chelidonate obtained according to the present invention and promoting increased survival of bone cells is a technical solution that is novel and inventive and aimed at increasing safety and enhancing the osteogenic activity of the agent in its biomedical application.

Bibliography

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2. Kofron M.D., Laurencin C.T. 2006. Bone tissue engineering by gene delivery // Adv. Drug Deliv. Rev. V. 58. P. 555-576. https://doi.org/10.1016/j.addr.2006.03.008.

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4. Yasodha V., Govindarajan S., Low JN, Glidewell C. Cationic, neutral and anionic metal(II) complexes derived from 4-oxo-4H-pyran-2,6-dicarboxylic acid (chelidonic acid). Acta Cryst. 2007, C63, m207-m215. https://doi.org/10.1107/S010827010701459X

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Claims (2)

1. A method for producing an osteogenic agent in the form of calcium chelidonate, comprising dissolving chelidonic acid synthesized from diethyl oxalate and acetone by heating in distilled water at a ratio of 1 g of acid per 100 ml of water, adding a 10% sodium hydroxide solution to the resulting solution, dissolving anhydrous calcium chloride in distilled water, gradually adding the chelidonic acid solution to an aqueous solution of calcium chloride, maintaining the resulting mixture at a temperature of 20-25 ° C until a precipitate forms, then for 12 hours at a temperature of 3-4 ° C, filtering off the resulting crystals, washing them with water and drying, characterized in that chelidonic acid is used that has not been recrystallized after its synthesis, a sodium hydroxide solution is added until a pH of 5-6 is achieved, calcium chloride is dissolved in water at a ratio of 1 g of calcium chloride per 75 ml of water, and the weight ratio of chelidonic acid and calcium chloride is 1:3. 2. The method according to paragraph 1, characterized in that the resulting mixture is maintained at a temperature of 20-25°C for 30 minutes.