BG111326A - Nanosized polyelectrolyte associates with antitumor activity, processes for their preparation and use thereof - Google Patents

Abstract

The invention relates to nanosized polyelectrolyte associates with formula (IV), process for their preparation and use thereof as drug-polymer system with antitumor activity, which is intended for use in pharmacy and nanomedicine.

Description

NANOSIZED POLYELECTROLYTIC ASSOCIATES, WITH ANTI-TUMOR ACTION, METHOD FOR THEIR

RECEIPT AND THEIR APPLICATION

Field of technology

The present invention relates to nanosized polyelectrolyte associates, a method for their preparation and their use as a drug-polymer system with antitumor action, intended for use in pharmacy and nanomedicine.

BACKGROUND OF THE INVENTION

The use of platinum complexes in anti-cancer chemotherapy is well known to those skilled in the art. The action of cis-dichlorodiaminoplatin (II) (cisplatin) is known in this respect.

H ₃ N Cl

H ₃ N Ci cisplatin

However, it has been clinically established that treatment with it can lead to severe nephrotoxicity and other side effects, as well as the acquisition of resistance in cancer cells.

Subsequent studies have synthesized more than 3,000 other platinum complexes, but only a small number of them, such as carboplatin and oxaliplatin, have advantages over cisplatin that could lead to their inclusion in clinical trials - some reduction in toxic side effects and overcoming of resistance [1].

For most cisplatin analogues, no improvement in efficacy or broadening of the spectrum of action was observed [2], which is explained by similar adducts - intrachain GG and AG adducts [3], which form the classical complexes of platinum (P) with DNA.

Subsequent studies are aimed at creating a new generation of cytostatics, such as polynuclear platinum (II) complexes, containing 2 or more metal nuclei, and their cytotoxicity has been determined against various tumor cell lines, as is known, for example, from patent descriptions of [4]. , [5] and [6].

From [7] is known polynuclear complex BBR3464, which has reached clinical trials, has a positive load (+4) and two coordination centers capable of binding to DNA for a time significantly less than necessary for cisplatin [8].

Also known [9] are platinum (II) diadrene complexes whose coordination centers are linked to a polyamine, for example spermine or spermidine. They are extremely active compounds that bind to DNA, a mechanism similar to that of BBR3464. The cytotoxic profile of BBR3571 (diamine complex with spermidine ligand) is similar to that of BBR3464 [10].

These complexes, as well as BBR3464, have excellent solubility in aqueous media, which is an advantage when taken as a drug. Another important feature is that they exhibit cytotoxic activity at nanomolar concentrations in contrast to cisplatin and its analogues active in the micromolar concentration range [10, 11].

The difference in the structures of cisplatin and polynuclear complexes determines the differences in the way of binding to DNA, as well as in the type of adducts formed [8,12].

The main feature that distinguishes polynuclear complexes from cisplatin is their ability to bind to distant DNA bases, but due to the large distance between the reaction centers, adducts of different ranges can be formed - from 1,2- to 1,6-interchain or intrachain crosslinks, some of which cannot be removed by the nucleotide repair mechanism [13, 14, 15, 16].

«*

However, clinical trials of BBR3464 in phase 2 patients have not shown satisfactory results to be registered for use. This is associated with the irreversible interaction of the complex with plasma proteins and its inactivation under the action of glutathione [17, 18, 19,].

Three main pharmacological factors are known to those skilled in the art, on which the clinical success of platinum coordination chemotherapy depends, namely: cell entry, frequency and type of DNA adducts formed, and metabolic deactivation of the complex. The latter is due to the interaction of the agent with biomolecules, mainly with sulfur-containing peptides and proteins, and these interactions can lead to an increase in the toxic side effects of the drug [20, 21].

Albumin is the protein with the highest concentration in blood plasma 30-50 g / Ι. It has been found that albumin-related platinum complexes are deactivated and eliminated from the body, which is why they do not reach their main pharmacological target - genomic DNA [22].

Glutathione is a sulfur-containing tripeptide whose concentration in cells is 5-10 μM, which makes it very likely to interact with platinum complexes having readily leaving groups, for example chloride ligand [23].

By replacing the chloride ligand with an "inert" amino ligand, new complexes have been synthesized [24, 25] that do not have a reaction center for covalent binding to plasma proteins or glutathione. They retain their cytoxic profile despite the presence of serum proteins, indicating a lack or low degree of inactivation compared to BBR3464 or cisplatin [26]. These BBR3464 analogs do not bind covalently to DNA, but through interactions based on electrostatic attraction or hydrogen bonds. However, they have been shown to have a less pronounced cytotoxic effect than BBR3464 [25].

[27, 28, 29] two liposomal preparations SPI-77 and Lipoplatin containing cisplatin are known to have reached clinical trials.

Although SPI-77 showed increased accumulation in tumor tissue, no improvement in clinical efficacy was observed due to the low degree of cisplatin release from liposomes [30].

Lipoplatin has successfully passed phase 3 clinical trials. In addition to reducing side effects such as damage to the kidneys, peripheral nervous system, hearing, etc., the application of lipoplatin is characterized by a 10-200-fold greater accumulation in tumor tissue compared to neighboring healthy tissues, as well as efficiency close to that of cisplatin. [31].

Also known is a poly-hydroxypropyl methacrylamide drug-polymer system designed for the targeted transport of oxaliplatin, which is in phase 2 of clinical trials against ovarian cancer [32].

The person skilled in the art is aware of the biological inertness of polyethylene glycol, as well as its ability to form a hydrophilic shell around the active substance, thus protecting it from enzymatic degradation and attack of macrophages, while extending the circulation time of this active substance in the circulatory system [33].

That is why pegylated platinum complexes have been developed and are already available in the prior art. However, it has been experimentally proven that pegylation of carboplatin and its analogues does not give the expected result. Despite the slower elimination from the body, there is a difficulty in cell internalization and a lower level of adduct formation between the modified complexes and DNA [34].

Polyphosphoresters are another class of polymers that have been relatively little studied as drug carriers. They are structurally diverse polymers that degrade under physiological conditions and are characterized by negligible cytotoxicity. For example, poly (propylene phosphate) showed low toxicity to COS-7, HEK293 cell lines and good biocompatibility (in an in vivo study of mice), which is explained by the low toxicity of degradation products [35].

Poly (hydroxy phosphates) they contain in their chain hydroxyphosphate groups, due to which they are water-soluble and in aqueous medium have polyacidic relations. These polymers have low cytotoxicity [36].

The use of polyphosphoesters as carriers of polynuclear complexes of platinum (II), as well as nanocolloid systems containing polynuclear complexes of platinum (II) or their polymer conjugates are not known.

Technical essence of the invention

A problem of the invention is the creation of a drug system comprising biocompatible carriers and polynuclear complexes of platinum (II) with high antitumor activity, for protection of the drug substance from metabolic degradation upon its delivery to the tumor tissue.

The problem of the invention is solved by nanosized polyelectrolyte associates, representing systems of polymeric carrier and polynuclear complex of platinum (II), a method for their preparation and their application as a drug-polymer system with increased efficiency against a spectrum of human tumor cell lines.

As a polymer component in the system, acting as a carrier of the biologically active substance, polyacids are suitable - poly (hydroxyalkylene phosphate) and whose linear chains are composed of segments connected by hydroxyphosphate groups. The polyphosphates were prepared by polymeranological conversion of poly (alkylene H-phosphonate) and (I) 100%. I Ί— (I- I ₍ p _v ,! 1! Р I

.......! Ъ:

(I) where

R is an alkylene or oxyalkylene segment,

R 1 is CH ₃ , C ₂ H ₅ or H; and

R ₂ eH.

Poly (alkylene H-phosphonates) with different degrees of polymerization were synthesized by polytransesterification of dialkyl H-phosphonates and diols according to the procedure described in [37].

The conversion of (I) to poly (hydroxyalkylene phosphates) (II)

(N) where

R is an alkylene or oxyalkylene segment, and

R 1 is CH ₃ , C ₂ H ₅ or H, carried out by the Atherton-Todd reaction [38] using water as the reagent (Scheme 1).

H ₂ O / CC1 ₄ / (C ₂ H ₅ ) N ch ₂ ci ₂ / ch ₃ cn

R = alkyl, oxyalkylene

R 1 is H, CH ₃ or C ₂ H ₅

R ₂ = H

R1O - ρ —Ο — R — ο - Ρ — OR!

OH

- ρ ¹

OH

II

Scheme 1

Poly (hydroxyalkylene phosphates) they (II) are prepared and purified according to the modification procedure described in [37].

In the present invention, a mixture of anhydrous solvents, dichloromethane and acetonitrile, is used as the reaction medium. The ratio of the two solvents dichloromethane: acetonitrile can vary from 1: 7 (v / v) to 7: 1 (v / v), with the recommended range from 3: 5 (v / v) to 5: 3 (v / v). ).

To the reaction mixture of carbon tetrachloride and dichloromethane, poly (alkylene H-phosphonate) (I) and triethylamine, previously dissolved in acetonitrile, as well as the required amount of water in acetonitrile are separately invited. The recommended molar ratio of reagents - H-phosphonate groups in the starting polymer water: triethylamine. Tetrachloromethane is 1: 1: 1: 5. The reaction mixture was stirred at room temperature until complete conversion of the H-phosphonate groups to hydroxyphosphate.

The isolation and purification of poly (hydroxyalkylene phosphates) te (II) involves several steps:

(a) evaporate the dichloromethane and cool the reaction mixture to - 12 ° C, then filter to separate the crystals from triethylamine hydrochloride as a by-product;

(b) the organic solvents are evaporated off under reduced pressure and the crude product obtained is dissolved in water;

(c) the solution is passed through a cation exchange column;

(d) dialyzed through a membrane with a pore size <3500 Da; and (e) being lyophilized.

The resulting products are colorless, transparent and soluble in water.

Biologically active component (III)

X = (CH ₂ ) ₆ ; (CH ₂ ) ₃ NH ₂ (CH ₂ ) ₄

L = C1

Lf = Cl; NH ₃ ; NH ₂ (CH ₂ ) ₆ NH ₃ ⁺ ;

NH ₂ (CH ₂ ) ₆ NH ₂ Pt (NH ₃ ) ₂ Cl 3_n_8

III is from the group of polynuclear complexes of platinum (P) and has a positive charge> 3.

H ₃ N

NH ₃ H ₃ N ^Pt \

NH ₂ XH ₂ N

L1 n + t

^X NH ₃

Coordination compounds contain two or more platinum (II) nuclei and ligands such as chloride ion and amino derivatives (1,6-hexandiamine, spermidine and NH ₃ ).

• ·

The complex with formula Sha, where

X is (CH ₂ ) ₆ ; L is Cl; Li is NH ₂ (CH ₂ ) ₆ NH ₂ Pt (NH ₃ ) ₂ Cl, and η is 4 synthesized by the procedure described in [4] is known as BBR3464. The three focal points are in a trans-configuration linked by two 1,6 hexanediamine molecules. The central metal nucleus carries a 2+ charge, and the end nuclei have a 1+ charge and have one chloride ligand - reaction centers for binding to proteins or DNA.

The binuclear complex of formula IIIb where

X is (CH ₂ ) ₃ NH ₂ (CH ₂ ) ₄ ; L is Cl; L 1 is Cl and η is 3 known as BBR3571, obtained by the method described in [9]. The two platinum nuclei are connected by a spermidine bridge and each have a chloride ligand and a charge of 1+. The total charge of the complex is 3+.

The complex Shs where

X is (CH ₂ ) ₃ NH ₂ (CH ₂ ) ₄ ; L is Cl; N is NH ₂ (CH ₂ ) ₆ NH ₃ ⁺ , and η is 5 is also dinuclear, but combines structural fragments of BBR3464 and BBR3571. A similar approach was used to obtain it, including:

(a) activation with silver nitrate of one of the coordination centers of the complex IIIb, the secondary amino group of the spermidine bridge of which is protected,

b) interaction with a monoprotective hexandiamine, and

c) release of the protected amino groups in an acidic medium.

The Shs complex has only one chloride ligand and a total charge of 5+.

The object of the present invention is a method for preparing polyelectrolyte associate (IV) by mixing aqueous solutions of polymer II and complex III at pH <7.

Polyelectrolyte Associate (IV)

1 _Ч ХЩ Sun Sun ¥ 2

II, χ ^ ХЩХЩХ ^ 'XII,

Ζ-

p: y = 4: 1 to 12: 1 z = p-yn + l

IV is formed as a result of the interaction of oppositely charged polyionic compounds - negatively charged polyphosphate (II) and polynuclear complex of platinum (II) with a positive charge> 3 (III).

The initial charge ratio of the two components can vary from 1: 1 to 4: 1 in favor of the negative charge of the polymer carrier. The recommended pH range for the association is between pH 5 and pH 6. The mixed solution is stirred for 10 to 20 hours at room temperature, then dialyzed against deionized water.

The resulting product is isolated either by evaporation of the water under reduced pressure and temperature <30 ° C or by lyophilization.

The molar ratio of phosphate groups to complex III varies from 4: 1 to 12: 1.

The components of the system interact in an aqueous medium and form intermolecular associations based on electrostatic forces and hydrogen bonds. In aqueous solution, the intermolecular associates form stable nanocolloid systems, the size of the nanoparticles depending on the molecular weight of the carrier.

According to the present invention, the polyelectrolyte associate (IV) is dispersed in an aqueous medium to obtain nanoparticles.

The nanoparticles were characterized by diffusion NMR spectroscopy (DOSY) and the light scattering method.

The diffusion coefficient is determined from the DOSY spectra, and the hydrodynamic radius of the particles is calculated from the Stokes-Einstein equation and the value of the diffusion coefficient.

The particle size distribution is obtained from the measurement by dynamic light scattering. The size of the nanoparticles depends on the molecular weight of the polymer carrier and varies slightly depending on the structure of complex III and the ratio between the components of the polymer carrier system and the polynuclear complex of platinum (II).

The nanocolloid system is stable in the temperature range 20 + 37 ° C.

The present invention also relates to the preparation of a nanosized drug-polymer system with increased efficiency of the active ingredient compared to that of the non-immobilized agent.

The efficacy of polynuclear complexes of platinum (II) (non-immobilized and immobilized to polyphosphate (II) was evaluated by determining their cytotoxicity to human tumor cell lines of different origin and cell type, namely:

> HL-60 - acute myeloid leukemia;

> HL-60 / CDDP - acute myeloid leukemia, with induced cisplatin resistance;

> BV-173 - chronic myeloid leukemia;

> MDA-MB-231 - adenocarcinoma of the mammary gland.

The obtained 1C ₅₀ values indicate that the polyelectrolyte associates show more pronounced cytotoxicity compared to the non-immobilized platinum complexes.

• # · * · ·· ··· ... ....

Brief description of the attached figures

Figure 1. 1 H NMR spectrum of poly (hydroxyoxyethylene phosphate) - products Pa and IIb.

Figure 2. P NMR spectrum of poly (hydroxyoxyethylene phosphate) - products Pa and Pb.

Figure 3. DOSY NMR spectrum of poly (hydroxyoxyethylene phosphate) - product Pa.

Figure 4. DOSY NMR spectrum of poly (hydroxyoxyethylene phosphate) - product IIb.

Figure 5. DOSY NMR spectrum of the particles of product Va.

Figure 6. Distribution curve of the hydrodynamic radius (determined by the light scattering method) of the particles of product Va.

Figure 7. DOSY NMR spectrum of Vb product particles.

Figure 8. Curve of distribution of the hydrodynamic radius (determined by the method of light scattering) of the particles of product Vb.

Figure 9. Distribution curve of the hydrodynamic radius (determined by the light scattering method) of the particles of product Via.

Figure 10. DOSY NMR spectrum of Via particles.

Figure 11. Distribution curve of the hydrodynamic radius (determined by the light scattering method) of the particles of product VIb

Figure 12. DOSY NMR spectrum of the particles from product VIb

Figure 13. Distribution curve of the hydrodynamic radius (determined by the light scattering method) of the particles of product VII.

Figure 14. DOSY NMR spectrum of the particles of product VII.

Figure 15. Cytotoxic effects of complex IIIa and its polyelectrolyte associates Va and Vb on HL-60 cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

* ft ··· ♦ · ··. ... ....

Figure 16. Cytotoxic effects of complex IIIa and its polyelectrolyte associates Va and Vb on HL-60 / CDDP cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 17. Cytotoxic effects of complex IIIa and its polyelectrolyte associates Va and Vb on BV-173 cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 18. Cytotoxic effects of complex IIIa and its polyelectrolyte associates Va and Vb on MDA-MB-231 cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 19. Cytotoxic effects of complex IIIb and its polyelectrolyte associates Via and VIb on HL-60 cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 20. Cytotoxic effects of complex IIIb and its polyelectrolyte associates Via and VIb on HL-60 / CDDP cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 21. Cytotoxic effects of complex IIIb and its polyelectrolyte associates Via and VIb on BV-173 cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 22. Cytotoxic effects of complex IIIb and its polyelectrolyte associates Via and VIb on MDA-MB-231 cells (human tumor cell line) determined by MTT assay after 72 hours of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 23. Cytotoxic effects of complex IIIc and its polyelectrolyte associate VII on HL-60 cells (human tumor cell line) were determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 24. Cytotoxic effects of complex IIIc and its polyelectrolyte associate VII on HL-60 / CDDP cells (human tumor cell line) determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 25. Cytotoxic effects of complex IIIc and its polyelectrolyte associate VII on BV-173 cells (human tumor cell line) were determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Figure 26. Cytotoxic effects of complex IIIc and its polyelectrolyte associate VII on MDA-MB-231 cells (human tumor cell line) were determined by MTT assay after 72 h of incubation. The curves were constructed on the basis of 8 independent experiments.

Advantages of the invention

The advantages of nanosized polyelectrolyte associates according to the invention are:

- Increased inhibitory effect on the growth of human tumor cell lines compared to the neimobilized complex;

- Increased inhibitory effect on the growth of HL-60 / CDDP cells (acute myeloid leukemia, is induced resistance to cispaltin);

- Increased stability to enzymatic degradation and interaction with sulfur-containing plasma proteins;

- Increased stability of the active substance in physiological environment;

- The nanoscale of the associations allows the creation of an injectable form or solution for infusion.

An exemplary embodiment of the invention

The invention is illustrated by the following examples, which illustrate it without limiting its scope of protection.

Materials

Poly (oxyethylene H-phosphonates) (I), wherein R = (CH ₂ CH ₂ O) ₄ ; R 1 = H or CH ₃ ; and R ₂ = H, with an average degree of polymerization p = 25 and p = 12, respectively with an average molecular weight M _n = 6 700 g / mol and M _n = 3 300 g / mol, were obtained by polytransesterification of dimethyl H -phosphonate (Merck) and polyethylene glycol with a molecular weight of 200 g / mol (Fluka) according to the procedure described in [37].

1 H NMR (CDCl 3, δ, ppm): 6.95 (d, 1j (PH) = 715.5 Hz, 1H, PH repeating unit); ^{6.87 (d,] J (PH} ) = 708.2 Hz, 1H, -P (H) OCH _3); 6.82 (d, 1j (PH) = 687.8 Hz, 1H, -P (H) OH); 4.25 ^ 1 .14 (m, 4H, -CH ₂ OP (O) (H) OCH ₂ -); 3.66-3 .64 (m, 50H, -CH ₂ OCH ₂ -).

¹ C C 1 H NMR (CDCl 3, δ, ppm): 70.43 (-CH 2 OCH 2 -); 70.03 (d, ³ J (PC) = 6.6 Hz, POCH ₂ CH 2 -); 64.57 (d, ² J (PC) = 6.2 Hz, -POCH ₂ CH ₂ -).

¹ H NMR (CDCl 3, δ, ppm): 10.46 (dq, * J (PH) = 715.5 Hz, ³ J (PH) = 9.9 Hz, CH 2 OP (O) (H) O CH 2); 11.17 (ds, 'j (PH) = 708.2 Hz, ³ J (PH) = 10.5 Hz, -CH2O (O) P (H) OCH ₃ ); 8.29 (dt, * J (PH) = 691.3 Hz, ³ J (PH) = 10.97 Hz; -CH ₂ O (O) P (H) OH).

Complex Illa [{mpawc-PtCl (NH ₃ ) ₂ } ₂ {p-mp6ZHc-Pt (NH ₃ ) ₂ (H ₂ N (CH ₂ ) ₆ (NH ₂ )}] ⁴⁺ (BBR3464) was synthesized according to the procedure described in [4].

1 H NMR (D ₂ O) d (ppm): 2.8 ppm (4H, m, 2NH ₂ -CH 2), 1.8 ppm (2H, m, 2NH ₂ -CH ₂ -CH 2) and 1.5 ppm (4H, m, - CH ₂ -CH ₂ -).

¹⁹⁵ Pt (D ₂ O) d (ppm): -2409 ppm; -2660 ppm.

Complex IIIb [{m /? Ans-P1Cl (HH ₃ ) ₂ } ₂ {p-spermidine-H ¹ , H ⁸ }] ³⁺ (BBR3571) was synthesized according to the procedure described in [9].

1 H NMR (D ₂ O), δ (ppm): 2.97 (2t, SCHINDSH 2); 2.66 (2t, PtNH ₂ CH2-); 1.97 (q, NH ₂ ⁺ CH 2 CH 2 CH 2 NH 2); 1.63 (m, NH 2 ⁺ CH _{2 Cl 2 CH 2 CH 2} NH ₂ );

¹⁹⁵ Pt (D ₂ O) δ (ppm): -2434 ppm.

1S * * * * * ·· «. **’ · * · * «*« ·

Complex III [{mpaHc-Pt (Cl) NH ₃ } {m / 9 «Hc-Pt (NH ₃ ) H ₂ N (CH ₂ ) ₆ NH ₃ } {pspermidine-№, K ⁸ }] ⁵⁺ was synthesized by a procedure similar to that described in [9].

1 H NMR (D ₂ O), δ (ppm): 3.42-3.36 (m, 4bCH ₂ N (NH ₃ ) ₂ PtNH ₂ CH 2 -); 3.25 (t, NH ₃ ⁺ CH 2 -); 3.08-2.91 (Cl (NH3) 2PtNH2CH2- and CibNH2ab); 2.36 (q, ΝΗ / ΟΤΟΙ ^^ ΝΗι); 2.03-2.0 (m, NH 2 ⁺ CH 2 CH 2 CH 2 CH 2 NH 2); 1.93-1.89 (m, NH ₃ ⁺ CH ₂ CH2CH ₂ CH ₂ CH2CH ₂ NH _2); 1.65-1.62 (m, HH + CH 2 CH 3 CH 2 CH 2 CH 2;

^{, 95} Pt (D ₂ O) δ (ppm): -2404 and -2655.

Triethylamine (Fluka) is distilled before use.

The solvents tetrachloromethane, dichloromethane and acetonitrile were dried over phosphorous pentoxide and distilled before use.

Dialysis membranes with pore size MWCO 1000 and 3,500 were purchased from SpectraPor.

Methods' H, ¹³ C {H}, ³¹ P, ¹⁹⁵ Pt NMR spectra and DOSY spectra were recorded on a Bruker 600 MHz apparatus in a deuterated solvent (CDCl ₃ or D ₂ O), the latter being indicated for each specific measurement.

^!! H, ¹³ C {H}, ³¹ P, ¹⁹⁵ Pt NMR spectra were used to determine the structure and composition of the corresponding product, as well as to monitor the removal of reaction by-products.

DOSY spectra were used to demonstrate the formation of intermolecular associations as well as to determine diffusion coefficients.

Assuming that the particles are spherical in shape is calculated fines in the equation of Stokes-Einstein: Rh = kT / (67ir | D), where R _h is the hydrodynamic radius, k is the constant of Boltzmann (1.38065χ10 ^'23 m ² kgs' ² K ''), η is the viscosity of the solvent at the corresponding temperature (p (D ₂ O) = 0.83675 Pa.s at 310 K), T = 310 K and D (m ² s ^-1 ) is the diffusion coefficient at the corresponding concentration .

The measurements by the method of dynamic light scattering were made on a Brookhaven BI-200 device at a wavelength of 2 = 632.8 ps with a He-Ne laser source and a digital autocorrelator Brookhaven BI-9000 AT.

': ·::: ·; . · *

The experiments were performed at 37 ° C and a reflected light angle of 30 °.

The experimental data were processed with CONTIN software. The size of the particles and their size distribution are determined.

Example 1

Preparation of poly (hydroxyoxyethylene phosphate) of formula (II), wherein R = (CH ₂ CH ₂ O) ₄ , R 1 = H or CH ₃ .

Place dichloromethane (25 ml) and carbon tetrachloride (5 ml) in a three-necked flask equipped with a reflux condenser, thermometer, inert gas inlet and magnetic stirrer. Solutions of poly (oxyethylene H-phosphonate) (2,551 g, 0.0104 mol monomer units) in 10 ml acetonitrile and triethylamine (1.45 ml, 0.0104 mol) and distilled water (0.19 ml, 0.0104) were added dropwise with continuous stirring under an inert atmosphere. mol) in 5 ml of acetonitrile. The reaction mixture thus obtained was stirred for 24 hours at room temperature and an inert atmosphere.

about

The dichloromethane was evaporated and the solution was cooled to -12 C. The separated triethylamine hydrochloride crystals were filtered. The solvents are evaporated. The resulting product was dissolved in water and passed through an ion exchange column. After dialysis against deionized water, the product is isolated by lyophilization. Conversion rate 100% (according to MRI data). Product yield 80%. The structure of the obtained poly (hydroxyoxyethylene phosphate) (II) was detected by NMR spectroscopy (Figures 1 and 2).

1 H NMR (D ₂ O), δ (ppm): 4.00-3.89 (m, 4H, CO 2, 3.60-3.50 (m, 12H, CO 2);

³¹ P {H} NMR (D ₂ O), δ (ppm): 1.24,1.08 (phosphate structures);

¹³ C {H} NMR (D 2 O), δ (ppm): 70.43 (O 120 CH ₂ ^{), 70.03 (d, 3} Jm = 5.8 Hz, POCH ₂ CH 2), 64.57 (d, ² J ( _P , c) = 6.2 Hz , POCH ₂ CH ₂ );

IR (KBr): 1291 cm- ¹ and 1259 cm- ¹ v (P = O), 1102 cm- ¹ v (H2C-O-CH2), 1036 cm- ¹ v (POCH2).

From the starting poly (oxyethylene H-phosphonate) and two products Pa and IIb were obtained, on which the diffusion coefficients and the hydrodynamic radii were determined (Figures 3 and 4, Table 1).

Example 2

Preparation of polyelectrolyte associate Va

Complex IIIa (0.026 g, 0.02 mmol) was dissolved in 0.5 ml of water. Poly (hydroxyoxyethylene phosphate) Pa (0.045 g, 0.18 mmol monomer units) was dissolved in 1.0 ml of water. The two solutions were mixed and the pH of the medium was raised to 5.5 by the addition of 0.1N NaOH. Final volume of the solution 2.4 ml. The solution thus obtained was stirred for 18 hours and then dialyzed against deionized water for 24 hours and changed three times for deionized water using a 1000 Da permeability membrane. The polyone associate Va is isolated by evaporating the water under reduced pressure. The amount of isolated product was 0.050 g (71%).

Example 3

Preparation of polyelectrolyte associate Vb

The polyelectrolyte associate Vb was prepared as described in Example 2 using poly (hydroxyoxyethylene phosphate) IIb. Yield of the isolated product 73%.

Example 4

Preparation of polyelectrolyte associate Via

Complex IIIb (0.035 g, 0.045 mmol) was dissolved in 1.5 ml of water. Poly (hydroxyoxyethylene phosphate) IIb (0.070 g, 0.27 mmol monomer units) was dissolved in 1.0 ml of water. The two solutions were mixed and the pH of the medium was raised to 5.5 by the addition of 0.1N NaOH. Final volume of the solution is 3.5 ml. The solution thus obtained was stirred for 11h and then dialyzed against deionized water for 24 hours and the deionized water changed three times using a membrane having a permeability of 1000 Da. The polyelectrolyte associate Via is isolated by evaporating the water under reduced pressure. The amount of isolated product was 0.069 g (69%).

• «>

Example 5

Preparation of polyelectrolyte associate VIb

Polyelectrolyte Associate VIb was prepared as described in Example 4 using a different ratio of reagents: complex IIIb (0.025 g, 0.032 mmol) dissolved in 1.0 ml water and polymer Pb (0.100 g, 0.38 mmol monomer units) dissolved in 1.0 ml of water. Volume of solution before dialysis - 4.2 ml. The amount of isolated product was 0.094 g (77%).

Example 6

Preparation of polyelectrolyte associate VII

Polyelectrolyte Associate VII was prepared as described in Example 4 using complex IIIc (0.035 g, 0.038 mmol) dissolved in 2.0 ml of water and polymer IIb (0.100 g, 0.38 mmol monomer units) dissolved in 1.0 ml of water. Volume of solution before dialysis - 4.5 ml. The amount of isolated product was 0.097 g (76%).

The composition of the resulting polyelectrolyte associates Va, Vb, Via, VIb or VII is given in Table 1.

Example 7

Preparation of nanocolloid solutions from polyelectrolyte associates

To a certain amount of polyelectrolyte associate Va, Vb, Via, VIb or VII, distilled and filtered (pore size 0.02 pm) water was added to obtain a solution with a concentration of 10 mg / ml. After stirring the system for 24 hours, the particles were characterized by DOSY NMR spectroscopy and the light scattering method (Figures 5 to 14).

Table 1. Composition of polyelectrolyte associates and particle sizes in aqueous solution at 37 ° C and concentration 10 mg / ml.

Product Composition * Mall. Relation to [polymeric structural units]: [platinum complex] Results of the DOSY experiment Light scattering measurement results DxlO ^llJ [m7s] R _h [nm] Rh [nm] DI ** Well - 0.38 6.96 - - ПЬ - 0.98 2.77 - - Sha - 4.16 0.65 - - Va 8.0: 1 0.39 6.96 7.11 0.19 Vb 5.1: 1 1.57 1.73 1.77 0.16 Via 4.6: 1 1.35 2.01 2.20 0.15 VIb 11.2: 1 1.78 1.53 1.68 0.08 VII 7.9: 1 1.58 1.72 1.70 0.24

* Determined by 1 H NMR spectra;

** Size distribution

Example 8

The cytotoxicity of the complexes III, IIIb, IIIc and of the polyelectrolyte associates Va, Vb, Via, VIb and VII were determined by the standard MTT test [39] on a spectrum of human tumor cell lines: HL-60 (acute myeloid leukemia, chemosensitive); HL-60 / CDDP (acute myeloid leukemia, with induced cisplatin resistance); BV-173 (chronic myeloid leukemia); MDA-MB-231 (mammary adenocarcinoma).

The cell suspension with a density of 1.5-3.10 ⁵ cells / ml (HL-60, HL-60 / CDDP, BV173) or 2.5.10 ⁴ cells / ml (MDA-MB-231) was instilled into 96-well plates (100 μΐ / pit). After 24 hours of incubation, the cells are treated with serial dilutions of the test complexes or the corresponding polyelectrolyte associates and are::: ·::: ·: · ··· ....... .......

incubate for 72 hours under standard conditions - humidified atmosphere with 5% CO ₂ at 37 ° C. At the end of the treatment period, 10 μΐ sterile MTT solution (5 mg / ml in phosphate buffer) was added to each well and the plates were re-incubated at 37 ° C for 4 hours. The resulting MTT-formazan crystals were dissolved by adding 110 μΐ 5% formic acid in 2-propanol to each well and stirring vigorously with a 12-channel pipette. Spectrophotometric readings of MTT-formazan absorption were performed at 580 nm using a universal microplate reader (Beckman-Coulter DTX800). By means of nonlinear regression analysis of the MTT data, sigmoidal “concentration / effect” curves were constructed (Figures 15 to 26) and the corresponding equivalent 1C ₅₀ values were determined, summarized in Table 2.

Table 2. Cytotoxic effects of complexes III, IIIb, and IIIc compared to the polyelectrolyte associates Va, Vb, Via, VIb and VII on a spectrum of human tumor cell lines.

Compounds IC50 (gmol / L) HL-60 ¹ HL-60 / CDDP ² BV-173 ³ MDA-MB-231 ⁴ Sha complex 5.2 ± 1.1 10.4 ± 0.6 9.6 ± 1.2 5.2 ± 1.1 Polyelectrolyte Associate Va 1.8 ± 1.0 8.6 ± 1.0 2.1 ± 1.3 1.9 ± 1.4 Polyelectrolyte Associate Vb 2.9 ± 1.1 5.9 ± 1.1 6.2 ± 1.1 3.3 ± 1.3 Complex ШЬ 4.4 ± 1.0 14.8 ± 1.2 3.7 ± 0.8 20.2 ± 2.0 Polyelectrolyte Associate Via 2.1 ± 0.7 6.4 ± 1.4 2.3 ± 1.1 11.9 ± 1.9 Polyelectrolyte Associate VIb 2.9 ± 0.9 7.9 ± 1.2 2.8 ± 0.7 14.4 ± 2.1 Complex Shs 17.1 ± 1.3 23.1 ± 2.0 27.2 ± 3.1 40.9 ± 3.2 Polyelectrolyte Associate VII 10.2 ± 2.2 12.6 ± 1.3 14.3 ± 1.7 20.4 ± 2.6

'acute myeloid leukemia;

acute myeloid leukemia with induced cisplatin resistance;

with chronic myeloid leukemia;

⁴ adenocarcinoma of the mammary gland

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Claims (6)

1. Nano-sized polyelectrolyte associates of general formula (IV) ZK | O O - P — OR L, XII, Η, X, L | I '/ j> / ll _t \ hi, IV where, R - alkylene, oxyalkylene R 1 is H, CH ₃ or C ₂ H ₂ X - (CH ₂ ) b; (CH ₂ ) ₃ NH ₂ (CH ₂ ) ₄ L - Cl L - Cl; NH ₃ ; NH ₂ (CH ₂ ) ₆ NH ₃ ⁺ ; NH ₂ (CH ₂ ) ₆ NH ₂ Pt (NH ₃ ) ₂ Cl p: y - from 4: 1 to 12: 1 z = p - yn + 1 Process for the preparation of nanosized polyelectrolyte associates, characterized in that an aqueous solution of negatively charged poly (hydroxyalkyl phosphate) (II) is mixed with an aqueous solution of a polynuclear complex of platinum P is a positive charge η> 3 (III), at pH <7 and an initial charge ratio of the two components of 1: 1 to 4: 1, after which the reaction mixture was stirred for 10 to 20 hours at room temperature, dialyzed against deionized water and isolated polyelectrolyte associate (IV) was molarly ratio of phosphate groups of poly (hydroxyalkylene phosphate) (II) to the polynuclear complex of platinumP (III) from 4: 1 to 12: 1, followed by conversion of the isolated polyelectrolyte associate (IV) into nanoscale by dispersion in aqueous medium and stabilization of the nanocolloid system at a temperature of 20 to 37 ° C. 24::: · ·:: · · + • · · · · ··· · · ··· ♦ «.. Method according to claim 2, characterized in that the isolation of the polyelectrolyte associate (IV) is carried out by evaporating the water at low pressure and a temperature <30 ° C. Method according to claim 2, characterized in that the isolation of the polyelectrolyte associate (IV) is carried out by lyophilization. 5. Nano-sized polyelectrolyte associate (IV) for use as a medicament. 6. Nano-sized polyelectrolyte associate (IV) for use in the treatment of acute myeloid leukemia; acute myeloid leukemia with induced cisplatin resistance; chronic myeloid leukemia; adenocarcinoma of the mammary gland.