RU2468804C2 - Gel-forming mixed dextran ester phosphates and carbamates, method for preparing them - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and represents gel-forming mixed dextran esters containing phosphate and carbamate groups of general formula: {C₆H₇O₂(OH)_3-x-y{[(OP(O)ONa)_mONa)]_xl[(O₂P(O)ONa)_k]x₂}_x(OCONH₂)_y}_n, wherein x=x₁+x₂ is a degree of substitution in phosphate groups (mono- and diesters), x=0.47-1.09; X₁ is a degree of substitution in monoesters, X₁=0.01-0.48; m is a number of phosphates in monoesters, m=1-2; x₂ is a degree of substitution in diesters, x₂=0.01-1.09; k is a number of phosphates in diesters, k=1-2; y is a degree of substitution in carbamate groups, y=0.39-1.23; n is a degree of polymerisation, 20≥n≤1000.

EFFECT: invention provides producing low-toxic low- and high-substituted dextran phosphates in the form of hydrogels containing additionally carbamate groups and possessing antiproliferative activity with respect to cancer cells.

2 cl, 3 dwg, 14 ex

Description

The invention relates to medicine, in particular to high molecular weight compounds, in particular, dextran esters containing phosphate and carbamate groups, having biological activity and is intended to create prolonged drugs.

Dextran is a polysaccharide consisting of glucopyranose units, linked mainly by an α-1,6 bond, and also, to a much lesser extent, α-1,2 and α-1,3 bonds. Dextran is soluble in water, has biocompatibility and the ability to biodegrad in the tissues of a living organism without releasing toxic substances. It is known that the introduction of a certain type of functional groups into the composition of dextran (sulfate, carboxyl, aldehyde, phosphate, etc.) tells him a set of additional physicochemical and biomedical properties [1-9]. For example [1], dextran sulfate has anticoagulant activity, low toxicity and is used in the clinic. In addition, it is known [2] that dextran sulfates have an antiproliferative effect against various viruses, as well as HIV infection. In contrast to dextran sulfates, a relatively small number of studies have been devoted to the synthesis and study of the biomedical properties of dextran phosphates [3, 6–8]. [3] describes a method for producing low-substituted dextran phosphates by esterification with a solution of polyphosphoric acid in formamide and found that dextran phosphate (phosphorus content - 1.7%) has immunomodulating activity and is able to induce the immunomodulating effect of interferons (IL - 10 and γ-IFN )

Dextran phosphates are derivatives of dextran in which, through esterification, part of the hydroxyl groups of the polysaccharide is replaced by phosphate groups, mainly with the formation of mono- and diesters. The formation of diesters determines the crosslinking of dextran macromolecules, i.e. the possibility of obtaining it not only in the form of solutions, but also hydrogels.

Obtaining hydrogels capable of absorbing a significant amount of water (or other liquid) and retaining it without dissolution is one of the promising and intensively developing areas in the chemistry of high molecular weight compounds. Hydrogels are used in the development of compositions for the manufacture of soft contact lenses, dosage forms with prolonged release of the active substance, transdermal therapeutic systems, sorbents, materials for the manufacture of endoprostheses, etc. The reason for the wide and diverse application of hydrogels is their unique porous structure, which provides a high swelling rate in water, high permeability for low and high molecular weight compounds, and good biocompatibility [10, 11].

The ratio of mono- and disubstituted phosphates of dextran depends on the type and composition of the esterifying mixture, the conditions for the phosphorylation reaction. The literature describes a variety of methods for the esterification of dextran. It is known [3, 7–9] that, to obtain highly substituted dextran phosphates (the degree of substitution of hydroxyl groups by phosphate groups in one glucopyranose unit is equal to or greater than 0.8), highly toxic and aggressive reagents, such as phosphorus oxychloride, phosphorus oxide ( V). The use of less aggressive reagents, for example, sodium or potassium phosphates, polyphosphates, phosphoric, polyphosphoric acids, etc., contributes to the production of low substituted dextran phosphates. The low substituted dextran phosphates obtained by the above methods are water soluble compounds.

The prototype of the invention are mixed starch ethers in the form of hydrogels containing phosphate and carbamate groups, the method of production thereof [12, 13]. The obtained mixed starch esters can be used as sorbents for water purification from multicharged metal cations, as thickening agents. The inclusion of carbamate groups in the structure of the polysaccharide phosphate contributes to the appearance of additional intermolecular interactions (hydrogen bonds, chemical crosslinking) and, as a result, to the production of esterification reactions in the form of hydrogels in a wide range of changes in the degree of substitution for phosphate groups. The known process of phosphorylation of starch is carried out with phosphoric acid in a urea melt at a temperature of 110-140 ° C under vacuum (0.01-0.13 atm) for 1-3 hours. After cooling, distilled water is added to the reaction mass to swell the hydrogels obtained, washed 3 times with a solution of methyl alcohol (80% solution), dried under vacuum at 50 ° C for three hours.

The specified method cannot be used to obtain dextran phosphate hydrogels for the following reasons:

- the intensively ongoing process of destruction of dextran leads to the production of dark-colored phosphate esters exclusively in the form of viscous solutions;

- the unsuitability of this method of esterification to obtain hydrogels of polysaccharides having a low degree of polymerization.

The objective of the invention is to obtain low-toxic low- and highly substituted phosphates of dextran in the form of hydrogels, additionally containing carbamate groups and exhibiting an antitumor effect.

The problem is solved in that, as a low-toxic anti-tumor high molecular weight compound, dextran esters containing phosphate and carbamate groups are proposed according to the formula:

{C ₆ H ₇ O ₂ (OH) _3-xy {[(OP (O) ONa) _m (ONa)] _x1 [(O ₂ P (O) ONa) _k ] _x2 } _x (OCONH ₂ ) _y } _n where

x = x ₁ + x ₂ is the degree of substitution for phosphate groups (mono - and diesters), x = 0.47-1.09;

x ₁ is the degree of substitution on monoesters, x ₁ = 0.01-0.48;

x ₂ is the degree of substitution on diesters, x ₂ = 0.01-1.09;

m is the number of phosphates in monoesters, m = 1-2;

k is the number of phosphates in diesters, k = 1-2;

y is the degree of substitution for carbamate groups, y = 0.39-1.23;

n is the degree of polymerization, 20≥n≤1000.

The method of producing dextran phosphate, including phosphorylation of dextran with orthophosphoric acid in a urea melt at 110-140 ° C and residual pressure, adding water to a paste-like state of the reaction mass, followed by washing and drying of the final product, is characterized in that the initial dextran is additionally dried and phosphorylated before modification carried out at a higher residual pressure of 0.05-0.27 atm, followed by treatment of the reaction mass with a 0.75 M solution of sodium chloride in a 70% solution of ethyl alcohol, bringing lowering the pH of the solution to 11.0-12.0, washing the precipitate with a 70% solution of ethyl alcohol.

The method for producing dextran esters containing phosphate and carbamate groups, in comparison with the method [13] is modified as follows:

- for the predominant formation of dextran diphosphate esters and to obtain products in the form of hydrogels, the initial dextran is preliminarily dried at 50 ° C and a residual pressure of 0.1 atm for 5-16 hours before the esterification reaction;

- the process of phosphorylation of dextran with phosphoric acid in the urea melt is carried out at a residual pressure of 0.05-0.27 atm. This parameter of the phosphorylation process has a significant effect on the yield of dextran phosphates in the form of a gel fraction. At a residual pressure of 0.5 atm, the yield of the gel fraction is significantly reduced (about 2 times). At atmospheric pressure, only low-substituted water-soluble dextran phosphate samples can be obtained;

- to replace a toxic solvent (methanol) during washing of dextran phosphates, shortening its period, and also obtaining the Na-form of phosphate esters, the modification product after cooling the reaction mass, adding distilled water is precipitated with a 0.75 M solution of sodium chloride in ethyl alcohol-water mixture ( 70% solution), the pH of which was adjusted with sodium hydroxide solution to 11.0-12.0. The precipitate obtained is washed in a Soxhlet apparatus with a 70% solution of ethyl alcohol and dried at 50 ° C in a vacuum oven at a residual pressure of 0.1 atm.

As starting material for the preparation of the claimed compounds, dextran with a molecular weight in the range of 40-1000 kDa is used.

The method allows to obtain low-toxic gelling dextran phosphates with antitumor activity. The pH of the swollen hydrogels is in the range of 7.2-7.4, which corresponds to the pH of the blood.

The yield of reaction products, calculated from the theoretically possible, is quantitative.

The invention is illustrated by drawings, tables, examples.

Figure 1. Electronic micrographs of granules of the starting (1) and phosphates (2, 3) dextrans: 2 - SZ _P = 0.48, SZ _N = 0.42; 3 - SZ _P = 0.68, SZ _N = 0.47.

Figure 2. The dependence of the amount of absorbed water 1 g of dextran phosphate (SZ _P = 1.09; CZ _N = 1.23) on time (Q, g / g).

Figure 3. Sarcoma M-1 growth dynamics (V, cm ³ ) in rats in the control and after intraperitoneal administration of dextran phosphate (SZ _P = 0.48, SZ _N = 0.42) at a dose of 2.0 g / kg.

Example 1. To 20 g of pre-dried at 50 ° C and a residual pressure of 0.1 atm for 16 hours of dextran (Mw = 40,000 Da) with constant stirring, 29.76 g of urea (hch) and 5.2 ml of 85% orthophosphoric acids. The molar ratio of d-anhydroglucopyranose unit (GPZ): phosphoric acid (H ₂ PO ₄ ): urea [(NH ₂ ) ₂ CO] is 1.0: 0.6: 4.0. It is maintained at a temperature of 125 ° C and a residual pressure of 0.06-0.25 atm for 3 hours. At the end of the phosphorylation reaction, the reaction mixture is cooled to room temperature. Then add distilled water to obtain a pasty mass, pour 400 ml (bath module (g / ml) - 1:20) of a NaCl solution (30 g per 1 liter of a solution of 70% ethyl alcohol, the pH of which is adjusted with sodium hydroxide to 11.5) and left at room temperature for 24 hours. The precipitate was washed in a Soxhlet apparatus with 70% ethanol, dried at a temperature of 50 ° C in a vacuum oven at a residual pressure of 0.1 atm. The yield of gel fraction is 96.7%. The phosphorus content in the obtained sample is 7.0%, nitrogen - 2.7%. The degree of substitution on phosphate groups (SZ _P ) is 0.49; carbamate groups (SZ _N ) - 0.42. The amount of water absorbed 1 g of dextran phosphate is 174.6 g / g.

Examples 2-14. Samples of dextran phosphates are obtained analogously to example 1 at various process parameters. The conditions for the phosphorylation process for all examples are shown in table 1.

Samples of dextran phosphates obtained in accordance with examples 1-14 were characterized by the content of phosphate and carbamate groups, structural features, degree of swelling, acid-base properties by elemental analysis, IR spectroscopy, scanning electron microscopy, and potentiometric titration [15].

Elemental analysis. The content of phosphate groups in dextran phosphate samples is determined by spectrophotometric method [14], nitrogen - by the Kjeldahl method [15].

The obtained compounds are characterized by the degree of substitution for phosphoric acid (C3 _P ) and carbamate (C3 _N ) groups (the number of phosphate or carbamate groups per anhydroglucopyranose unit (GPZ) of the polysaccharide). The data obtained are shown in table 1.

IR spectroscopy In the IR spectrum of the claimed compound, an absorption band near 790 cm ^-1 (non-planar deformation vibrations of the P-O-P groups), a shoulder near 950 cm ^-1 and an absorption band near 1020 cm ^-1 (stretching vibrations of the P-O-P and P-O-N), a shoulder near 1210 cm ^-1 (stretching vibrations of the phosphoryl group P = O), indicating the formation of phosphate groups. The IR spectra of all dextran samples esterified with orthophosphoric acid in the urea melt contain an intense absorption band near 1720 cm ^-1 , due to asymmetric stretching vibrations of C = O bonds of carbamate groups. With an increase in the degree of esterification, these spectral changes increase.

Scanning electron microscopy. As a result of phosphorylation (Figure 1), the dextran granules break up into fragments that are not uniform in length and width, which have a loose, porous structure (micropore sizes range from 2 to 10 nm). An increase in the concentration of phosphoric acid in the esterifying mixture promotes a gradual densification of the structure of dextran, an increase in the size of fragments and the disappearance of micropores.

Water absorbing ability. Water absorption capacity (Q, g / g) is determined by the gravimetric method and calculated by the formula:

,

,

where Q is the water absorption capacity (Q, g / g);

m ₁ is the mass of the sample swollen in water, g;

m ₂ - the mass of the sample, dried to constant weight, g

Dextran phosphate hydrogels are separated from excess water by centrifugation on a glass filter with a pore size of 160 μm with a centrifugal force of 2400 g. Drying to constant weight is carried out in the presence of phosphorus oxide (V) at a temperature of 50 ° C, a residual pressure of 0.1 ATM.

The values of the water absorption capacity of hydrogels based on dextran phosphates are shown in table 1 and figure 2.

The obtained hydrogels of dextran phosphates are characterized by a high rate of water absorption: the water absorption of hydrogels with different contents of phosphate and carbamate groups reaches its maximum value in less than 5 minutes and practically does not change over time (Figure 2) - using phosphate with M.m. 60 kDa (SZ _P = 1.09; SZ _N = 1.23).

Potentiometric titration. From the data of table 2 it follows that

table 2 Potentiometric titration of hydrogels based on dextran phosphates Sz _P CZ _N Potentiometric titration data OE ₁ , mEq / g POE, mEq / g pK ₁ pK ₂ one 0.48 0.42 1,5 3.2 2.9 7.0 2 0.78 0.83 1.9 1.9 3,1 - 3 1.09 1.23 1.3 1.3 3.0 -

the obtained phosphates of dextran have a total exchange capacity in the range of 1.3-3.2 mEq / g. According to the values of the apparent ionization constants (pK ₁ and pK ₂ ), dextran phosphates contain one or two acidic functional groups that dissociate mainly in the pH range of 1.8-5.4 and 5.0-9.0 .

In vivo study of the acute toxicity of hydrogels of polysaccharide phosphates. The acute toxicity of PD hydrogels was determined in rats (each series of 4 rats weighing 200-250 g). Animals were administered 10 g of a 50% hydrogel intraperitoneally. It was established (table 3) that dextran phosphates belong to the class of low toxic substances.

Table 3 Acute Toxicity of Dextran Phosphate Hydrogels Polysaccharide Sz _P C3 _N LD ₅₀ Dextran (Mw = 60,000) 1.09 1.22 ~ 5000 mg / g Dextran (Mw = 500,000) 0.85 1.16 > 5000 mg / g

Assessment of the antitumor activity of dextran phosphate hydrogels. A comparative study of the antitumor activity of the proposed dextran phosphates was carried out in vitro and in vivo.

In vitro study of the antitumor activity of hydrogels of dextran phosphates.

The antitumor effect of hydrogels based on dextran phosphates was evaluated on a monolayer culture of Hela tumor cells (human cervical epithelioid carcinoma, clone M) by comparing the number of surviving cells with their initial number before exposure to dextran phosphates.

The decrease in the number of cells (N) after exposure to the claimed compounds below the initial level indicates the predominance of the cytotoxic effect, and above the initial, but lower than the control, the prevalence of the cytostatic effect. The efficacy of the antitumor effect of hydrogels IK ₅₀ (concentration causing inhibition of proliferation by 50%) was calculated by the method of regression analysis of the data. Statistical processing of the results was carried out using the Origin 7 program.

The results are presented in table 4. It is seen that with an increase in the content of phosphate groups in dextran, the ability of the claimed esters to inhibit the proliferation of tumor cells increases. The cytostatic activity of the studied compounds is dose dependent.

2 460 ± 20 280 45

In vivo study of antitumor activity. The in vivo efficacy of the antitumor effect of hydrogels based on dextran phosphates was tested on outbred white rats of both sexes (16 animals), which were transplanted sarcoma M-1 subcutaneously into the region of the left thigh.

On days 7–10 after transplantation of the tumor, rats received dextran phosphate hydrogel once intraperitoneally (dose 2000 mg / kg). Measurements of the size of the tumors in the control and experimental groups were performed 3 times a week, and the tumor volume (V, cm ³ ) was calculated by the Shrek formula:

V = (a × b × c) × π / 6, where a, b, c are the linear dimensions of the tumor (cm).

Inhibition of tumor growth was determined by the formula:

(Vcp. (Control) -Vavr. (Experience)) / Vavr. (Control)

The results obtained (Figure 3) reveal the antitumor activity of the claimed compounds. Two days after the administration of dextran phosphates, the difference in the growth rate of tumor cells of the experimental batch compared with the control becomes obvious; on the 7th day - the tumor volume compared with the control is reduced by about half.

Thus, the modification of dextran with a mixture of phosphoric acid and urea leads to the formation of low toxic compounds with antitumor effects. The claimed compound provides, in comparison with dextran and existing dextran phosphates, the following advantages:

- compounds can be obtained in the form of hydrogels in a fairly wide range of changes in the content of phosphate groups and the molecular weight of dextran;

- hydrogels based on dextran phosphates have a high swelling rate;

- upon receipt, relatively non-toxic reagents are used, which makes the purification step of the target product simpler and more economical.

The proposed phosphate dextran can be obtained in the conditions of enterprises producing chemical and pharmaceutical preparations.

INFORMATION SOURCES

1. WO Pat. 90/04970, MKI 5 A61K 31/725, 1990.

2. Baba M., Pauwells R., Balzarini J., Arnout J., Desmyter J. at al. Mechanism of ingibitory effect of dextran sulfate and heparin on replication of human immunodeficiency virus in vitro. Proceeding of the National Academy of the United States of America. 1988. Vol. 85. P.6132-6136.

3. US Pat. 20060154896, MKI A61K 31/721, 2006.

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6. Suzuki M., Mikami T., Malsumoto T., Suzuki S. Preparation and antitumor activity of o-palmitoyldextran phosphate, o-palmitoyldextrans and dextran phosphate. Carbohydrate Reseach. 1977. Vol. 53. P.223-229.

7. US Pat. 2970141, MKI C12P 19/08, C12P 19/00, 1961.

8. Kirci B., Kaplan H., Rzaev Z.M., Guner A. Preparation conditions and swelling equilibria of dextran hydrogels prepared by some crosslinking agents. Polymer 2004. Vol. 45. P.6431-6435.

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Claims (2)

1. Gel-forming mixed esters of dextran containing phosphate and carbamate groups of the general formula: {C ₆ H ₇ O ₂ (OH) _3-x-y {[(OP (O) ONa) _m ONa)] _x1 [(O ₂ P (O) ONa) _k ] _x2 } _x (OCONH ₂ ) _y } _n , where x = x ₁ + x ₂ is the degree of substitution for phosphate groups (mono and diesters), x = 0.47-1.09;
x ₁ is the degree of substitution in monoesters, x = 0.01-0.48;
m is the number of phosphates in monoesters, m = 1-2;
x ₂ is the degree of substitution on diesters, x = 0.01-1.09;
k is the number of phosphates in diesters, k = 1-2;
y is the degree of substitution for carbamate groups, y = 0.39-1.23;
n is the degree of polymerization, 20≤n≤1000, with antiproliferative activity against tumor cells. 2. The method of producing dextran phosphate according to claim 1, including phosphorylation of dextran with phosphoric acid in a urea melt at 110-140 ° C and residual pressure, adding water to a paste-like state of the reaction mass, followed by washing and drying of the final product, characterized in that the initial dextran before modification, they are additionally dried, phosphorylation is carried out at a higher residual pressure of 0.05-0.27 atm, followed by treatment of the reaction mass with a 0.75 M solution of sodium chloride in a 70% solution of ethyl alcohol that, bringing the pH of the solution to 11.0-12.0, washing the precipitate with a 70% solution of ethyl alcohol.

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