RU2742196C1 - Pharmaceutical composition for preparing injection solution when used in treating magnetic hyperthermia and method for preparing thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for preparing a solution for injections used in treating oncological diseases by magnetic hyperthermia and a method for preparing it. Essence of the invention is that the pharmaceutical composition for preparing the injection solution when used in treating magnetic hyperthermia is a lyophilisate of a solution of magnetic nanoparticles of cobalt ferrite (CoFe₂O₄) with size of not more than 20 nm, coated with sorbitol molecules, with following ratio of components, wt. %: cobalt ferrite 70–75, sorbitol 25–30.

EFFECT: technical result: enabling re-dissolution of the declared pharmaceutical composition and repeated drying with preservation of its stability and pharmacological properties, longer storage life of the solution prepared from the declared pharmaceutical composition.

8 cl, 6 tbl, 37 dwg

Description

The technical field to which the invention relates

The claimed invention relates to the field of chemistry and medicine, namely, to a new pharmaceutical composition for the preparation of a solution for injection used in the treatment of cancer by the method of magnetic hyperthermia and a method for its production.

State of the art

One of the promising directions for the development of hyperthermia is the creation of targeted ferromagnetic drugs [Motoyama J., Yamashita N., Morino T., Tanaka M., Kobayashi T. and Honda H. Hyperthermic treatment of DMBA-induced rat mammary cancer using magnetic nanoparticles. Biomagn Res Technol, 2008, 6, 2], containing magnetic nanoparticles (MNP), followed by treatment of the tumor with an alternating magnetic field. Due to physical phenomena, the particles are heated, raising the temperature of the tumor tissue to 43-47 ° C.

The disadvantages of the known magnetic particles are the difficulty of obtaining nanoparticles of the same size and composition, their low stability in solution, the need for a shell to increase biocompatibility.

After a series of studies [Gilchrist RK, Medal R., Shorey WD, Hanselman RC, Parrott JC, Taylor CB Selective inductive heating of lymph nodes. Ann. Surg., 1957, 146, 596-606], which first demonstrated the use of magnetite nanoparticles (Fe ₃ O ₄ ) for magnetic hyperthermia, various groups of researchers clinically tested them for the treatment of patients with brain tumors. It is well known that magnetic hyperthermia using ferrites makes a promising therapeutic method for treating malignant tumors upon excitation by an alternating current field [Lee JH, Jang JT, Choi JS, Moon SH, Noh SH, Kim JW, Kim JG, Kim IS, Park KI, Cheon J. Nat Nanotechnol, 2011, 6 (7), 418-422]. The ease of use of iron oxide nanoparticles in this regard is quite high [Parsian M., Unsoy G., Mutlu P., Yalcin S., Tezcaner A., Gunduz U. Loading of gemcitabine on chitosan magnetic nanoparticles increases the anticancer efficacy of the drug. Eur J Pharmacol. 2016. 784, 121-128].

There are also disadvantages in their use, which consist in the complexity of the synthesis of nanoparticles of a certain composition, without impurities of other iron oxides and with a narrow size distribution, which, in turn, negatively affects the magnetic properties of such nanoparticles.

From publication EP1883425, injectable superparamagnetic nanoparticles for hyperthermia treatment are known, which are nanoparticles of maghemite, magnetite, or a mixture thereof, having an average diameter of not more than 20 nm, immobilized in silica granules, which preferably have an average diameter in the range from 20 nm to 1 μm ... These particles are dissolved in any solution of the precipitating polymer in a water miscible solvent.

The main disadvantage of such particles is the ambiguous magnetic properties of a mixture of iron oxides, as well as the impossibility of varying the concentration of the solution without affecting the stability of the suspension of nanoparticles.

In the publication US 6514481B1 disclosed so-called "nanoclinics", which consist of nanoparticles of iron oxide in a shell of silicon dioxide and coated with a targeting agent. It is known that the use of a constant magnetic field destroys target cells through magnetically induced lysis - in contrast to the heat generated in an alternative magnetic field.

US Pat. No. 6,541,039 (B1) discloses iron oxide particles enclosed in at least two shells. The outer shell, which has neutral and / or anionic groups, provides an appropriate distribution in the tumor tissue. The inner shell contains cationic groups that promote adsorption / absorption by cells. Nanoparticles are injected in the form of a suspension ("magnetic fluid").

However, the known particles during use do not allow reaching a controlled temperature at moderate temperatures in a certain volume and repeating the heating procedure in a certain volume without reintroducing the composition.

According to the publication of the Russian Federation No. 2633918 for the treatment of malignant neoplasms using magnetic hyperthermia with known Zn-substituted magnetic nanoparticles based on manganese ferrite of the formula Zn _x Mn _1-x Fe ₂ O ₄ (x = 0-0.9), the average size of which is less 40 nm, the Curie temperature is from 39 to 550 ° C, and the coercive force is from 5 to 250 E. Nanoparticles can be a biocompatible polymer material, which is polyethylene glycol (PEG), polypropylene, dextran, polyvinyl difluoride (PVDF), polyvinylpyrrolidone (PVP ) or polyacrylic acid.

These coatings provide high biocompatibility and increase the circulation time of nanoparticles in the body, but, according to studies, do not increase the shelf life of drugs. The service life of such magnetic fluids usually does not exceed two months, which leads to the need to constantly synthesize new batches of the drug in limited quantities.

JP 2013256405 discloses Ni-Zn ferrite-based magnetic nanoparticles having a shell containing amorphous SiO ₂ , the average particle size being 10-20 nm. These nanoparticles have a high manufacturing cost and low heat generation temperature, which limits their use in hyperthermia [Bae ST, Chung KW. Method for preparing engineered Mg doped ferrite superparamagnetic nanoparticle exhibiting AC magnetic induction heating at high temperature and Mg doped ferrite superparamagnetic nanoparticles engineered by the method (2011)]. In addition, the shelf life of such nanoparticles does not exceed several months due to the loss of stability.

Disclosure of invention

Thus, there is a need to develop magnetic nanoparticle compositions with improved time stability and magnetic properties. To realize magnetic hyperthermia as an alternative stand-alone therapeutic procedure for cancer treatment, magnetic nanoparticles with optimal performance within biologically safe limits must be created using simple, reproducible and scalable methods.

The technical problem to be solved by the claimed invention consists in overcoming the disadvantages inherent in analogues of the technical solution, namely, in overcoming the low stability of the solution of nanoparticles after reconstitution of the pharmaceutical substance or during a long shelf life, which as a result leads to the need to manufacture drugs in small batches with a short shelf life.

The technical result achieved when using the claimed invention is to provide the possibility of reconstitution of the claimed pharmaceutical composition and its repeated drying while maintaining its stability and pharmacological properties, increasing the shelf life of the solution prepared from the claimed pharmaceutical composition. The technical advantages of the claimed invention are also

- the possibility of preparing an injection solution with the required concentration of the active substance due to the final form of the product in the form of a lyophilisate-dry powder;

- the osmolarity of the solution for injection is not less than 300 mOsm / l, which corresponds to the osmolarity of the blood plasma.

The technical problem is solved by the fact that the claimed pharmaceutical composition for the preparation of an injection solution when used in the treatment of magnetic hyperthermia, according to the technical solution, is a lyophilisate of a solution of magnetic nanoparticles of cobalt ferrite (CoFe ₂ O ₄ ) with a size of no more than 20 nm, coated with sorbitol molecules, with the following ratio of components, wt%:

cobalt ferrite 70-75 sorbitol 25-30

The average diameter of these magnetic nanoparticles is 16 nm.

The technical problem is also solved by the fact that the method of obtaining the claimed pharmaceutical composition is characterized by the sequential execution of the following steps:

a) a suspension of magnetic nanoparticles of cobalt ferrite is prepared by adding aqueous solutions of iron (III) chloride (FeCl ₃ ) and cobalt dichloride hexahydrate (CoCl ₂ ⋅ 6H ₂ O) to a boiling solution of sodium hydroxide (NaOH) with constant stirring and further cooling the mixture to room temperature temperature;

b) nanoparticles of cobalt ferrite in the form of a precipitate in the suspension is collected using a permanent magnet, after which the supernatant liquid is decanted;

c) an aqueous solution of cobalt ferrite nanoparticles with pH 2-3 is prepared by adding deionized water to the resulting precipitate.

d) stabilize the surface of nanoparticles in solution with citrate ions;

e) adjust the pH of the solution to 7.3-7.4 by adding dropwise with continuous stirring and constant monitoring of the pH level, the sodium hydroxide solution;

f) the nanoparticle solution obtained in step f) is washed using centrifugal filters with a pore size of not more than 100 kDaltons.

g) sorbitol is added to the obtained aqueous solution of cobalt ferrite nanoparticles in an amount of 50-60 mg for every 100 ml of cobalt ferrite solution;

h) the resulting solution with an osmolarity of not more than 300 mOsm / l is filtered, followed by lyophilization at a residual pressure of not less than 0.64 mbar.

In step a), an aqueous solution of iron (III) chloride (FeCl ₃ ) at a concentration of 81 g / l, cobalt (II) chloride hexahydrate (CoCl ₂ ⋅6H ₂ O) at a concentration of 860 g / l, sodium hydroxide solution (NaOH) at a concentration of 1 mol / l, while for the preparation of the suspension the components are taken in the following ratio, wt%:

iron (III) chloride (FeCl ₃ ) 25 cobalt dichloride (CoCl ₂ ) eighteen sodium hydroxide solution (NaOH) 57

In step b) and c), wash the precipitate with deionized water 2-3 times. In step d), to form citrate ions on the surface of the particles, 0.01M citric acid solution and 1M sodium hydroxide solution are added to an aqueous solution of cobalt ferrite, while 80 ml are taken for 40 ml of cobalt ferrite solution, and 1.3 ml of these components, respectively ... These magnetic nanoparticles are Co-substituted magnetic nanoparticles based on magnetite of the formula CoFe ₂ O ₄ . After step b), additional oxidation of the nanoparticle surface can be carried out, for which

- to the obtained nanoparticles of cobalt ferrite, add solutions of nitric acid at a concentration of 2 mol / l and nanohydrate of iron (III) nitrate with a concentration of 85 mg / ml in a volume of 30 ml each for 40 ml of nanoparticle solution and boil with constant stirring, followed by cooling the mixture to room temperature temperature,

- after which the precipitate of cobalt ferrite nanoparticles obtained as a result of synthesis is collected at the bottom of the flask using a permanent magnet, and the resulting supernatant liquid is decanted, followed by washing the resulting precipitate with nitric acid.

The claimed technical result is achieved due to the use of cobalt ferrite nanoparticles in the composition and their magnetic properties. The special advantages of cobalt ferrite MNPs are that Co in the +2 state and Fe in the +3 state are highly stable, and air oxidation usually does not occur in such materials. To overcome some of the limitations, CoFe ₂ O ₄ nanocubes were synthesized and their magnetic properties were studied for promising methods for the treatment of hyperthermia and drug delivery [Manuchehrabadi N., Gao Z., Zhang, J. et al. Improved tissue cryopreservation using inductive heating of magnetic nanoparticles. Sci Transl Med, 2017, 9 (379)]. Cobalt ferrites are one of the most promising materials due to their high magnetization capacity. They have a partially reverse spinel structure, where both cationic sublattices are occupied by manganese / cobalt and iron ions. To create an ideal hyperthermal agent, it is important to modulate the structural and magnetic properties of nanoparticles. One of the approaches to increasing the stability and performance of nanoparticles is the use of a surface coating on a magnetic nanoparticle.

Brief Description of Drawings

The claimed invention is illustrated by the following drawings and images.

Figure 1 shows a photo of cobalt ferrite nanoparticles obtained by coprecipitation of salts during boiling.

Figure 2a-b shows a chemical scheme for the synthesis of the claimed pharmaceutical composition.

FIG. 3 presented

(A) Nanoparticle solution after adding deionized water,

(B) Sonication in an ultrasonic bath of a solution of cobalt ferrite nanoparticles in deionized water.

Figure 4 presents

(A) A solution of cobalt ferrite nanoparticles after redispersion in deionized water,

(B) Sediment of cobalt ferrite nanoparticles obtained after adding 0.01 M citric acid solution,

(B) Adding 1M sodium hydroxyl solution and raising the pH to 7.4.

Figure 5 shows a centrifugal filter with nanoparticles of cobalt ferrite after centrifugation.

Figure 6 shows the preparation of a lyophilisate of cobalt ferrite nanoparticles.

Figure 7 shows a lyophilisate of nanoparticles in a sterile vial.

Figure 8 shows an X-ray diffraction pattern of cobalt ferrite.

Figure 9 shows the results of measuring the hydrodynamic size of nanoparticles by dynamic light scattering (DLS).

Figure 10 is a transmission electron microscopy image (left) and an atomic scanning microscopy (AFM) image of the nanoparticles.

Figure 11 shows a graph of the dependence of the signal intensity on the hydrodynamic size of nanoparticles of cobalt ferrite from the 1st day of testing.

Figure 12 presents a graph of the dependence of the signal intensity on the hydrodynamic size of nanoparticles of cobalt ferrite from the 10th day of testing.

Figure 13 presents a graph of the dependence of the signal intensity on the hydrodynamic size of nanoparticles of cobalt ferrite from the 20th day of testing.

On Fig presents a histogram of the distribution of nanoparticles in height for the sample from the 1st day of testing.

Figure 15 shows an atomic scanning microscopy (AFM) image of nanoparticles from day 1 of testing.

On Fig presents a histogram of the distribution of nanoparticles in height for the sample from the 10th day of testing.

Figure 17 shows an atomic scanning microscopy (AFM) image of nanoparticles from day 10 of testing.

On Fig presents a histogram of the distribution of nanoparticles in height for the sample from the 20th day of testing.

Figure 19 shows an atomic scanning microscopy (AFM) image of nanoparticles from day 20 of testing.

Figure 20 shows the results of X-ray diffraction analysis of a sample of nanoparticles.

FIG. 21 shows a control photo of an intratumoral injection of MNPs on a tumor preparation.

FIG. 22 shows the main stages of a hyperthermia session:

A - Intratumoral administration of particles

B - The mouse in the holder is placed in an alternating magnetic field generator

B - Thermal imager attached to a smartphone aimed at the tumor

D - Measurement of tumor temperature in real time.

Figure 23 is a graph showing the growth of individual tumors in group 1 (control).

On Fig presents a graph of the growth of individual tumors in group 2 (MNP without magnetic field).

Figure 25 shows a graph of the growth of individual tumors in group 3 (MNP + alternating magnetic field).

On Fig presents a graph of the growth of individual tumors in group 4 (cisplatin).

On Fig presents a graph of the growth of individual tumors in group 5 (MNP + alternating magnetic field + cisplatin).

On Fig presents a graph of the growth of tumors in the study groups 1-5.

On Fig presents a graph of survival in study groups 1-5.

Figure 20 is a graph of tumor growth in Group 4 and effectively warmed tumors in Group 5.

On Fig presents a graph of changes in body weight in the study groups 1-5.

Figure 32 is a representative photo of tumors in animals treated and not treated with hyperthermia (12th day after initiation of therapy).

Figure 33 is a graph showing the growth of individual CT-26 tumors in group 1 (control).

Figure 34 is a graph showing the growth of individual CT-26 tumors in group 2 (sorbitol).

Figure 35 shows a graph of the growth of individual CT-26 tumors in group 3 (MNP without field).

Figure 36 is a graph of the growth of individual CT-26 tumors in group 4 (MNP + field).

On Fig presents a graph of changes in the volume of tumors on average in each of the groups.

Implementation of the invention

The method of obtaining the claimed pharmaceutical composition is described below using an example of a specific implementation, providing 1.5 g of the claimed lyophilisate of the pharmaceutical composition (figure 1). The implementation of the method is possible for any technologically acceptable volumes, with a proportional increase in the amount of all components of the synthesis. Under laboratory conditions, in a maximum of one cycle, a pharmaceutical composition was obtained in an amount of 15 g with a tenfold loading of the components (Figs. 2a, 2b).

To obtain the claimed pharmaceutical composition, the following reagents and materials were used:

anhydrous iron (III) chloride (FeCl ₃ , 97%), cobalt (II) chloride hexahydrate (CoCl ₂ × 6H ₂ O, 97%), iron (III) nitrate nonahydrate (Fe (NO ₃ ) ₃ × 9H ₂ O, ≥98.0%), hydrochloric acid (HCl, 36%), nitric acid (HNO ₃ ; ≥65.0%), sodium hydroxide (NaOH, ≥98.0%) and citric acid (C ₆ H ₈ O ₇ , 99%) were purchased from Sigma-Aldrich, Sorbitol (C ₆ H ₁₄ O ₆ , ≥98.0%) was purchased from Sigma-Aldrich. Deionized water was produced using the Millipore Milli-Q Academic System. All reagents were used further without any additional purification. All laboratory glassware used for the reactions was washed with hot aqua regia (HCl (36%) / HNO ₃ (65%) = 3/1) and then with deionized water.

1. Preparation of salt solutions

A 50 ml glass beaker is charged with 3.24 g of iron (III) chloride, and 40 ml of deionized water is added with continuous stirring on a magnetic stirrer, after which the solution is heated to a temperature of 50 ° C.

Similarly, 4.30 g of cobalt (II) chloride hexahydrate is loaded into a 10 ml glass beaker, and a solution of 1 ml of hydrochloric acid in 4 ml of deionized water is added with continuous stirring on a magnetic stirrer, and then heated to a temperature of 50 ° C. After that, the resulting salt solutions are mixed.

2. Preparation of a suspension containing nanoparticles of cobalt ferrite

Then, 8.12 g of sodium hydroxide is placed in a three-necked flask with a volume of 1 liter, and 203 ml of deionized water is added with continuous stirring on a magnetic stirrer to prepare 1M sodium hydroxide solution. The resulting solution is heated to boiling, and then the prepared salt solution is added dropwise to it. As a result, the color of the solution changes to dark brown and then to black, which indicates the progress of the reaction and the formation of nanoparticles of cobalt ferrite.

3. Decanting

The cobalt ferrite nanoparticles obtained after synthesis are collected at the bottom of the flask using a permanent magnet, and the supernatant is decanted. The remaining precipitate is washed with deionized water (3 x 250 ml). Further, the resulting precipitate is dissolved in 30 ml of 2M nitric acid solution and then 30 ml of 0.35M solution of iron (III) nitrate nonahydrate is added. The resulting solution was heated to 100 ° C and stirred for 1 hour. After the reaction was completed, the solution was cooled to room temperature by removing the heating source, and the nanoparticles were collected at the bottom of the flask using a permanent magnet. The supernatant liquid is decanted, and the formed precipitate is washed with 2M nitric acid solution (2 × 300 ml). After washing, the nanoparticles are redispersed in 40 ml of deionized water (Fig. 3, 4A).

1. Stabilization of nanoparticles

To give stability to nanoparticles under physiological conditions (pH = 7.4), their surface is coated with citrate ions. For this, 40 ml of 0.01M citric acid solution is added to 40 ml of an aqueous solution of nanoparticles, which leads to their precipitation (Fig. 4B). Then add dropwise 1.3 ml of 1M sodium hydroxide solution, increasing the pH of the solution from 2.3 to 7.4, which leads to the coating of nanoparticles with citrate ions and stabilization (Fig.4B). The resulting solution of nanoparticles is poured into centrifugal filters (6 pieces) of 100 kDa, equilibrated on a balance and centrifuged for 20 min at 6000 rpm (Fig. 5). The lower pale layer is discarded. To the sediment remaining on the filters, add 20 ml of a solution for washing nanoparticles, equilibrate on a balance, and centrifuge again for 20 min at 6000 rpm. The above procedure is repeated twice. The formed precipitate is redispersed in 10 ml of deionized water and the resulting solution is collected with a pipette into a 15 ml plastic tube, after which it is sonicated in an ultrasonic bath for 5 minutes. The concentration of cobalt ferrite nanoparticles is determined using atomic emission spectroscopy by measuring the intensity of the emission line of the test substance and calibration standards with a known concentration of iron and cobalt.

2. Coating with sorbitol

The sample is diluted with a sorbitol solution (300 mOsm / L) so that the concentration values fall within the range of 5-50 μg / ml. The iron concentration in the test sample was 48.6 μg / ml.

Determination of the hydrodynamic diameter of cobalt ferrite nanoparticles is carried out using the method of dynamic light scattering (Fig. 9). The concentration of the dispersed phase of nanoparticles in the measuring cuvette during the measurement is 40 μg / ml. When preparing samples, the sample is filtered through a filter with a pore diameter of 0.22 µm to separate small aggregates, dust and particulates. Next, 3 measurements of the hydrodynamic diameter of cobalt ferrite nanoparticles are carried out. As can be seen from the graphs (Fig. 10, 11), the average value of the hydrodynamic diameter is 45 nm.

1. Lyophilization

The resulting solution is lyophilized; for this, the nanoparticle solution is filtered through a syringe filter with a pore size of 0.22 μm, and then frozen at a temperature of -20 ° C.

The vial with the frozen solution is placed in a 1 L flask fixed to a lyophilizer (Fig. 6). Lyophilization is carried out at a residual pressure of 0.64 mbar for 2 hours.

2. Conducting studies of the composition and morphology of the obtained nanoparticles

To conduct studies of nanoparticles by X-ray phase analysis, 100 mg of the lyophilisate (Fig. 7) is placed in a special cuvette and diffraction patterns are recorded in the θ / 2θ mode with constant time at a point using CoKα radiation (Fig. 8). The positions of the 3 most intense diffraction peaks of CoFe ₂ O ₄ correspond to the following diffraction angles:

Thus, the data obtained by X-ray phase analysis confirm the fact that the nanoparticles obtained contain pure cobalt ferrite.

To determine the morphology of nanoparticles, atomic force microscopy is used, in which the linear dimensions of the particles are determined. The concentration of nanoparticles in the test sample during the measurement is 40 μg / ml. FIG. 15, 17, 19 are photomicrographs obtained by atomic force microscopy and the corresponding histograms of the nanoparticle size distribution (Fig. 14, 16, 18). The average nanoparticle size was 16 nm.

The therapeutic activity of controlled hyperthermia using the claimed pharmaceutical composition was carried out on two tumor models:

the 4T1 mouse breast cancer model, a highly aggressive model characterized by resistance to chemotherapy and a high rate of metastasis;

the CT-26 mouse rectal cancer model, a widely used immunogenic tumor that does not exhibit spontaneous metastases.

The following parameters were chosen for the therapy:

1. Variable magnetic field parameters in the range from 220 kHz-10 mT to 260 kHz-24 mT.

2. The concentration of the drug is 90 mg / ml.

3. The volume of intratumoral injection is 50 µl.

4. The duration of the hyperthermia session is 30 minutes.

5. The number of sessions of hyperthermia - threefold hyperthermia with an interval of 24 hours for modes 260 kHz-24 mT / 390 kHz-16 mT; single hyperthermia for 220 kHz-10 mT.

In addition, a comparison was made of the temperature detected by the thermal imager from the outside of the tumor (through the skin) and from the inside. For this, the animal under anesthesia was made a skin incision along the spine to isolate a skin fold with a tumor. Next, the temperature of the outer and inner surfaces of the tumor was measured. It turned out that the performance of the thermal imager in both cases does not differ significantly. Thus, it was found that the temperature detection from the outside of the tumor is not distorted by the thermal insulation properties of the skin, and the temperature detected from the outside of the tumor adequately reflects the temperature inside the tumor. In addition, in this series of experiments, the technique of intratumoral injection was developed, which ensures the infiltration of the tumor with magnetic nanoparticles. Thus, in FIG. 21 shows that MNPs are located in and around tumor tissues.

Evaluation of specific antitumor activity of controlled hyperthermia in a 4T1 mouse breast cancer model

In the first experiment, 4T1 tumors were treated under conditions of moderate hyperthermia (mode 220 kHz-10 mT) (Fig. 22).

The day before the start of therapy, 50 animals were divided into 5 groups:

Group 1. Control group - mice not receiving therapy (no treatment, n = 10).

Group 2. Group receiving magnetic nanoparticles without exposure to an alternating magnetic field (NP only, n = 10).

Group 3. Group receiving magnetic nanoparticles of cobalt ferrite with sorbitol, followed by exposure to an alternating magnetic field (NP + AFM; n = 10).

Group 4. Group receiving cisplatin therapy (cis; n = 10).

Group 5. Group receiving magnetic nanoparticles followed by exposure to an alternating magnetic field in combination with chemotherapy with cisplatin (NP + AFM + cis; n = 10).

Group 3 was the main experimental group.

It is known that hyperthermia is capable of exerting both a direct damaging effect on a tumor and an indirect one when used in combination with chemotherapy and radiation therapy. Presumably, improved blood flow in the area of local heating contributes to more efficient accumulation of chemotherapy (Sohail et al., 2017). To study the effectiveness of magnetic hyperthermia in combination therapy, two additional groups were introduced into the experimental design: group 4 received only cisplatin (chemotherapy control), and group 5 received chemotherapy in combination with cisplatin.

50 μl of nanoparticles with a concentration of 90 mg / ml (Co + Fe) were injected.

5 minutes after the injection of nanoparticles, the mouse in a special holder was placed in an alternating magnetic field generator and a controlled hyperthermia session was performed with the following parameters: 220 kHz - 10 mT, session duration - 30 min.

Intravenous administration of cisplatin was performed based on literature data. A dose of 5 mg / kg was chosen, twice with an interval of 4 days.

Evaluation of the efficiency of heating tumor tissues was carried out using a Seek Thermal thermal imager and / or an IR thermometer. To increase the detection efficiency, the area around the tumor was shaved again before the experiment. The temperature of the tumor and adjacent tissues was measured before placing the animals in the magnetic field generator, after 10 min and 30 min in the field. Along with the absolute temperature values, the following parameters were evaluated:

1. The maximum temperature difference in the tumor during 30 minutes of hyperthermia (Δ max swell)

2. The maximum temperature difference in adjacent tissues within 30 minutes of hyperthermia (Δ max skin)

3. The maximum temperature difference between the tumor and adjacent tissues within 30 minutes of hyperthermia (Δ max swollen skin).

Tables 1 and 2 show the data obtained with the thermal imager in groups 2 and 5, respectively. It can be seen from the above data that the body temperature of animals under anesthesia is lower than normal (on average 31 ° C), which is associated with a violation of thermoregulation. It is also seen that, despite the heating of the skin areas adjacent to the tumor, hyperthermia is local in nature with a maximum in the area of intratumoral injection of particles. To assess the temperature difference in the tumor and normal tissues, it is necessary to measure the temperature in the area of the skin distant from the heating focus, which was difficult due to the heat-insulating properties of wool. Based on the thermal imager data, groups with different tumor heating efficiency were identified:

1. Ineffective hyperthermia (T swelling≤37 ° ΔC)

2. Suboptimal hyperthermia (37 ° C <T swelling≤40 ° C)

3. Effective hyperthermia (T swelling> 40 ° C).

Table 1 - Temperature measurement data for tumors and surrounding tissues in group 2 (NP + AFM).

Animal cipher 0 minutes 10 minutes 30 min Δ max swollen Δ max leather Δ max swelling
leather T is swollen T leather T is swollen T leather T is swollen T leather 18-2 31 34 ND ND 31 29.5 -3 -4.5 1.5 19-2 31 31 34 31 31 28.5 3 0 3 20-2 31 31 36 31 33 thirty five 0 five 21-2 thirty thirty 33 29 34 31 4 one 4 22-2 thirty 32 36 33.5 37 34.5 7 2.5 2.5 23-2 31 31 33 29.5 33 30.5 2 -0.5 3.5 24-2 31 31 36 32.5 ND ND five 1.5 3.5 25-2 31 33 38 32 41 36 ten 3 five 26-2 31 29 40 35 38 34 9 6 five 27-2 thirty 27 37 34 40 34 ten 7 6

Table 2 - Temperature measurement data for tumors and surrounding tissues in group 5 (NP + AFM + cis).

Animal cipher 0 minutes 10 minutes 30 min Δ max swollen Δ max leather Δ max swelling
leather T is swollen T leather T is swollen T leather T is swollen T leather 18-1 31 31 41 35 40 36 9 five 4 18-5 31 32 39 33.5 42 36 eleven five 6 19-5 31 32 41 36 42 37 eleven five five 20-5 32 31.5 45 35 45 37 thirteen 5.5 ten 21-5 thirty 32 41 34 41 36 eleven 4 7 23-5 thirty 31.5 37 33 41 37 eleven 5.5 4 24-5 31 31 41 36 - - ten five five 25-5 thirty 31 38 35 38 36 8 five 3 26-5 31 31.5 37 34 38 35 7 3.5 five 27-5 31 30.5 38 34 39 36 8 5.5 4

Subsequently, to analyze the survival and growth of tumors in groups 2 and 5, subgroups with effective heating of tumors (HT> 40C) were identified: n = 3 in group 2, n = 7 in group 5.

The animal was taken out of the experiment when the area of the measured tumor surface was more than 100 mm ² . Data on the dynamics of growth of individual tumors in groups 1-5 are shown in Fig. 23-28, where the tumor size was calculated using the formula S = A × B.

A pronounced therapeutic effect was observed in the group receiving combination therapy (NP + AFM, Table 3; Starting from the 23rd day after implantation, inhibition of tumor growth was observed compared to the control (p <0.005, ttest) (Fig. 29).

Table 3 - Statistical analysis of differences in survival in groups 1 and 5.

Options Group 1 Group 5 Median survival, days 26 37.5 Log-rank (Mantel-Cox) Test p = 0.0001 Gehan-Breslow-Wilcoxon Test p = 0.0005

If you look at the graph of tumor growth in the first few days after starting therapy 20, you can see a sharp increase in tumor size in this group, as well as in groups 2-3 (no treatment, NP only, NP + AFM). This is due to the fact that intratumoral injection of 50-100 μl of particles creates an additional volume of tissue. In addition, the inflammatory infiltrate that occurs after the tumor is chipped is also contributing. Despite the initial increase in tumor size in the combination therapy group, tumor shrinkage is observed starting from 12-16 days (4-8 days after initiation of therapy). By the 26th day, the size of tumors in this group was lower than with cisplatin monotherapy (p = 0.09). Survival analysis revealed a trend (p = 0.057) for improved survival in this group of mice compared to the group receiving cisplatin monotherapy (Table 4).

Table 4 - Statistical analysis of differences in survival in groups 4 and 5.

Options Group 4 Group 5 Median survival, days 32.5 37.5 Log-rank (Mantel-Cox) Test p = 0.057 Gehan-Breslow-Wilcoxon Test p = 0.084

It should be noted that when analyzing a subgroup (from group 5) with effective heating indicators, the above trend acquires statistical significance (Fig. 30). Thus, starting from the 26th day after tumor implantation, the average size of effectively heated tumors in group 5 was significantly lower than in group 4 (p <0.02).

The therapeutic effect in this group may be associated with the potentiation of the chemotherapeutic effect by hyperthermia. It is known that vasodilation and increased tumor oxygenation promotes more efficient penetration of cisplatin into the tumor (Sohail et al., 2017). It may also be due to the direct effect of hyperthermia, which, as discussed above, in this group reached higher temperatures inside the tumor. Finally, a more pronounced therapeutic effect can be a consequence of the summation of the cytotoxic effect of chemotherapeutic and thermal effects on tumor tissues.

Figure 31 shows data on the change in the weight of animals on average in the five study groups. Cisplatin, both in monotherapy and in combination with hyperthermia, causes a sharp weight loss in the first 10 days after the initiation of therapy (p <0.001), after which the animals regain the lost weight.

Thus, hyperthermia did not lead to an additional aggravation of the side effects well described for cisplatin, while increasing the effectiveness of inhibition of tumor growth.

The low efficiency of hyperthermia as a monotherapy for tumors may be associated with:

1.with a low efficiency of heating tumors at the selected parameters of the alternating magnetic field;

2. with the aggressiveness of the selected tumor model.

Evaluation of specific antitumor activity of controlled hyperthermia in the CT-26 mouse colocarcinoma model

Taking into account the results obtained, the second experiment was carried out on a model of murine colocarcinoma CT-26, using the maximum permissible parameters of an alternating magnetic field - 260 kHz-24 mT and 390 kHz - 16 mT. Also, unlike the first experiment, 3 sessions of hyperthermia were used to achieve a therapeutic effect.

The day before the start of therapy, the animals were divided into 4 groups:

Group 1. Control group - mice not receiving therapy (n = 10).

Group 2. Group receiving intratumoral injection of sorbitol (n = 9).

Group 3. Group receiving intratumoral injection of magnetic nanoparticles as part of the claimed composition without exposure to an alternating magnetic field (n = 10)

Group 4. Group receiving magnetic nanoparticles as part of the claimed composition, followed by exposure to an alternating magnetic field (NP + AFM; n = 15).

50 μl of nanoparticles with a concentration of 90 mg / ml (Co + Fe; groups 3 and 4) or 50 μl of sorbitol (group 2) were injected. 5 minutes (day 0) after the injection of nanoparticles, as well as 24 hours (day 1) and 48 hours (day 2), a session of controlled hyperthermia was performed for 30 minutes. The parameters of the alternating magnetic field were varied (260 kHz - 24 mT, 390 kHz - 16 mT, 390 kHz - 13 mT, 390 kHz - 14 mT, 390 kHz - 15 mT) to maintain the temperature in the tumor in the range of 43-50 ° С.

As in the previous experiment, the initial body temperature of the animals under anesthesia was lower than normal (on average 30 ° C) and on the contralateral skin area of the tumor remained at this level throughout the hyperthermia session. The use of high-energy modes of heating the tumor made it possible to quickly (on average, within 60 s) warm up the tumor to a temperature of 50 ° C and maintain the temperature in the range of 43-50 ° C throughout the hyperthermia. In this case, the 260 kHz-24 mT mode was used for warming up, and the alternation of 390 kHz-16 mT, 390 kHz - 13 mT, 260 kHz-24 mT (depending on the individual characteristics of tumor heating) was used to maintain the temperature. It is important that the target heating values using these modes were achieved for all animals, which significantly differs from the heating efficiency when using moderate hyperthermia. Moreover, the second (Table 5) and third (Table 6) hyperthermia sessions were also effective in terms of warming up tumor tissues. Analysis of literature data indicates that 2- and 3-fold hyperthermia increases the therapeutic effect, but each subsequent session demonstrates less heating of the tumor (Kolosnjaj-Tabi et al., 2014; Espinosa et al., 2016). In our case, repeated sessions of hyperthermia practically did not differ in tumor heating temperature from the first session.

Table 5 - Data of temperature measurements of tumors and surrounding tissues in group 4 during the second session of hyperthermia.

Cell code Animal number tumor Healthy skin Δ max swollen Δ max leather Δ max swollen skin early Max early end v-46 2 29 fifty 32,7 31.5 21 1,2 22.2 v-46 3 thirty 46 29.2 28.9 16 0.3 16.3 v-46 4 32 51 31.7 29.3 19 2.4 21.4 v-46 five thirty fifty 35.1 33.4 20 1.7 21,7 v-47 2 29 55 32 31 26 one 27 v-48 2 29 51 31.6 30.2 22 1.4 23.4 v-48 4 32 52 31 29.1 20 1.9 21.9 v-49 2 thirty fifty 32 30.1 20 1.9 21.9 v-49 4 29 fifty 37.6 35.9 21 1.7 22.7 v-49 five 28 fifty 33.2 31.7 22 1.5 23.5 v-50 one 32 fifty 29.6 28.8 eighteen 0.8 18.8 v-50 3 29 51 30.5 29.2 22 1.3 23.3 v-50 five 31 fifty 32.8 30.6 19 2.2 21.2 v-51 3 29 fifty 31.5 30.1 21 1.4 22.4 v-51 4 29 fifty 31.7 31 21 0.7 21,7

Table 6 - Measurement data of the temperature of tumors and surrounding tissues in group 4 during the third session of hyperthermia.

Cell code Animal number tumor Healthy skin Δ max swollen Δ max leather Δ max swollen skin swollen early Max early end v-46 2 27 fifty 32 30.7 23 1.3 24.3 v-46 3 28.5 48.5 28.5 27.9 20 0.6 20.6 v-46 4 thirty fifty 32.9 27 20 5.9 25.9 v-46 five 28 fifty 32 30.5 22 1.5 23.5 v-47 2 29 fifty 30.3 28.1 21 2.2 23.2 v-48 2 29 fifty 32 28.9 21 3.1 24.1 v-48 4 29 fifty 32,7 30.9 21 1.8 22.8 v-49 2 29 fifty thirty 28.9 21 1.1 22.1 v-49 4 thirty fifty 30.6 27.9 20 2.7 22.7 v-49 five 28 fifty 29 27.5 22 1.5 23.5 v-50 one 29 32 31 29.2 3 1.8 4.8 v-50 3 29 fifty 31 29.4 21 1.6 22.6 v-50 five 29 36 thirty 29.3 7 0.7 7,7 v-51 3 29 38 29 27.8 9 1,2 10.2 v-51 4 29 fifty 31 28.7 21 2,3 23.3

The animal was taken out of the experiment when the area of the measured tumor surface was more than 100 mm ² . Data on the dynamics of growth of individual tumors in groups 1-4 are shown in Fig. 33 - 36, where the tumor volume was calculated using the formula V = A ^2/2 × B, where A - the smallest two orthogonal dimensions. Conversion to tumor volume seems to be rational due to the fact that after hyperthermia, tumor flattening was often observed, which is not reflected when calculating the surface area, but means a decrease in the tumor mass. Thus, FIG. 32 clearly illustrates the reduction in tumor volume after hyperthermia in comparison with the control (MNP without field), while the measured area decreases slightly.

From the presented graphs 33-37 it can be seen that the volume of tumors in the group of animals treated with hyperthermia 2 weeks after therapy is significantly less than in each of the control groups (152 mm ³ versus 508 mm ³ in group 1 (p = 0.018); 396 mm ³ in group 2 (p = 0.056); 460 mm ³ in group 3 (p = 0.0017).

A study of the stability of the claimed lyophilizate of nanoparticles was carried out by the method of dynamic light scattering.

Dynamic light scattering (DLS) is a universal and generally accepted method for the analysis of nanohybrid materials, therapeutic agents, and polymer stabilizers.

The hydrodynamic diameter of the obtained nanoparticles, measured by the DLS method, is 45-50 nm, which indicates the absence of aggregates and the stability of the nanoparticles (Fig. 11-13). Based on the results obtained, it can be said that the sample of the lyophilizate of nanoparticles has high stability in aqueous solution, which is confirmed by the data of dynamic light scattering. The average size remains within 45 ± 5 nm, and the polydispersity index Pdi, which does not exceed 0.35, and the absence of aggregates indicate the monodispersity of nanoparticles in solution and their high aggregative stability.

A study of the stability of the lyophilizate of nanoparticles was also carried out by atomic force microscopy (Fig. 14). 1 μl of lyophilizate of nanoparticles diluted 100 times with water was applied to freshly cleaved mica until completely dry. Based on the data obtained by atomic force microscopy, it can be said that the nanoparticles have an average size of 15 ± 5 nm and a narrow size distribution (Fig. 14-19), which is consistent with the data obtained by the method of transmission electron microscopy, and confirms the fact of monodispersity of particles. In addition, the particles are evenly distributed over the substrate and do not form aggregates.

The study of the stability of test samples of lyophilisate of nanoparticles by the method of "accelerated aging" for 20 days at a temperature of + 60 ° C indicates that no significant changes in the structure and physicochemical properties of these samples are observed. The phase composition of the samples does not undergo any changes (100% cobalt ferrite phase); in addition, the hydrodynamic diameter of nanoparticles remains within 45 ± 5 nm, which indicates a high stability of nanoparticles in aqueous solutions. Atomic force microscopy shows that there is no change in the size of the cobalt ferrite nanoparticles.

Thus, as a result of the studies carried out, it was concluded that the stability and pharmacological properties of the inventive composition are preserved upon reconstitution, as well as an increase in the shelf life of the solution prepared from the inventive pharmaceutical composition.

Claims (20)

1. A pharmaceutical composition for the preparation of an injection solution for use in the treatment of magnetic hyperthermia, characterized in that it is a lyophilisate of a solution of magnetic nanoparticles of cobalt ferrite (CoFe ₂ O ₄ ) with a size of not more than 20 nm, coated with sorbitol molecules, with the following ratio of components, wt .%: cobalt ferrite 70-75 sorbitol 25-30 2. A composition according to claim 1, characterized in that the average diameter of said magnetic nanoparticles is 16 nm. 3. A method of obtaining a pharmaceutical composition according to claim 1, characterized in that the following steps are sequentially performed: a) a suspension of magnetic nanoparticles of cobalt ferrite is prepared by adding aqueous solutions of iron (III) chloride (FeCl ₃ ) and cobalt dichloride hexahydrate (CoCl ₂ ⋅ 6H ₂ O) to a boiling solution of sodium hydroxide (NaOH) with constant stirring and further cooling the mixture to room temperature temperature; b) nanoparticles of cobalt ferrite in the form of a precipitate in the suspension is collected using a permanent magnet, after which the supernatant liquid is decanted; c) an aqueous solution of cobalt ferrite nanoparticles with pH 2-3 is prepared by adding deionized water to the resulting precipitate. d) stabilize the surface of nanoparticles in solution with citrate ions; e) adjust the pH of the solution to 7.3-7.4 by adding dropwise with continuous stirring and constant monitoring of the pH level, the sodium hydroxide solution; f) the nanoparticle solution obtained in step f) is washed using centrifugal filters with a pore size of not more than 100 kDaltons. g) sorbitol is added to the obtained aqueous solution of cobalt ferrite nanoparticles in an amount of 50-60 mg for every 100 ml of cobalt ferrite solution; h) the resulting solution with an osmolarity of not more than 300 mOsmol is filtered, followed by lyophilization at a residual pressure of not less than 0.64 mbar. 4. The method according to claim 3, characterized in that in step a) an aqueous solution of iron (III) chloride (FeCl ₃ ) at a concentration of 81 g / l, cobalt (II) chloride hexahydrate (CoCl ₂ ⋅6H ₂ O) in concentration of 860 g / l, sodium hydroxide (NaOH) solution at a concentration of 1 mol / l, while for preparing the suspension, the components are taken in the following ratio, wt%: iron (III) chloride (FeCl ₃ ) 25 cobalt dichloride (CoCl ₂ ) eighteen sodium hydroxide solution (NaOH) 57 5. The method according to claim 3, characterized in that in step b) and c) the precipitate is washed with deionized water 2-3 times. 6. The method according to claim 3, characterized in that in step d) for the formation of citrate ions on the surface of the particles, 0.01M citric acid solution and 1M sodium hydroxide solution are added to an aqueous solution of cobalt ferrite, while 40 ml of cobalt ferrite solution take 80 ml and 1.3 ml of these components, respectively. 7. A method according to claim 3, characterized in that said magnetic nanoparticles are Co-substituted magnetic nanoparticles based on magnetite of the formula CoFe ₂ O ₄ . 8. The method according to claim 3, characterized in that after step b) additional oxidation of the surface of the nanoparticles is carried out, for which - to the obtained nanoparticles of cobalt ferrite, add solutions of nitric acid at a concentration of 2 mol / l and nanohydrate of iron (III) nitrate with a concentration of 85 mg / ml in a volume of 30 ml each for 40 ml of a solution of nanoparticles and boil with constant stirring, followed by cooling the mixture to room temperature temperature, - after which the precipitate of cobalt ferrite nanoparticles obtained as a result of synthesis is collected at the bottom of the flask using a permanent magnet, and the resulting supernatant liquid is decanted, followed by washing the resulting precipitate with nitric acid.

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