RU2644332C1 - Method for obtaining a vegetable ml1 lecture from a missile white and a pharmaceutical composition on its basis (variants) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: inventions relate to the preparation of biologically active preparations. Method for isolating the ML1 protein from mistletoe white and a pharmaceutical composition (variants) are provided. This involves extracting the protein from the dried leaves of mistletoe white in 0.25M NaCl solution, carrying out three purification steps, including affinity chromatography with lactosyl-containing sorbent and ion exchange chromatography with a monoS-type cation exchange resin, and concentrating up to 2 mg/ml on a membrane with pore sizes from 10 to 30 kDa. Composition contains 0.04–0.15 mg/ml of the ML1 protein and aqueous isotonic saline solution or 0.1–10 mcg/ml ML1 protein, 0.1–1 mg/ml ferromagnetic nanoparticles and aqueous isotonic saline.

EFFECT: invention provides a lectin ML1 from mistletoe white with a purity of at least 95 % and a pharmaceutical composition based on it, having an immunostimulating, antitumor effect and stability for two and a half years.

8 cl, 5 dwg, 4 tbl, 2 ex

Description

Technical field

The invention relates to pharmaceuticals and relates to a method for the isolation (preparation) of plant viscumin lectin (mestletoe lectin I, MLI) from white mistletoe (VISCUM ALBUM), as well as a pharmaceutical composition based on it having antitumor and immunomodulatory effects.

State of the art

Malignant tumors are one of the most serious diseases, are one of the main causes of human death. At least 10 million cases of diseases of this kind are recorded annually, a significant part of which lead to death. Given the high mortality rate from cancer (according to WHO, over 7.5 million people per year, which makes up more than 13% of all deaths), as well as the annual increase in the incidence rate, the need for the development of effective new-generation anti-cancer drugs and diagnostic tools is increasing . The knowledge accumulated to date on the mechanisms of malignant transformation, the functioning and molecular composition of cells that have undergone such transformation, as well as certain successes in the study of tumor markers - molecules that distinguish between normal and tumor cells - form a solid theoretical basis for the creation of agents capable of selective exposure to cancer cells.

, Kaveri S.V. (ed.). Mistletoe: From Mythology to Evidence-Based Medicine. 2015), смогли показать терапевтические эффекты при использовании, в частности, так называемых лектинов омелы белой (вискумин, агглютинин Viscum album, VAA). В настоящее время помимо их цитотоксического действия обсуждается, в частности, неспецифическая стимуляция иммунного ответа, положительные эффекты которого используют для сопутствующей терапии и для долечивания больных с опухолями (

, Kaveri S.V. (ed.). Mistletoe: From Mythology to Evidence-Based Medicine. 2015).White mistletoe extracts have been used for therapeutic purposes for centuries. Since the beginning of this century, preparations of mistletoe white, with one way or another used in the treatment of cancer (

Kaveri SV (ed.). Mistletoe: From Mythology to Evidence-Based Medicine. 2015), were able to show therapeutic effects when using, in particular, the so-called white mistletoe lectins (viscumin, agglutinin Viscum album, VAA). Currently, in addition to their cytotoxic effects, in particular, non-specific stimulation of the immune response is being discussed, the positive effects of which are used for concomitant therapy and for treating patients with tumors (

Kaveri SV (ed.). Mistletoe: From Mythology to Evidence-Based Medicine. 2015).

As the active principle of the mistletoe extract, white mistletoe lectin protein (ML) weighing 63 kDa, which can be obtained biochemically from extracts (Franz N. Viscaceae lectins. // Advances in lectin research (Franz H., ed. ), Berlin, VEB, Verlag volk und Gesundheit, 2, 28-59.). ML protein consists of two subunits covalently linked by a disulfide bridge, whose A chain is responsible for the enzymatic inactivation of ribosomes, and the B chain is responsible for the binding of carbohydrates. Biological activity according to currently known data is reduced mainly to the lectin activity of the B-chain.

The heterogeneity of plant preparations of mistletoe lectin white is, among other things, the result of post-translational treatment of MLI in isoforms of MLII and MLIII, so that in ML preparations, depending on the isolation technique or the duration of fermentation, different contents of MLI, MLII and MLIII, which differ in the specificity of the carbohydrate binding fragment (Schumacher U., Pfuller U. From Berlin and Witten to Southampton and Hamburg: 25 Years of Mistletoe Research Cooperation // Mistletoe: From Mythology to Evidence-Based Medicine. - Karger Publishers. 2015. Vol. 4. P. 39-47.) .

, Kaveri SV (eds): Mistletoe: From Mythology to Evidence-Based Medicine. Transl. Res. Biomed. Basel, Karger, 2015, Vol 4, P. 11-23].The proportion of MLI in the composition of white mistletoe extract is usually the highest, in addition, this lectin has the highest biological activity, which is attributed to its ability to bind and activate lymphocytes, and therefore is considered the most valuable component of the extract and is of the greatest therapeutic interest [Urech K., Baumgartner S. Chemical Constituents of Viscum album L .: Implications for the Pharmaceutical Preparation of Mistletoe. //

, Kaveri SV (eds): Mistletoe: From Mythology to Evidence-Based Medicine. Transl. Res. Biomed. Basel, Karger, 2015, Vol 4, P. 11-23].

The extract also contains MLII and MLIII - other isoforms of toxic lectins. MLI, MLII and MLIII differ in molecular weight as well as carbohydrate specificity. MLI is specific for galactose, MLIII for N-acetylgalactosamine, and MLII has the same affinity for both sugars. The use of mistletoe extracts as additional drugs in the treatment of cancer significantly reduces the side effects of chemotherapy.

, Kaveri S. V. (ed.). Mistletoe: From Mythology to Evidence-Based Medicine. 2015). Проведение исследований и оценка полученных результатов осложняются невозможностью стандартизации экстрактов, поскольку последние представляют собой комплексные многокомпонентные смеси, в состав которых входит множество различных веществ, обладающих фармакологической активностью (Urech K., Baumgartner S. Chemical Constituents of Viscum album L.: Implications for the Pharmaceutical Preparation of Mistletoe. //

, Kaveri SV (eds): Mistletoe: From Mythology to Evidence-Based Medicine. Transl. Res. Biomed. Basel, Karger, 2015, Vol 4, P. 11-23).Currently, white mistletoe extracts are quite widely used in such countries of Western Europe as Switzerland, Germany, France, Austria. Most clinical trials of the effectiveness of mistletoe extracts are conducted against the background of the current treatment of patients, i.e. as a means of additional drug therapy. This makes it difficult to draw conclusions about the antitumor effectiveness of white mistletoe extracts. Nevertheless, as indicators of the effectiveness of white mistletoe extracts, an increase in the duration and quality of life of cancer patients, an improvement in the tolerance of standard conventional treatment (

Kaveri SV (ed.). Mistletoe: From Mythology to Evidence-Based Medicine. 2015). Research and evaluation of the results are complicated by the impossibility of standardizing extracts, since the latter are complex multicomponent mixtures, which contain many different substances with pharmacological activity (Urech K., Baumgartner S. Chemical Constituents of Viscum album L .: Implications for the Pharmaceutical Preparation of Mistletoe. //

, Kaveri SV (eds): Mistletoe: From Mythology to Evidence-Based Medicine. Transl. Res. Biomed. Basel, Karger, 2015, Vol 4, P. 11-23).

In recent years, the main attention has been paid to the study of the drug based on the recombinant analogue MLI (Aviskumin drug), due to which the problem of standardization of drugs could be solved. Recombinant MLI has been shown to have the same biological properties as the natural MLI found in white mistletoe extracts. This allowed the start of clinical trials of Aviskumin (Bergmann L., Aamdal S., Marreand S. et al. Phase I trial of rviscumin (INN: aviscumine) given subcutaneously in patients with advanced cancer: a study of the European organization for Research and Treatment of cancer (EORTC protocol number 13001) // Europ. J. Cancer. - 2009. - Vol. 44. - P. 1657-1662). However, recombinant MLI does not have post-translational modifications characteristic of a natural protein, which affects the stability of the protein molecule. The absence of glycosylated fragments can also adversely affect the immunomodulatory properties of MLI. Moreover, preparations based on recombinant viscumin have a shelf life of about 1.5 years, which indicates the insufficient stability of the active component used.

Mistletoe preparations were previously used for internal or parenteral use in various forms (in the form of tea, powder, drops, injections), mainly aqueous or alcoholic extracts, or juices. However, the use of such forms caused many problems. It is known that parenteral use of drugs, even if they have significant immunostimulating and antitumor effects, caused many undesirable side effects and general allergic reactions, such as chills, fever, headache, shortness of breath, etc. In addition, oral use of drugs caused pressure surges. dizziness, narrowing of coronary vessels, cardiac arrhythmias.

When isolated from a plant source with the current level of technology, as a rule, get a heterogeneous mixture of substances. The heterogeneity of herbal preparations of mistletoe lectin is among other things the result of post-translational treatment of MLI in isoforms of MLII and MLIII, so that in ML preparations, depending on the isolation technique or the duration of fermentation, different contents of MLI, MLII and MLIII are detected.

The prior art method for producing viscumin disclosed in security document DE 4221836. To isolate galactosyl-specific lectin (MLI), dried mistletoe leaves are soaked overnight in phosphate buffer, then the resulting suspension is mixed and centrifuged. After centrifugation, lectin was isolated from the supernatant by affinity chromatography on lactosyl-Sepharose. After passing, the resulting lectin is washed several times. After chromatography, the solution was concentrated by ultrafiltration. As a result of this isolation method, a mixture of MLI, MLII and MLIII lectins is obtained with a predominant MLI content (85-90%). The disadvantage of this method is that MLI lectin is obtained from white mistletoe with a purity of not more than 90%.

From protection document DE 4229876 it is known to isolate mistletoe lectins from plant materials by homogenizing fresh or dried ground parts of mistletoe with distilled water, stirring, filtering and acidifying the resulting extract with acetic acid. After centrifugation, the supernatant is placed on a suitable cation exchange column, preferably SP-Sephadex. The disadvantage of this method is that aggressive solutions are used to extract lectins.

WO 9600239 discloses the isolation of MLI lectin from mistletoe by grinding freshly picked mistletoe plants and mixing with distilled water. The aqueous suspension is filtered and the pH of the solution is adjusted to <7 by the addition of acetic acid. The resulting suspension is centrifuged, the supernatant is passed through an SP-Sephadex cation exchange column. The collected fractions are washed with a buffer solution. The disadvantage of this method is that the use of freshly collected raw materials limits the application of the method in time, and the access of raw materials is limited.

The closest set of essential features to the developed method is the method disclosed in document WO 9711967, which consists in obtaining an aqueous extract of mistletoe white, containing mistletoe lectins in a ratio of MLI: MLII: MLIII from 0.1-90: 90-0.1: 0- 3, according to which all parts of the plant are soaked in a cold aqueous solution, preferably isotonic saline or phosphate buffered saline, then the mixture is filtered, the proteins from the filtrate are salted out with ammonium sulfate, and the filtration is repeated until low molecular weight ponents having a molecular weight of less than 1 kD, and the extract is further diluted with water. In order to standardize the ratio of the content of lectin isomers in the composition, their content is further adjusted by adding pure substances. The disadvantage of this method is that the resulting composition of the extract is not uniform in composition and requires adjustment by adding pure substances. The resulting composition includes mistletoe lectin isoforms having different specificities, as well as a different mechanism of attachment to tumor cells. In addition, the content of MLI lectin obtained from mistletoe white is not more than 90%.

Thus, it remains relevant to develop a method for the production of such a mistletoe preparation, which would have a significant immunostimulating and antitumor effect and would not cause unwanted side effects.

For the purpose of a detailed study of the therapeutic effect of mistletoe lectin white MLI, it seems necessary to obtain it in its pure form for use as an active component of drugs in the industrial production of the latter.

Disclosure of invention

The present invention is to develop a method for producing MLI lectin from white mistletoe with a purity of not less than 95% and a pharmaceutical composition based on it.

The problem is solved by the claimed method for the isolation of MLI protein (mistletoe lectin I) from white mistletoe (Viscum album), including:

- obtaining a finely divided suspension of dried leaves of white mistletoe in a 0.25 M NaCl solution, taken in an amount sufficient for extraction,

- carrying out the first stage of purification of the suspension from impurities not belonging to the ML protein group by introducing ammonium sulfate into the suspension at a rate of 60-80 g per 100 ml of extract, keeping the suspension obtained to precipitate plant proteins, including the ML protein group (with a content of at least 95% MLI protein), followed by separation of the precipitate;

- dissolution of the obtained precipitate in a NaCl solution, which is taken in an amount sufficient to completely dissolve the precipitate;

- conducting a second purification step to isolate ML group proteins from the resulting plant protein solution, by affinity chromatography using a 0.25 M NaCl solution as a buffer;

- the third stage of purification for the isolation of MLI protein from a solution of a mixture of proteins of the ML group using ion-exchange chromatography, using a cation exchange resin of the monoS type with a sorbent particle size of not more than 10 microns as a sorbent;

- transferring the obtained fraction of purified MLI to phosphate-buffered saline with physiological pH and isotonic composition using dialysis using Slide-A-Lyzer ™ Dialysis Cassettes (Thermo Fisher) dialysis membranes with a molecular weight cut-off of 10-20 kDa or analogs, or gel filtration using a HiTrap Desalting Column (GE Helthcare).

Preferably, incubation of the suspension in the first stage of purification for at least 16 hours

The problem is also solved by the method of obtaining the MLI protein concentrate, including the concentration of the MLI protein solution obtained by the above method to a value of 2 mg / ml with an acceptable deviation of up to 20%. Concentrating systems are used for this purpose, including those such as Amicon® Ultra manufactured by Millipore with a membrane with a low sorption capacity (pore size of the filtering membrane from 10 to 30 kDa).

Also, the problem is solved by a pharmaceutical composition for injection, including an active component and a buffer, where the MLI lectin obtained by the above method is used as the active component, and an aqueous isotonic saline solution with physiological pH is used as the buffer, while the content of plant lectin is viscumin (MLI) in 1 ml of the solution is 0.04-0.15 mg.

Preferably, when the pharmaceutical composition additionally contains auxiliary substances (additives), for example, to stabilize the protein, reduce protein sorption, such as pyridoxine hydrochloride, and boric acid (1-2% by weight), sugars, polyol alcohols, amino acids and their derivatives, surfactants, for example, Tween-80 and others, in a generally accepted amount (it is preferable that the total amount of additives by weight be no more than 10% by weight of the solution with the active component). In this case, to obtain an aqueous isotonic saline, sodium hydrogen hydrate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, and also sodium chloride are used.

The problem is also solved by a pharmaceutical composition for injection, including a buffer and an active component, where the active ingredient is MLI lectin concentrate obtained as described previously, conjugated with ferromagnetic nanoparticles (low-pass filter), and an aqueous isotonic saline solution with physiological pH is used as a buffer while the content of plant lectin viscumin (MLI) in 1 ml of solution is 0.1-10 μg, ferromagnetic nanoparticles - 0.1-1 mg.

The technical result of the claimed group of inventions is that the developed isolation method allows to obtain MLI lectin with a purity of not less than 95% (an admixture of isoforms MLII and MLIII - not more than 5%), while the resulting pharmaceutical composition has a stability of at least 2.5 of the year. In addition, the use of a pharmaceutical composition containing a conjugate of plant lectin and ferromagnetic particles, allows to increase the residence time of viscumin in the tissue without increasing the dose of the drug, which leads to a decrease in side effects.

Brief Description of the Drawings

In FIG. 1 presents examples of typical electrophoregrams obtained by confirming the claimed technical result, regarding the purity of the drug lectin ML obtained by the claimed method, at least 95%.

In FIG. 2 presents the results of the analysis of the drug lectin MLI obtained by the claimed method, mass spectrometric method (MALDI).

In FIG. Figure 3 shows histograms showing the change in gene expression involved in the cell response to an increase in the amount of unfolded proteins in ER as a result of MLI exposure at concentrations of 1⋅10 ^-7 M (black), 1⋅10 ^-9 M (gray), 1⋅10 ^-11 M (white) for CaCo2 and Molt4 cells.

In FIG. 4 is a graph of the survival of mouse B-cell lymphomas MLA G3 and MLB H8 at various concentrations of MLI and low-pass filter conjugate-MLI. The results of processing the cells with equivalent amounts of a control LPF preparation indicated that there was no effect of the LPF on cell viability in the studied quantities of the LPF-MLI conjugate.

In FIG. Figure 5 shows the dynamics of the ratio of the intensity of the MRI signal in tumors to the signal in the muscle during intratumoral administration of preparations containing the control preparation of ferromagnetic nanoparticles (LPF) and ferromagnetic nanoparticles conjugated with MLI (LPF-MLI).

The implementation of the invention

The antitumor effect of viscumin is due to both its cytotoxic properties and the ability to non-specifically activate the immune system. The proposed mechanism involves the direct interaction of viscumin with NK cells and macrophages localized in the upper layers of the skin in the area of drug administration, leading to their activation. Activation is manifested in the form of cytokine induction, as well as in a change in the expression profile of surface cell receptors. Activated cells of the immune system are characterized by a higher lysing potential and are able to overcome barriers that shield tumor cells from the immune system.

Unlike the previously obtained extracts of white mistletoe, the developed method allows to obtain highly purified plant viscumin lectin containing all natural modifications.

Isolation of ML1 protein (mistletoe lectin I) from white mistletoe (Viscum album) is carried out in the following sequence.

First, the dried leaves of the white mistletoe are crushed to a powdery state, after which they are poured with a 0.25 M NaCl solution, taken in an amount sufficient for effective extraction. In this case, as a rule, this amount can vary from 400 ml to 1 l per 50 g of crushed white mistletoe leaves.

Then, the obtained finely divided suspension of dry mistletoe leaves is purified in several stages to obtain a lectin concentrate ML1 with a content of at least 95%.

At the first stage, the suspension is purified from impurities not belonging to the ML protein group by introducing into the suspension ammonium sulfate at a rate of 60-80 g per 100 ml of extract, maintaining the suspension obtained to precipitate plant proteins, including the ML protein group, followed by separation of the resulting draft. In this case, the suspension is usually incubated for at least 16 hours. A separation of the precipitate formed is carried out by known methods, for example, by centrifugation for 20-30 minutes at 4500-5500 g.

The resulting precipitate is then dissolved in a solution of 0.5 M NaCl, which is taken in an amount sufficient to completely dissolve the precipitate, for example 30-40 ml per 50 g of the original dry ground leaves.

At the second stage of purification, ML group proteins are extracted from the resulting plant protein solution by affinity chromatography using a 0.25 M NaCl solution as a buffer. In this case, affinity chromatography can be carried out using a lactosyl-containing sorbent, for example, lactosyl-Sepharose 4B or α-Lactose-Agarose (Sigma).

At the third stage of purification, MLI protein is extracted from a solution of a mixture of proteins of the ML group using ion-exchange chromatography, and a cation exchange resin of the monoS type with a particle size of the absorbent of not more than 10 μm is used as a sorbent. Ion exchange chromatography is carried out in 15 mm nitrate buffer at a pH of 4.0-5.0, the target protein is eluted in a concentration gradient from 250 to 500 mm NaCl. MonoS type cation exchange resin can be used, for example, Mono S 5/50 GL brand (GE Healthcare). After the third stage of purification, the concentration of the obtained protein (active component) is 0.5-2 mg / ml.

The fraction of purified MLI protein obtained in the third stage is transferred to phosphate-saline buffer with physiological pH and isotonic composition using dialysis, using, for example, dialysis membranes of the Slide-A-Lyzer ™ Dialysis Cassettes (Thermo Fisher) type with a molecular weight cut-off of 10 -20 kDa. It is also possible to use gel filtration using a column, for example, such as HiTrap Desalting Column (GE Helthcare). The concentration of the obtained protein (active component) after the introduction of phosphate-saline buffer is 0.25-1.5 mg / ml.

Thus, the product obtained by the claimed method with a given concentration (0.25-1.5 mg / ml) can be used for therapeutic purposes (as an antitumor and immunomodulating agent), while for injection administration of the drug, the patient is recommended to dilute it with phosphate-salt a buffer with physiological pH and isotonic composition to a concentration of the active component of 200-800 ng / ml

At the same time, pharmaceutical companies are recommended to produce a pharmaceutical composition with an active ingredient content of MLI 0.04-0.15 mg per 1 ml of solution. To obtain an aqueous isotonic saline solution, sodium hydrogen hydrate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, and also sodium chloride can be used.

In addition, the pharmaceutical composition may further comprise adjuvants for stabilizing the protein, reducing protein sorption, etc.

In addition, to ensure the prolonged action of the active component in the tumor, the pharmaceutical composition for injection can contain MLI lectin conjugated with ferromagnetic nanoparticles with a content of 1 to 10 μg of plant MLI lectin in ferrous nanoparticles, from 0.1 to 10 μg of ferromagnetic nanoparticles 1.0 mg A method for conjugating MLI with ferromagnetic nanoparticles is presented in the work (Khutornenko A.A., Gerasimov V.M., Sakharov D.A. Obtaining and studying the internalization of the conjugate of viscumin-ferromagnetic nanoparticles with human glioblastoma cells A172 // Bulletin of Experimental Biology and Medicine. 2015 Vol. 160. No. 12 / P. 797-801). In the process of obtaining the MLI-LPF conjugate, an MLI protein of 1.6 to 2.4 mg / ml is used. For this purpose, the protein solution is concentrated using a concentration system with a membrane with a low sorption capacity, characterized by a pore size of 10 to 30 kDa.

A viscumin-based drug can be used to treat patients with metastatic or locally advanced colorectal cancer as an independent drug if chemotherapy is not possible.

Adding stabilizing substances to protein solutions is the easiest way to maintain their activity. As stabilizing additives can be used solutions of pyridoxine hydrochloride (5-10% by weight) and boric acid (1-2% by weight) (RU 2020154, 1991), sugars, polyol alcohols, amino acids and their derivatives (EP 0821058, 2002 ), surfactants, for example Tween-80. In this case, to obtain an aqueous isotonic saline, sodium hydrogen hydrate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, and also sodium chloride are used.

The following are examples of specific implementations of the invention, detailing each step of the proposed method, which illustrate, but do not limit the scope of the claims.

Isolation of viscumin - plant MLI lectin from dry leaves of white mistletoe (Viscum album)

From 100 g of dry leaves, ~ 14 mg of a mixture of lectins (MLI, MLII, MLIII) was obtained; the following steps were taken:

1) To obtain 100 g of dried leaves, the leaves were separated from the branches, which were then ground to a powder state. The resulting powder from white mistletoe leaves was poured into 800 ml of a 0.5 M NaCl solution and stirred for 1 hour at room temperature, then for 48 hours at 4 ° C.

2) The resulting suspension was filtered through cheesecloth (more conveniently under vacuum). The volume of suspension after filtration was 650-700 ml.

3) The filtrate was centrifuged at 10,000 g for 20 minutes. The supernatant was transferred to a separate tube, the pellet was discarded. Repeated the procedure two more times. The total volume of the supernatant after three rounds of centrifugation was 550-600 ml.

4) The resulting suspension was filtered through 0.45 μm. The filtrate volume was approximately 550 ml.

5) Conducted the first stage of purification of the target protein. Solid ammonium sulfate (60 g per 100 ml of extract) was added portionwise to the filtrate (approximately 550 ml) with stirring. The mixture was stirred for 16 hours at room temperature, then incubated for 1 hour at room temperature without stirring.

6) The suspension was centrifuged for 20 min at 5000 g. Carefully drained the supernatant (approximately 420 ml). The precipitate was dissolved in a minimum volume of a 0.5 M NaCl solution (approximately ~ 60-70 ml). The mixture was centrifuged twice at 5000 rpm for 20 min to remove insoluble sediment. The volume of the supernatant after centrifugation was approximately 60 ml.

7) The supernatant was filtered through a 0.45 μm filter with a non-viscumin binding membrane (type of PVDF or PES membrane). The filtrate volume was approximately 60 ml.

8) For the second stage of purification of the target protein, an affinity column (lactosyl-Sepharose 4B, binding capacity of at least 6 mg / ml) with a volume of 50 ml (resin volume of 10 ml), which was filled with a sorbent, and washed with 15 volumes of solution 0, was prepared. 5 M NaCl.

9) The entire volume of the transparent supernatant (about 60 ml) was applied to the prepared affinity column, after which a slip containing unbound proteins was collected.

10) The column was washed with 10 volumes of 0.25 M NaCl solution, and an aliquot of the sample was retained for analysis.

11) The column was washed with 10 volumes of a 0.25 M NaCl solution with 20 mM D-galactose.

12) The target protein was eluted using an elution buffer (0.25 M NaCl, 200 mM D-galactose). The first volume, corresponding to the column volume (~ 10 ml), did not contain the target protein. The first volume was collected in a separate tube. After that, 8 fractions of 2 ml were collected. The optical density in each of the fractions was measured. Combined all fractions containing protein (usually 0.2-0.6 p.u./ml). The total volume of the eluate containing the protein was about 12 ml.

13) Washed column 5 with volumes of 0.25 M NaCl, 400 mM D-galactose to remove all bound proteins. The column was washed with 5 volumes of 50% alcohol, stored under alcohol at 4 ° C.

14) We analyzed the eluate on SDS-PAGE according to Laemmli for the content of the target protein.

15) The eluate was concentrated after affinity chromatography about 3-4 times, so that the concentration of the solution did not exceed 4 pu / ml at 280 nm (volume about 4 ml).

16) The eluate was filtered after concentration through a 0.45 μm filter. The filtrate volume was about 4 ml.

17) For the third purification step, a 20 ml HiTrap Desalting Column (GE Helthcare) column was prepared (4 consecutively connected 5 ml columns) to transfer the filtrate to citrate buffer, equilibrating it with 5 volumes of 15 mM citrate buffer pH 4.2.

18) Using a 20 ml HiTrap Desalting Column (GE Helthcare) balanced citrate buffer column (4 consecutively connected 5 ml columns), the total filtrate volume obtained (about 4 ml) was transferred to 15 mM citrate buffer pH 4.2 for subsequent ion exchange chromatography.

19) The protein was collected in citrate buffer, a volume of about 6 ml (diluted 1.5-2 times). Permissible concentration of the solution is 1.5-2 times.

20) After transferring the protein to citrate buffer, the solution was filtered through a 0.45 μm filter. The filtrate volume was 3-6 ml.

21) The HiTrap Desalting Column (GE Helthcare) column was washed with 5 volumes of 15 mM citrate buffer pH 4.2, then with 5 volumes of 20% ethanol, stored at 4 ° C.

22) A monoS 5/50 GL cation exchange column was prepared (1 ml sorbent volume, 50 mg / ml binding capacity) for final purification of the target protein. For this, the column was balanced by washing it with 5 volumes of 15 mM citrate buffer pH 4.2.

23) Put the entire filtered protein solution in citrate buffer onto a monoS 5/50 GL column.

24) Washed column 8 volumes of 15 mm citrate buffer pH 4.2.

25) The column was washed with 12 volumes of 15 mM citrate buffer pH 4.2 with 300 mM NaCl.

26) Bound proteins were eluted with a NaCl concentration gradient of 300 to 800 mM over 8 column volumes. 1.5 ml fractions were collected.

27) Washed column 5 volumes of 15 mm citrate buffer pH 4.2 with 1 M NaCl.

28) The column was washed with 5 volumes of 15 mM citrate buffer pH 4.2, then with 5 volumes of 20% ethanol, stored at 4 ° C.

29) After ion-exchange chromatography in Laemmly SDS-PAGE, fractions were analyzed which contained the target protein and which corresponded to the second part of the peak in the chromatogram (fractions 3-4 from the beginning of the peak during protein elution). After combining the fractions, the total volume of the purified protein solution was about 4.5 ml.

30) A phosphate-salt buffer was prepared to store pH 8.0 for dilution of the resulting protein and subsequent storage of the solution.

The composition of the buffer:

NaCl - 7.5 g / l;

Na ₂ HPO ₄ × 2 H ₂ O - 3.4 g / l;

NaH ₂ PO ₄ × 2 H ₂ O - 0.16 g / l.

In a pyrogen-free glass measuring container with a capacity of 2 l, 1 l of deionized water was poured, suspended components were added: 15 g of NaCl, 6.8 g of Na ₂ HPO ₄ × 2 H ₂ O, 0.32 g of NaH ₂ PO ₄ × 2 H ₂ O and stirred on a magnetic stirrer until they are completely dissolved. The pH value was set in the solution to 8.0 ± 0.02 by the pH meter by adding a 2M NaOH solution in a volume of ~ 10 ml. The total volume of water was adjusted to 2 liters.

31) The purified protein solution was transferred to phosphate buffered saline to store pH 8.0 using a HiTrap Desalting Column (GE Helthcare) or dialysis. The protein concentration was measured spectrophotometrically in the resulting solution at a wavelength of 280 nm.

Confirmation of receipt of MLI lectin with a purity of not less than 95%

The purity and molecular weight of the MLI protein were confirmed by electrophoretic separation in 10% SDS-polyacrylamide gel under non-reducing conditions (without mercaptoethanol).

We performed determinations of the% content and molecular weight of the protein for three series of the MLI preparation obtained by the claimed method. For each series of the MLI preparation, 2 gels were poured; 4 test samples were applied to each gel with two different analysts. Examples of the obtained electrophoregrams are presented in FIG. 1 Precision Plus Protein Unstained Standards (Bio-Rad) were used to determine molecular weight. Electrophoregrams were scanned, and the content and molecular weight of the target protein were determined using ImageLab Software (Bio-Rad). Using Microsoft Excel, the standard deviation and relative standard deviation were calculated. The results are presented in table 1.

Conclusion: The relative standard deviation, determined for tests performed by different analysts, does not exceed 3% when determining the molecular weight, and is 0.6% when determining the purity of the drug. The purity of the MLI preparations obtained by the claimed method is 96.2% and higher.

Comparison of the results of determining the molecular weight of the MLI protein obtained by electrophoretic and mass spectrometric methods.

Mass spectrometric analysis (MALDI) showed the presence of a basic compound with a molecular weight of about 63 kDa (see FIG. 2). The mass of the target protein is 63-65 kDa.

The average molecular weight determined by the electrophoretic method is about 54 kDa for a full-sized glycosylated protein. Moreover, the addition of molecular weights obtained for the A- and B-subunits of the protein gives a value of about 63 kDa. The abnormal mobility of a full-sized protein in a polyacrylamide gel with respect to standard proteins (markers) can be explained by the presence of glycosylated fragments.

Example 1 demonstrating the possibility of using MLI as an active component of lectin.

From the lectin obtained by the above method, a preparation concentrate of the following composition was prepared:

Viscumin Plant Lectin (MLI) 0.1 mg Excipients: Sodium hydrogen phosphate, dihydrate 3,382 mg Sodium dihydrogen phosphate dihydrate 0.156 mg Sodium chloride 7.498 mg Water for injections up to 1 ml

For this, using the prepared phosphate-salt buffer for storage of pH 8.0, the resulting purified protein solution was diluted to a concentration of 0.1 mg / ml.

The subcutaneous injection solution concentrate is a colorless, clear solution. For subcutaneous injections, the concentrate is pre-diluted.

The dilution solution is a colorless, clear solution of the following composition (1 ml):

Sodium hydrogen phosphate, dihydrate 3,382 mg Sodium dihydrogen phosphate dihydrate 0.156 mg Sodium chloride 7.498 mg Water for injections up to 1 ml

Example 2, demonstrating the possibility of using MLI conjugated with magnetite ferromagnetic nanoparticles as the active component of lectin.

PEG-modified ferromagnetic nanoparticles (LPFs) with covalently immobilized MLI lectin were prepared as follows. To a 3 ml solution of PEG acid modified low-pass filter (1.67 mg / ml) in a MES buffer (pH 6.0), a solution of 1.53 mg of EDC and 4.34 mg of sulfo-NHS in 1 ml of MES buffer (pH 6.0) was added. Carboxyl groups were activated for 15 min, after which the low-pass filter was precipitated by centrifugation and resuspended in MES buffer (pH 6.0) three times, the last time the low-pass filter was resuspended in phosphate-saline buffer (pH 8.0) and concentrated to 250 μl. Then, 3 ml of an MLI solution with a concentration of 2 mg / ml and 400 μl of 100 mM lactose in 1x PBS, pH 8.0, was added to the LPF with activated carboxyl groups. Incubated for 2 hours. After that, NPs were precipitated by centrifugation at 18000 g and resuspended in phosphate-buffered saline (pH 8.0). Precipitation and resuspension in phosphate-buffered saline (pH 8.0) was repeated five times to eliminate unreacted lectin from the low-pass filter. (Khutornenko A.A., Gerasimov V.M., Sakharov D.A. Obtaining and studying the internalization of conjugate of viscumin-ferromagnetic nanoparticles with human glioblastoma cells A172 // Bulletin of experimental biology and medicine. 2015. Vol. 160. No. 12 / P 797-801).

Studies of the immunomodulating and antitumor effects of the developed drug were carried out on cell cultures (in vitro study) and xenograft animal models.

In vitro study of the cytotoxic properties of the studied MLI preparation

As a result of in vitro experiments, it was shown that the cytotoxic properties of the developed MLI preparation are different for different types of cells. 70% inhibition of tumor cell growth is achieved on average at concentrations of 0.2 ng / ml for the active substance (plant lectin viscumin). It was established that the formation of modified 28S ribosomal RNA in tumor cells - a manifestation of the specific N-glycosidic activity of viscumin - is detected upon exposure to the drug after 15 minutes, indicating a high activity of the test drug.

For four cell lines MV3, THP-1, Caco2 and MOLT4, MTT was used to assess cell viability after interacting with the claimed drug over a wide range of doses. It was shown that in the case of the MV3 line, the drug exhibits toxic properties even at a concentration of ^100-24 M. Thus, incubation of the test substance at a given concentration with MV3 cells for 72 hours leads to a decrease in viability to 65%. For cell lines of differentiated THP-1 monocytes and Caco2 colorectal cancer, IC ₅₀ values differ, indicating a lower sensitivity of these cells to the drug. The calculated IC50 values for the MV3, THP-1, Caco2, and MOLT4 cell lines were 5 × 10 ^-12 nM, 3 × 10 ^-9 nM, 1.0 × 10 ^-6 M, and 4.6 × 10 ^-10 M, respectively.

A specific reaction of the claimed preparation is the hydrolysis of the N-glycosidic bond at the 4324 position of the sarcin loop of the 28S ribosomal RNA (in the case of human cells at position 4605). The method for assessing this activity of viscumin is based on determining the fraction of destroyed ribosomes in a cell using real-time PCR.

The results of determining the N-glycosidase activity of the drug correlate with the MTT test data: at higher concentrations, the viability of THP-1 and Caco2 cells decreases and the proportion of modified ribosomes increases, reaching 52.0% and 9.5%, respectively.

When studying the kinetics of the formation of modified 28S ribosomal RNAs, the detection of enzymatic activity is detected after 15 minutes for the studied concentrations of 10 ^-7 M, 10 ^-9 M and 10 ^-11 M. At the initial exposure times, a higher rate of accumulation of the modified product in MOLT4 cells was noted. which is consistent with data on their greater sensitivity to the drug according to the MTT test.

The results of evaluating the development of the reaction of unfolded proteins under the influence of the developed drug MLI

Activation of the immune system is considered as the main mechanism of antitumor action of MLI. It has been shown that the elimination of immunocompetent cells as a result of the cytotoxic effect of MLI in their direct interaction is accompanied by the release of signals that promote the activation of other immune cells and increase their antitumor activity [Schumacher U., Pfuller U. From Berlin and Witten to Southampton and Hamburg: 25 Years of Mistletoe Research Cooperation // Mistletoe: From Mythology to Evidence-Based Medicine. - Karger Publishers. 2015. Vol. 4. P. 39-47.]. Activation of the immune system can be facilitated by the initiation of immunogenic apoptosis and endoplasmic reticulum stress (ER-stress) [Aaes T. L., Kaczmarek A., Delvaeye T., et al. Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity // Cell reports. 2016. Vol. 15. No. 2. P. 274-287; Grootjans J., Kaser A., Kaufman, R. J., Blumberg R. S. The unfolded protein response in immunity and inflammation // Nature Reviews Immunology. 2016. Vol. 16. P. 469-484]. MLI is a type II ribosome inactivating protein and is able to block protein synthesis in the cell [Sweeney E.C. Tonevitsky, A.G., Palmer, R.A., Niwa, N., Pfuller, U., Eck, J., Lentz H., Agapov I.I., Kirpichnikov, M.P. Mistletoe lectin I forms a double trefoil structure // FEBS letters. 1998. Vol. 431. No. 3. P. 367-370], a similar mechanism of activation of the immune system is possible for this lectin.

To test the hypothesis about the role of ER stress and trigger the cell response to an increase in the number of unfolded protein response (UPR) in the mechanism of antitumor and immunomodulating activity of MLI, we studied the change in gene expression involved in these processes in Molt4 and CaCo2 cell lines. with different sensitivity to MLI (IC ₅₀ is 4.6⋅10 ^-10 M and 1.0⋅10 ^-6 M, respectively). From the data presented in FIG. Figure 3 shows that the profile of changes in the expression of genes involved in UPR depends on cell type, MLI concentration, and duration of exposure. However, a general tendency is observed for both types of cells: lower MLI concentrations (1⋅10 ^-9 M, 1⋅10 ^-11 M) contribute to the start of adaptive processes in the cell rather than to the start of apoptosis. Thus, an increase in the level of the spliced form of the mRNA of the XBP1 gene (XBPls), the mRNA of thaler protein genes (HSPH1, HSPB1, HSPA5) and the transcription factor ATF6 is apparently due to the activation of IRE1α and ATF6-dependent signaling pathways [Grootjans J., Kaser A ., Kaufman, RJ, Blumberg RS, The unfolded protein response in immunity and inflammation // Nature Reviews Immunology. 2016. Vol. 16. P. 469-484], aimed at reducing the total number of proteins, on the one hand, and increasing the number of proteins necessary for removing from the ER incorrectly folded proteins, increasing its size and capacity, as well as increasing the efficiency of protein folding, on the other hand . Changes in the number of transcripts of the ATF4 transcription factor genes and its CHOP target (DDIT3 gene) are dose-dependent. For both genes, a wavelike pattern of expression changes is traced. At the same time an increase in the expression of DDIT3, mediator of ER-stress-dependent apoptosis in the case Caco2 observed almost exclusively to concentrate MLI 1⋅10 ^-7 M, and in the case Molt4 - for all test concentrations. This is consistent with the different sensitivity of these cell types to MLI. Note that the mRNA of the EIF2AK3 gene encoding the transmembrane protein of the endoplasmic reticulum PERK, which is responsible for triggering the PERK-dependent signaling cascade, the third cascade associated with UPR, is quite well represented in CaCo2 cells and is very little or practically absent in Molt4 cells.

The most important marker of the immunogenicity of the process is the release of calreticulin encoded by the CALR gene. Calreticulin belongs to the so-called molecular patterns associated with cell damage (DAMPs). In the case of CaCo2 cells CALR increase in gene expression is observed for concentrations MLI 1⋅10 ^-9 and 1⋅10 ^-11 M, and in the case MOLT4 - even a drop of expression (Fig 3.).

The data obtained allow us to conclude that the impact of MLI really leads to ER-stress and the launch of UPR. Moreover, the release of an important immunogenic component of calreticulin is dependent on the type of cell, dose and duration of exposure to MLI. Lower concentrations of MLI (1⋅10 ^-9 and 1⋅10 ^-11 M) are associated with a high content of the CALR transcript and activation of adaptive processes in the cell. Thus, for the manifestation of the immunomodulating properties of MLI, the time of the presence of lectin in the tissue is apparently important, while an increase in the local concentration of MLI can offset this effect.

In vitro study of the immunological activity of MLI

In the framework of this study, the effectiveness of the developed drug was evaluated to induce the release of TNFα, IL-6 and the total form of IL-12 on the fractions of mononuclear cells obtained from the blood of healthy people. At a concentration of 10 ng / ml, there was a significant increase in TNFα (p <0.05), and at 1 ng / ml, IL-6 (p <0.05). The expression of IL-12 produced mainly by activated monocytes has also been investigated. A significant increase in total IL-12 was detected in a mononuclear culture after 24 hours of incubation with 10 ng / ml of the drug (2.1 times; p <0.05).

It is assumed that the activation mechanisms of blood cells in the immune system include not only an increase in cytokines, but also an increase in phagocytic activity, the level of surface molecules involved in adhesion and recognition, as a result of which various macrophage-mediated native and adaptive forms of the immune response are regulated. The results obtained in the framework of the conducted studies suggest that one of the possible mediators of changes in the phenotype of cells under the influence of the developed drug on them may be the activation of the reaction of unfolded proteins (see above).

In vivo study of the antitumor and antimetastatic effects of the developed MLI preparation in xenograft mouse models

Studies of the antitumor and antimetastatic effects of the developed drug were carried out using xenograft models based on SCID immunodeficient mice with transplanted human tumor cells of several types of cancer: ovarian cancer, melanoma, colorectal cancer. The drug was administered to animals subcutaneously in three doses of 30, 150 and 500 ng / kg. Tumor growth and administration of the developed drug to animals in all three studied doses of 30, 150 and 500 ng / kg did not adversely affect the viability, behavior and physiological reactions, appearance, and food and water intake of animals. In some cases, with the introduction of the maximum dose of 500 ng / kg, a local irritant effect was observed, peeling at the injection site, two animals showed a change in skin pigmentation,

In animals in the ovarian cancer group receiving the test drug at doses of 30 and 500 ng / kg, there was a statistically significant increase in life expectancy compared to the control group (p <0,0002).

In animals in the melanoma group receiving a dose of 150 ng / kg, the average weight of primary tumors was 1.5 times greater than in the group with a dose of 30 ng / kg (p <0.04). In the group of mice treated with 30 ng / kg of the claimed drug, there was a decrease in the number of metastases in 2.3 compared with the control group (p <0.0001). For all experimental groups, a statistically significant increase in the number of dendritic cells infiltrated into the primary tumor was detected (for all groups, p <0.05).

In animals in the colorectal cancer group treated with the drug at a dose of 30 ng / kg, survival was significantly better (p <0.01). Moreover, the average weight of the primary tumor in animals in the 30 ng / kg group was statistically significantly lower than in the control group (p = 0.04). Also in this group there was a significant decrease in the number of metastases (3.7 times) compared with the control group (p <0.0001). The number of dendrin cells for animals of this group was 1.8 times more.

Immunomodulatory effect of MLI

The immunomodulating properties of the developed preparation were studied using an in vivo model of Wistar rats. An increase in the absolute number of mononuclear cells in the peripheral blood (PBMC) of rats after subcutaneous administration of the drug by 15-22% depending on the administered dose of the drug (from 1 to 30 ng / kg) was shown. An increase in the absolute number of large granular lymphocytes (LGLs) was also shown to be 2–3.5 times after 24 and 48 hours in the “0.5 ng / kg” and “1 ng / kg” groups, respectively. Higher doses of the drug (10 and 30 ng / kg) led to a statistically significant decrease in the total number of LGLs by 1.5 and 2 times after 24 hours and 48 hours, respectively. An increased content of activated monocytes as well as NK cells was detected.

In addition, it has been shown that low doses (30 ng / kg or lower) increase the number of dendritic cells that infiltrate into the primary tumor of the mice. In addition, experiments on rats showed that even lower doses of the drug (1 ng / kg) lead to an increase in the number of large granular lymphocytes, as well as the content of activated cells of the immune system.

Acute Toxicity Study of MLI

The study of the acute toxicity of plant MLI lectin from Viscum album (hereinafter referred to as viscumin) in mice and rats after subcutaneous administration with the determination of tolerable, toxic and lethal doses was carried out on white outbred rat mice. The drug was administered to mice and rats subcutaneously once in prescribed doses of viscamine according to the experimental conditions. In a preliminary study, each dose was tested on one male and one female, and in the main study, 5 males and 5 females. The animals of the control groups were injected with a carrier - phosphate buffer with a pH of 8.0 (dilution solution) - in the same volume as the substance in the maximum dose. The injected volumes were adjusted taking into account the individual body weight of the animal.

During the observation period (within 14 days after administration), the general condition of the animals was assessed by their motor activity, feed and water consumption, the condition of the coat and mucous membranes, and body weight.

After the observation period, a postmortem autopsy was performed in all animals that received the solvent and the maximum dose of the drug that did not cause death. The killing of animals was carried out by inhalation of carbon dioxide. Postmortem examination was carried out within 1 hour after euthanasia of animals. The morphological state of the internal organs of animals was determined visually at autopsy.

The calculation of the acute toxicity parameters of the drugs (LD ₁₀ , LD ₁₆ , LD ₅₀ , 95% confidence interval LD ₅₀ , LD ₈₄ ) was carried out by the probit method.

Assessment of acute toxicity of the substance during subcutaneous administration was carried out on white outbred mice and rats in two stages.

At the first stage, a preliminary study determined the indicative range of tolerated, toxic, and lethal doses of the test substance in two animals per dose (1 male and 1 female). Doses of 0.5, 5, 50, 100, 200 and 300 mcg / kg were tested on mice. All animals at doses: 100, 200 and 300 mcg / kg died. At rats, doses of 0.5, 5, 50, 100, 200 and 300 μg / kg were first tested. The death of animals within 14 days of experience was not. Therefore, additional doses of 400 and 800 μg / kg were tested. All animals at both doses died on the 3-4th day of the experiment.

According to the results of a preliminary study in mice and rats, dose ranges were established for the main study.

In the main study, the substance was administered to mice at doses of 50, 100 and 200 μg / kg, and to rats at doses of 125, 250 and 500 μg / kg (5 males and 5 females per dose).

On the first day after administration of the substance, clinical symptoms of poisoning were absent in mice and rats of both sexes and all experimental groups. On the 2nd day after administration at the maximum and average doses in animals of both species, dose-dependent nonspecific symptoms of intoxication (depressed state, rapid breathing, decreased feed and water intake) and a local irritant effect were noted.

Local irritant effect was manifested as follows. In all animals at the maximum dose, swelling and redness of the skin at the injection site, and necrotic changes in the subcutaneous tissue at the injection site were observed from the second day of the experiment. In animals that received the minimum and average doses, reddening of the skin at the injection site was observed. And starting from 2-3 days of the experiment and until the end of the observation period (14 days), increasing dose-dependent necrotic changes in the subcutaneous tissue were observed with the formation of open nonhealing ulcers in some experimental animals receiving the test substance in doses of ≥50 μg / kg (mouse) and ≥ 125 mcg / kg (rat).

The death of mice and rats was observed starting from the 3rd day of the experiment. The death of the bulk of the animals occurred at night against the background of an oppressed state in 3-6 days.

The parameters of the acute toxicity of the substance for mice and rats are presented in tables 2 and 3. From the obtained results it follows that mice are more than rats sensitive to viscumin. At the same time, in mice and rats, sexual differences in sensitivity to viscumin are not expressed.

In all animals that received the test substance, weight loss was noted in the first week of observation. In the surviving animals, in the 2nd week of the experiment, the general condition improved slightly: they began to consume more food, and partially compensated for the loss of body weight.

A pathomorphological study of mice and rats was carried out in cases of death of animals during the observation period, as well as on the 15th day after the administration of viscumin (substance of the claimed drug, solution for injection, 0.1 mg / ml) (animals from groups in which there were no case and animals receiving the carrier).

During pathoanatomical autopsy of dead mice and rats receiving a subcutaneous preparation of viscumin, pronounced necrotic changes in the skin and subcutaneous tissue were found at the injection site, and in some cases, additional petechial hemorrhages in the adjacent muscle tissue.

In addition, in fallen mice that had previously received a dose of 200 μg / kg of viscumin, dark red contents, presumably blood, were found in the gastrointestinal tract. In this case, damage to the mucous membranes or vessels of the stomach and intestines was not detected.

During a planned autopsy of mice that received the test substance at a dose of 50 μg / kg, changes in the skin and subcutaneous tissue were established during external examination and examination of corpses. Small densities were determined in the skin areas at the injection site of viscumin in animals, sometimes with ulceration on the surface of the skin, as well as petechiae in the underlying subcutaneous tissue and muscle tissue. In mice that were injected with the media, changes in the skin and adjacent tissues were not detected. No other differences between experimental and control mice were found. With necropsy, the organs of the thoracic and abdominal cavities of mice in the experimental and control groups had an anatomically correct location and normal macrostructure; no macroscopically distinguishable pathological changes were found.

When a postmortem autopsy was performed on rats receiving a dose of 250 mcg / kg of viscumin, external examination and examination of corpses revealed significant changes in the size and severity of signs of changes in the skin and subcutaneous tissue. So, in the skin areas at the injection site of the test substance in animals, extensive post-necrotic lesions covered with dry crusts of color from dark gray to black and ranging in size from 4.5 × 1.5 cm to 7.0 × 3.5 cm were determined. males and two females under the described crusts were found foci with creamy contents that had an ichorous odor, which, apparently, was a consequence of infection of necrotic tissues with pyogenic bacteria. In rats to which the vehicle was injected, no changes in the skin and adjacent tissues were detected. No other differences between experimental and control rats were found. With necropsy, the organs of the thoracic and abdominal cavities of rats of the experimental and control groups had an anatomically correct location and normal macrostructure; no macroscopically distinguishable pathological changes were found.

Thus, a pathomorphological study found a pronounced local irritant effect of viscumin when administered subcutaneously in tested concentrations and doses to mice and rats, leading to deep necrosis of the skin and adjacent tissues. Damage to the macrostructure of the internal organs of animals has not been established.

On white outbred mice and rats, the acute toxicity of viscumin (the substance of a new drug) was studied with a single subcutaneous injection.

According to the results of clinical observations, dose-dependent nonspecific symptoms of intoxication (depressed state, rapid breathing, decreased feed and water intake) and local irritating effect were revealed. The death of mice and rats was observed starting from the 3rd day of the experiment. The death of the bulk of the animals occurred at night against the background of an oppressed state in 3-6 days.

According to animal death data, the parameters of acute toxicity of the substance (LD ₁₀ , LD ₁₆ , LD ₅₀ , 95% confidence interval LD ₅₀ , LD ₈₄ ) for mice and rats were established in experiments.

Mice were found to be more sensitive to viscumin than rats. In mice and rats, gender differences in viscumin sensitivity are not pronounced. The minimum LD ₅₀ values of viscumin for female mice (59 μg / kg) and female rats (266 μg / kg) exceed the expected single human therapeutic dose by subcutaneous administration (5 ng / kg) by more than 11,800 times and 53200 times, respectively.

A pathomorphological study found a pronounced local irritant effect of viscumin when administered subcutaneously in tested concentrations and doses to mice and rats, leading to deep necrosis of the skin and adjacent tissues. Damage to the macrostructure of the internal organs of animals has not been established.

Despite the presence of local irritant effects in excessive doses (≥50 μg / kg in mice and ≥125 μg / kg in rats), the substance has a high therapeutic index and has no contraindications for conducting clinical trials.

In the second part of the acute toxicity study of the MLI preparation, white outbred mice and rats were injected subcutaneously with doses of 50, 500 and 5000 ng / kg viscumin. The animals of the control groups were injected with a carrier - phosphate buffer with a pH of 8.0 (dilution solution) - in the same volume as the drug in the maximum dose.

A pathomorphological study was performed on the 15th day after drug administration. Necropsy was performed on mice and rats that received the study drug in the maximum dose that did not cause the death of the animals — 5000 ng / kg, as well as all animals that received the vehicle.

A pathomorphological study did not establish morphological signs of any damaging effects of the drug on the organs and tissues of experimental rats and mice. Also, at the injection site in no case were any signs of local irritant effect found.

The developed drug has a high therapeutic index and has no contraindications for conducting clinical trials.

The study of chronic toxicity of the developed drug MLI in rats after subcutaneous administration for one month

The experiments were carried out on white outbred rats to assess the nature and severity of the possible damaging effect of the drug on the rat organism with repeated subcutaneous administration.

The test drug was administered to rats for one month subcutaneously 2 times a week after 2-3 days at doses of 0, 5, 50 and 500 ng / kg for viscumin. Each dose of the drug was tested on 10 males and 10 females.

During the course of administration and 14 days of the recovery period, the general condition of the animals was assessed by their motor activity, feed and water consumption, the condition of the coat and mucous membranes, and body weight. 24 hours after the end of the administration course and after 14 days of the recovery period, biological samples were selected according to the experimental design at each time for hematological, biochemical and pathomorphological studies.

During the monthly course of administration and 14 days of the recovery period, there were no clinical symptoms of intoxication and death of the animals. The general condition of the animals in the experimental and control groups did not differ. In all groups, the animals were active, willingly ate food. The dynamics of changes in body weight in animals of all groups was positive. There were no statistically significant differences in the mean group values of body weight of animals of the experimental groups relative to the control.

Analysis of the data of hematological studies, biochemical studies of rat blood serum, pathomorphological studies, organ mass ratios, comparative microscopic studies of histological preparations of animal organs and tissues shows that the preparation, when administered subcutaneously for one month in the studied doses, does not cause signs of general toxic and local irritating effect after a monthly course of subcutaneous administration to rats, and at the end of the recovery period.

Study of the effect of the developed MLI drug on vital systems (nervous, respiratory, cardiovascular) with its subcutaneous injection

The study evaluated the effect of the substance of the drug “ViskulaN” - “Viscumin, plant lectin MLI from Viscum album” on the functions of the nervous, circulatory and respiratory systems with repeated subcutaneous administration of CD-I mice to males and females.

The test substance was administered in three doses (10 ng / kg, 100 ng / kg and 1000 ng / kg). A dose of 10 ng / kg (conditionally therapeutic) is close to the proposed therapeutic dose of a person and to the minimum doses at which the therapeutic effect is observed (taking into account conversion per unit surface area of the mouse body (mg / m ² )), a dose of 1000 ng / kg ( conditionally toxic) taken as subtoxic. A dose of 100 ng / kg was taken as an intermediate. The comparison drug was not provided, because the drug "Viskulan" is innovative and has no analogues, allowing comparisons.

As a control substance, a solvent was introduced - a special buffer intended for dilution of the original preparation.

The drug and the control substance were administered once every two days, for 30 days, that is, each animal received a total of 15 doses.

During the administration period, an increase in body weight, feed intake, mortality, locomotor activity, forefoot muscle strength (Grip strength test), central nervous system parameters (assessment of neurotoxicity), and respiration parameters were recorded in animals. On the 31st day after the first injection, the state of the circulatory system was evaluated, after which the animals were euthanized and necropsy; organs examined and weighed, skin samples taken at the injection site.

During the study, no signs of neurotoxicity were found, including no signs of abnormalities in locomotor activity, muscle strength, and other parameters of neurotoxicity. No deviations were found in the dynamics of body weight, feed intake, and respiratory system parameters.

When assessing the state of the cardiovascular system, there were no signs of an effect on blood pressure. There were no statistically significant signs of the effect of the drug on the heart rate in females in all groups and in males who received the drug in a conditionally therapeutic dose (10 ng / kg). Available heart rate data show a tendency to increase for animals that received the drug at doses of 100 ng / kg and 1000 ng / kg, but do not go beyond the acceptable values and cannot be unambiguously interpreted as a manifestation of pathology and, at the same time, cannot exclude the possibility of the effect of the drug on the cardiovascular system.

During the experiment, in individual animals treated with the drug at a dose of 1000 ng / kg, pronounced signs of local irritating effect were observed at the injection site (skin between the shoulder blades). The effect was also observed at the time of necropsy; skin samples were taken from animals. In the groups receiving the drug in doses of 10 ng / kg and 100 ng / kg, no signs of local irritating effect were observed. The death of animals associated with the action of the drug is not installed.

Thus, the study of the drug "Viscumin" did not reveal signs of influence on the nervous and respiratory systems. The possibility of the formation of a pronounced local irritant effect in individual animals with the administration of the drug at a dose of 1000 ng / kg was revealed. Available data on the parameters of the cardiovascular system do not exclude the effect of the drug on this link in physiology, but cannot be unambiguously interpreted as a manifestation of a pathological effect.

Specific Toxicity of MLI

Study of the effect of the developed drug on humoral immunity

The effect of the studied drug on humoral immunity was studied on the model of the immune response to EB. Used two routes of administration of the drug:

- a single intraperitoneal injection at a dose of 10 TD;

- course introduction: subcutaneously 2 times a week for 30 days in doses of 10 TD and 100 TD.

Humoral immunity was evaluated after completion of the course of administration of the drug and after the recovery period, according to the level of hemagglutinins in the blood serum of animals isolated on the 7th day after immunization with EB.

The suppressive effect of the drug at a dose of 100 TD on humoral immunity was shown, however, it had a reversible effect with recovery by 21 days after completion of the course of administration.

Study of the influence of the developed drug MLI on cellular immunity

The effect of the drug on cellular immunity was studied on the model of the immune response to trinitrobenzenesulfonic acid (TNBS). The study drug was administered subcutaneously at doses of 10 TD and 100 TD 2 times a week for 30 days. Hapten TNBS was administered on the day of completion of the drug administration and after the recovery period (14 days after the last injection). Cellular immunity was assessed in the test paw swelling of the mouse on the 6th day after injection of TNBS, according to the index of IR calculated by the formula. It was found that the claimed drug does not affect cellular immunity.

Study of the effect of the developed MLI preparation on the functional activity of peritoneal macrophages

The effect of the study drug on the functional activity of peritoneal macrophages (PMF) was studied with subcutaneous administration at doses of 10 TD and 100 TD 2 times a week for 30 days. PMF was isolated on the next day after completion of the drug. The indicator of phagocytosis activity (AF is a percentage indicator of the number of macrophages that have absorbed bacteria) and the phagocytosis intensity indicator (IF is an indicator of the average number of bacteria in one phagocyte) were evaluated.

There were no statistically significant differences in the values of the AF and IF indicators between the groups receiving the study drug and the negative control group. Since, after completion of the introduction course, the studied indicators in the experimental groups did not differ from the indicators in the control group, there was no need to evaluate them after the recovery period.

Thus, the MLI preparation does not affect the phagocytic activity of macrophages.

The study of the mutagenic effect of the developed drug MLI with a single subcutaneous injection

With a single subcutaneous injection, the mutagenic effect of two doses of the drug (according to the content of the active substance) was studied: 1.2 ng / individual (1 TD) and 20 ng / individual (1/5 LD ₅₀ ). Animals from the negative control group were injected once subcutaneously with phosphate buffer at pH 8.0 (dilution solution). Animals from the positive control group were injected once intraperitoneally with cyclophosphamide at a dose of 20 mg / kg. The study was conducted on males.

It was shown that with a single subcutaneous injection in mice at doses of up to 20 ng / individual (1000 ng / kg), the mutagenic effect of the inventive preparation in the test for accounting for bone marrow chromosome aberrations was not established in mice according to the content of the active substance.

The study of the mutagenic effect of the developed drug MLI with four subcutaneous administration

With four subcutaneous injections, the mutagenic effect of one dose of the drug was studied: 1.2 ng / individual by the content of the active substance (1 TD). The drug was administered at intervals of 24 hours. The animals from the negative control group were injected four times subcutaneously with phosphate buffer with a pH of 8.0 (dilution solution). The study was conducted on males and females.

In the analysis of cytogenetic preparations of the bone marrow of mice after four times subcutaneous administration of the studied drug, there was no statistically significant increase in the number of cells with structural chromosome abnormalities compared with the negative control groups. No cells with multiple aberrations were detected. Identified chromosomal aberrations are single or paired fragments of chromosomes. The proportion of damaged cells in mice did not reach 4%: 2.00 ± 0.71% (females) and 1.40 ± 0.55% (males) cells with chromosomal aberrations were detected in the experimental groups, in the corresponding negative control groups - 1 , 60 ± 0.89% and 1.80 ± 0.45% of cells with chromosomal aberrations.

Thus, with four subcutaneous injections of mice at a dose equivalent to the therapeutic one, calculated by the ratio of the surface area of the human and mouse body, the mutagenic effect of the drug in the test for accounting for bone marrow chromosome aberrations has not been established.

The study of the allergenic effect of the developed drug MLI in mice

The drug was administered to mice subcutaneously 2 times a week for 30 days, HRT was evaluated 5 after the completion of the administration course, and GNT was evaluated 14 days after the completion of the administration.

To detect HRT in mice, a paw edema test was used. The reaction index (IR) of HRT was determined by the difference in weight of the experimental and control paws 24 hours after the introduction of the resolving dose (RD).

As a result of the study, the allergenic effect of the claimed drug in mice was not detected.

The study of the allergenic effect of the developed drug MLI in guinea pigs

For the sensitization of guinea pigs, two administration schemes were used: 1) subcutaneous administration 2 times a week for 30 days; 2) three times administration: the first - subcutaneously, the second and third - in a day - intramuscularly.

To identify hypersensitivity in guinea pigs, the conjunctival test method and the general anaphylaxis reaction were used.

The conjunctival test method tested albino guinea pigs receiving the drug subcutaneously for 30 days. When conducting a conjunctival test, there were no signs of animal anxiety and changes in the condition of the lacrimal duct and conjunctiva during the first 30 minutes, as well as 24 and 48 hours after the introduction of the resolving dose. By the conjunctival test method, an allergic reaction to the claimed drug was not detected in any animal that received a course of subcutaneous administration of the drug in doses up to 10 TD.

The reaction of general anaphylaxis was carried out on guinea pigs sensitized according to the scheme: the first injection was subcutaneous, the second and third one day later intramuscularly. During the reaction of general anaphylaxis, the state of the animals was recorded within an hour after intracardiac administration of the resolving dose in excess of the total sensitizing dose. The reaction of general anaphylaxis was negative in all animals: 0 points on the Weigle index table.

Thus, the allergenic effect of the claimed drug in guinea pigs has not been identified.

Determination of the toxicity of the studied drug MLI on test strains of salmonella dilution method

When setting the Ames test in test tubes with selective semi-enriched agar (0.6%), 0.1 cm ³ suspensions of the test drug were added at the required concentrations, then 0.1 cm ³ suspensions of the studied bacterial strains in 2.5% DMSO solution were added. dissolved in physiological saline. After that, 0.5 cm ^{3 of a} microsomal activating mixture was introduced into the samples. In control variants, suspensions of the test substance were replaced with an equal volume of a 2.5% DMSO solution. The contents of the tubes were quickly mixed and poured onto the surface of the lower minimum agar. The time interval between introducing a microsomal activating mixture into the samples and pouring semi-liquid agar into plates did not exceed 5-10 seconds.

The cups were left at room temperature for 30-40 minutes and after complete solidification of the agar was transferred to a thermostat (37 ° C). Analysis was carried out after 48-72 hours of incubation. As positive controls, substances that induce mutations in the corresponding test strains in the presence or absence of activation conditions were used.

The results of the studies showed that the collection strains of Salmonella typhimurium correspond to their passport characteristics that are relevant for the work on assessing the mutagenicity of a drug in the form of film-coated tablets containing metformin and sibutramine as active substances.

Cytotoxicity

At the first stage of the study, the cytotoxicity of the test sample was evaluated. The results of the studies showed that in the range of all studied concentrations of 0.1-1000.0 μg / cup, the studied drug did not exert a cytotoxic effect on the strains of Salmonella typhimurium TA97, TA98, TA100.

Mutagenicity Assessment for MLI

When conducting the Ames test in each control and experimental variants, three cups were used. Mutants inducing mutations in the corresponding test strains were used as positive controls. When setting negative controls, instead of the test substance, equal volumes of a 2.5% DMSO solution were added to the plates.

Positive controls worked for both the strains and the performance of the microsomal mixture. The experimental variants containing the drug were not significantly (p = 0.95) did not differ from the variants without the drug (negative controls).

Thus, as a result of experimental studies evaluating the potential mutagenic activity of the claimed drug, it was found that the studied drug and its metabolites do not induce histidine prototrophy in his ^- Salmonella typhimurium revertants (strains TA97, TA98, TA100) as in conditions with metabolic activation by the S9 fraction of rat liver homogenate, and without it.

Monitoring the biological activity of MLI at a concentration of 0.1 mg / ml N-glycosidase activity

A specific activity of viscumin A-chain is the hydrolysis of the N-glycosidic bond at the 4324 position of the sarcin loop of the 28S ribosomal RNA. The method for assessing this activity of viscumin is based on the determination of destroyed ribosomes in the cell using real-time PCR. For this, Caco2 cells were cultured with an intact nutrient medium and containing MLI at a concentration of 1.04 × 10 ^-8 M for 6 hours in a CO ₂ incubator (37 ° C, 5% CO ₂ ). Then, the medium with the MLI preparation was taken from each cell well almost to dryness. Cells were lysed by the addition of Qiazol Lysis reagent (Qiagen, Germany), incubated for 5 min at room temperature. Next, RNA was isolated from the obtained lysates. Only high quality RNA (RIN ≥7) was taken for reverse transcription and subsequent analysis.

Reverse transcription was performed using the commercial SuperScriptVILO ™ cDNASynthesisKit kit (Invitrogen, USA). The quantification of the content of modified ribosome fragments in the samples was carried out using PCR-RV. They acted according to the following protocol:

1) The cDNA of each sample was diluted 1000 times.

2) Two pairs to different parts of the 28S RNA subunit were used as primers:

CLR_L: 5'-AGGTGGGGAGTTTGACTGGG-3 '

CLR_R: 5'- TCCACGGGAGGTTTCTGTCC-3 '

RIP_L: 5'-TGCCATGGTAATCCTGCTCAGTA-3 '

RIP_m_R: 5'-TCTGAACCTGCGGTTCCACA-3 '

CLR - primers that are annealed next to the modified nucleotide, but do not affect it - control primers of the distal fragment.

RIP_m - primers for the modified sequence, allow to generate a PCR product with modified 28S ribosomal RNA. The amount of RIP_m_R primer in the reaction mixture should be 3 times greater with respect to RIP_L.

3) To prepare the reaction mixtures, the 2.5x reaction mixture for PCR-RV PCR-RV kit was used (Synthol, Russia). Prepared 2 reaction mixtures for 24 wells (with a margin) for each of a pair of primers.

4) An analysis of the output of PCR-RV (values of threshold cycles Ct) was carried out.

For this:

A. Calculated averages

where i are the repetitions of the reactions (i = 1 ... n), j is the number of the biological repeat (j = 1 ... k), Ct (counter) is the threshold cycle for the control pair of primers, Ct (mode) is the threshold cycle for the pair of primers to the modified plot.

B. The average value was calculated for three biological repeats for each pair of primers:

The active substance in the composition of the test solution is considered active if the threshold cycle for the control pair of primers (for the distal fragment) lies in the range Ct (control) = 12 ± 2. The threshold cycle for a pair of primers to the modified portion of the ribosomal RNA Ct (mode) ≤22.

The developed preparation is a solution for subcutaneous administration containing plant-based viscumin lectin (MLI) and a buffer. The MLI protein isolated from white mistletoe leaves belongs to the ribosomine activating proteins, namely it removes the adenine in the sarcinoma loop of the ribosomal RNA, and thus blocks protein synthesis in the cell and causes apoptosis. The antitumor effect of the drug is caused not only by a direct effect on tumor cells, but also by the effect on the cytotoxic links of the immune system.

The studies conducted allow us to conclude that the drug has antitumor activity (inhibition of tumor growth) and immunomodulating effect.

It was established that all tested doses of the drug did not cause the death of experimental animals. The LD ₅₀ values of the preparation for mice and rats of both sexes exceed the maximum tested dose of 5000 ng / kg and, thus, exceed the single therapeutic dose for humans by subcutaneous administration (5 ng / kg) by more than 1000 times. Differences in sexual sensitivity to the drug in the tested doses have not been established. The drug does not have a local irritant effect with a single subcutaneous injection.

A study of the drug showed no signs of an effect on the nervous and respiratory systems. The possibility of the formation of a pronounced local irritant effect in individual animals with the administration of the drug at a dose of 1000 ng / kg was revealed. The available data on the parameters of the cardiovascular system do not exclude the direct or indirect effect of the drug on this link in physiology, but cannot be unambiguously interpreted as a manifestation of the pathological effect.

To confirm the stability of the obtained plant lectin, stability studies of the concentrate were carried out for the preparation of a solution for subcutaneous injection with a long-term storage test of +2 - + 8 ° C for 2.5 years in three series of the drug. The results are presented in table 4.

Over 2.5 years of storage of the drug - a concentrate for the preparation of a solution for subcutaneous injection, the studied parameters comply with the specifications.

Numerous data indicate that the antitumor effect of MLI is based on the launch of immunogenic apoptosis and endoplasmic reticulum stress (ER-stress) - stimulators, the so-called immunization in vivo (Aaes T.L., Kaczmarek A., Delvaeye T., et al. Vaccination with Necroptotic Cancer Cells Induces Efficient Anti-tumor Immunity // Cell reports. 2016. Vol. 15. No. 2. P. 274-287). In this case, as was shown above, the time of the presence of lectin in the tissue is apparently important, whereas an increase in the local concentration of MLI can offset the immunomodulating effect. In order to possibly increase the retention time of MLI protein by the tissue after administration, a conjugate of ferromagnetic magnetite and MLI nanoparticles (LPF-MLI) was obtained and characterized. As part of the study of the pharmacological properties of the low-pass filter, MLI, the elimination of the conjugate in mice with intratumoral administration was studied.

Methodology for the study of the drug LPF-MLI

Reagents and materials, L-15 cell culture medium (Invitrogen, USA), FBS (PAA, Austria), amniotic (antibiotic-penicillin, streptomycin and amphoteracin B; Invitrogen, USA), Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-Free (Coining, USA).

Obtaining plant lectin MLI and conjugate MLI with ferromagnetic magnetite nanoparticles. Native plant MLI lectin was obtained from the green parts of the white mistletoe (Viscum album) of the claimed method. Ferromagnetic nanoparticles and their conjugate with MLI с were also obtained as described in (Khutornenko A.A., Gerasimov V.M., Sakharov D.A. Preparation and study of the internalization of the conjugate of viscumin-ferromagnetic nanoparticles with human glioblastoma cells A172 // Bulletin experimental biology and medicine. 2015. Vol. 160. No. 12 / P. 797-801). The number of MLI molecules conjugated to a single nanoparticle and retaining activity was determined by competitive binding of the fetuin protein to immobilized ligand in the presence of various concentrations of lactose (Manevich E.M., Tonevitsky A.G., Bergelson LD The binding of the B-chain of ricin to Burkitt lymphoma cells // FEBS letters. 1986. Vol. 194. No. 2. P. 313-316.).

Cell culture cultivation MDA-MB231 model. MDA-MB231 human mammary adenocarcinoma cells were cultured for 5-7 passages on L-15 medium supplemented with 10% FBS and amniotic. Before injection into animals, tumor cells were removed from the substrate with trypsin / versene solution and after 5 min centrifugation at 1000 rpm, the pellet was carefully resuspended in serum-free medium, bringing to a concentration of 4-5 × 10 ⁶ cells in 100 μl.

The survival of hybridoma cells, as well as the MDA-MB231 line at various MLI concentrations, was assessed by MTT for 24 hours, as described in [Moisenovich, 2004].

Xenograft model. The model of orthotopic xenograft of human breast carcinoma cells MDA-MB231 to immunodeficient mice of the SCID line (Severe combined immunodeficient mouse) was used. Female mice were subcutaneously orthotopically injected with MDA-MB231 cells in a mixture with matrigel in a 1: 1 ratio in an amount of 100 μl (5 × 10 ⁶ cells). After administration for 42 days, the growth of the tumor node was observed. The study involved animals with tumor site sizes less than 500 mm ³ (small tumor).

Investigation of the biodistribution of the conjugate of ferromagnetic magnetite nanoparticles and MLI. Animals were injected intratumorally once with 10 μl of the LPF-MLI preparation (MLI concentration was 6 × 10 ^-9 M) or a control preparation of nanoparticles in an equivalent amount in terms of iron content. As a control preparation of nanoparticles, ferromagnetic magnetite nanoparticles coated with polyethylene glycol (LPF) were used. The distribution of the preparations was monitored by magnetic resonance imaging (MRI) by the change in the signal of the magnetic resonant substance (LPF) after 0.15 minutes, 2 and 24 hours after drug administration.

Some animals, 3 days after a single intratumoral injection of low-pass filter MLF-MLI, were subjected to eftanasia, tumor tissue was taken and fixed in 4% formalin solution. Then, standard tissue wiring was performed, followed by pouring into paraffin blocks for histological analysis.

Histological analysis. Sections 4 μm thick were mounted on positively charged glasses (SuperFrost Plus, ThermoFisher Scientific). Hematoxylin and eosin staining was performed according to standard procedures.

Research results

To assess the effect of conjugation of MLI with ferromagnetic nanoparticles on the properties of viscumin, the conjugate cytotoxicity for hybridoma cells was studied. The results presented in FIG. 4, indicate the preservation of the cytotoxic activity of MLI in the conjugate.

The MDA-MB-231 line was chosen as a model of tumor cells. This line is a thrice-negative human breast adenocarcinoma (without estrogen, progesterone receptors and with reduced expression of Her2 receptors) and has increased resistance to various antitumor drugs. The sensitivity of different cells to MLI is known to vary greatly. Therefore, the cytotoxic properties of MLI were first evaluated for MDA-MB-231 cells. The IC ₅₀ is shown to be 3.4 × 10 ⁻⁸ M.

Female SCID immunodeficient mice with orthotopically inoculated with a subcutaneous tumor MDA-MB231 were once injected intratumorally with 10 μl of a control preparation of ferromagnetic nanoparticles that did not contain the MLI vector ligand, or an LPF-MLI preparation containing an equivalent amount of iron. The concentration of MLI in the injected LPF-MLI sample was 6 × 10 ^-9 M. The preliminary data obtained indicate that the immunomodulating properties of MLI are manifested in concentrations lower than cytotoxic. The dose of the LPF-MLI preparation used for intratumoral administration satisfies this condition.

The change in the content of the low-pass filter-MLI and the control drug in the tumor was monitored using magnetic resonance imaging (MRI). The dynamics of changes in the MRI signal in the tumor with respect to the signal in the muscle, as the most stable magnetocontrast tissue, is shown in FIG. 5. The largest decrease in the relative intensity of the MRI signal in the tumor, which indicates the accumulation of magnetocontrast substance, was observed both for the control low-pass filter and for low-pass filter-MLI after 15 minutes. However, at the 2 hour time point, differences in the rate of excretion of the control preparation of nanoparticles and the preparation containing MLI were already manifested, when a high degree of accumulation for low-pass filter MLI was noted (p <0.08).

Previously, the biodistribution of native plant MLI in mice was studied with subcutaneous administration, it was shown that after 2 hours no more than 8% of the administered dose remained at the injection site, and after 24 hours less than 1%. In the case of the LPF-MLI, the content of the conjugate in the tumor node decreases 1.4 times after 2 hours, and 3.5 times after 24 hours, compared with the value after 15 minutes. In this case, the relative intensity of the tumor tissue by 10% remains lower than before the introduction of the drug. Thus, the elimination rate of the LPF-MLI conjugate is apparently much lower than that of native plant MLI. Histological analysis three days after the administration of the drug did not reveal significant differences in the state of the tissue of the tumor nodes in the case of injection of the control drug and the LPF-MLI conjugate.

Thus, the data obtained indicate that conjugation of MLI with ferromagnetic magnetite nanoparticles leads to a decrease in the rate of elimination of lectin from the tumor node. In addition, the presence of the conjugate in the doses used in the study does not lead to undesirable changes in the state of the tumor tissue. This suggests the possibility of fine regulation of the antitumor immune by triggering immunogenic apoptosis, the induction of which was demonstrated by us on cell lines.

Claims (13)

1. A method for isolating ML1 protein from white mistletoe Viscum album, comprising: - obtaining a finely divided suspension of dried leaves of white mistletoe in a 0.25 M NaCl solution, taken in an amount sufficient for extraction; - carrying out the first stage of purification of the suspension from impurities not belonging to the ML protein group by introducing into the suspension ammonium sulfate at a rate of 60-80 g per 100 ml of extract, keeping the suspension obtained to precipitate plant proteins, including the ML protein group, followed by separation of the resulting draft; - dissolution of the obtained precipitate in a NaCl solution, which is taken in an amount sufficient to completely dissolve the precipitate; - carrying out the second purification step to isolate the proteins of the ML group from the resulting vegetable protein solution by affinity chromatography using a lactosyl-containing sorbent and as a buffer of a 0.25 M NaCl solution; - the third stage of purification for the isolation of MLI protein from a solution of a mixture of proteins of the ML group using ion-exchange chromatography, using a cation exchange resin of the monoS type with a sorbent particle size of not more than 10 microns as a sorbent; then, the obtained fraction of purified MLI protein is transferred to phosphate-buffered saline with physiological pH and isotonic composition. 2. The method according to p. 1, characterized in that the aging time of the suspension is at least 16 hours 3. The method according to p. 1, characterized in that the translation of the purified MLI protein into phosphate-buffered saline with physiological pH and isotonic composition is carried out using dialysis using dialysis membranes of the type Slide-A-Lyzer ™ Dialysis Cassettes with molecular weight cutoff 10-20 kDa, or by gel filtration using a HiTrap Desalting Column column. 4. The method according to p. 1, characterized in that it comprises concentrating the MLI protein solution to a value of 2 mg / ml with an acceptable deviation of up to 20% using a concentrating system with a membrane having pores ranging in size from 10 to 30 kDa. 5. A pharmaceutical composition for injection, having an immunostimulating and antitumor effect, characterized in that it is a buffer and an active component, where the ML1 protein obtained by the method according to claim 1 is used as the active component, and an aqueous isotonic saline solution is used as a buffer physiological pH, while the content of plant protein ML1 in 1 ml of solution is 0.04-0.15 mg. 6. The pharmaceutical composition according to claim 5, characterized in that it further comprises auxiliary substances for stabilizing the protein, reducing protein sorption. 7. The pharmaceutical composition according to claim 5, characterized in that for the preparation of an aqueous isotonic saline, sodium hydrogen hydrate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, and also sodium chloride are used. 8. A pharmaceutical composition for injection, having an immunostimulating and antitumor effect, characterized in that it is a buffer and an active component, where the ML1 protein obtained by the method of claim 1, conjugated with magnetite ferromagnetic nanoparticles, is used as the buffer as an active component. - an aqueous isotonic saline solution with a physiological pH, while the content of MLI plant protein in 1 ml of solution is 0.1-10 μg, ferromagnetic nanoparticles - 0.1-1 mg.

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